copyright by Zackary Johnson

2000

iv

Abstract

Marine photosynthesis accounts for approximately half of all global primary

production. It is crucial in providing the base of the marine food chain and a

critical component of the global carbon cycle. Because all but the upper few

meters of the ocean is light-limited with respect to photosynthesis,

photosynthesis and photosynthetic efficiency are often described using the

photosynthesis-irradiance curve. This curve is convenient to measure and varies

significantly with the environment. Nevertheless, the photosynthesis-irradiance

relationship only provides an empirical description of photosynthetic efficiency

and has little diagnostic and prognostic capacity. To understand modes of

photosynthesis-irradiance variability, I focus on photosystem II-specific

processes as a potentially dominant determinant of photosynthesis-irradiance

magnitude and structure. I use the marine diatom Skeletonema costatum as a

model marine phytoplankter and probe the role that photosystem II has in

determining photosynthesis-irradiance relationships in the context of very low

background light, photoacclimation and nitrogen limitation. I also use the fresh

water green alga Chlamydomonas reinhardtii that is mutant in the xanthophyll

cycle to investigate non-photochemical quenching. I relate photosystem II-

specific (fluorescence) measures in the presence of a background irradiance

gradient of conversion efficiency and cross-sectional area to observed carbon

uptake and oxygen evolution-based measures of photosynthesis-irradiance,

photosynthetic unit cross section, photosynthetic unit size and photosynthetic unit

turnover. These results demonstrate that under a variety of environmental

v

situations characteristic of natural oceanic variability, photosystem II properties

describe well the relative structure of photosynthesis-irradiance curves.

However, other processes not associated with photosystem II (or characterized

by fluorescence) and unique to each environmental situation can significantly

impact the magnitude of photosynthetic rates and efficiency. Combined, these

results demonstrate the utility of photosystem II properties as a function of

background irradiance in describing the irradiance structure of photosynthetic

efficiency, but also demonstrate the limitations of fluorescence techniques.

vi

Acknowledgements

This dissertation greatly benefited from the advice, support and encouragement

of many people. My advisor Dr. Barber, who guided me as an apprentice, gave

me professional and financial latitude to explore new ideas, make mistakes (and

profit from them) and encouraged me to grow as a thinker and scientist. My

committee members, Ramus, Kamykowski, Siedow and Falkowski supported me

academically, technically and financially and their input greatly advanced this

dissertation. I thank Lisa Borden, Anna Hilting, Tim Boynton, Patty Nolin and Bill

Hunnings as well as many others for technical and logistic support. I also thank

Dr. Sallie Chisholm, Melanie and Ronald Johnson and many others who kept me

motivated and encouraged. And thank you to all of my graduate student friends

at the Duke Marine Laboratory - you were the salt on my french fry of graduate

school life.

Thank you.

vii

Table of Contents

Abstract

iv

Acknowledgements

vi

List of Figures

viii

List of Tables

xi

Chapter I

Introduction 1

Chapter II

On the reduction of photosynthetic quantum efficiency at low irradiances

12

Chapter III

Photophysiological characterization of wild type and non-photochemical quenching mutants of Chlamydomonas reinhardtii (WT, npq1, npq2) using standard and novel modulated fluorescence techniques

48

Chapter IV

Mechanisms and consequences of photoacclimation in Skeletonema costatum

86

Chapter V Mechanisms of reduction in photosynthetic efficiency under nitrogen-limitation in Skeletonema costatum

114

Chapter VI

Conclusions 159

Appendix 1

Definition of Symbols and Abbreviations 168

References

171

Biography 189

viii

List of Figures

Figure page II-1 Growth and Measurement Spectra

38

II-2 Contemporary models of photosynthesis-irradiance expressed in (a) P-E, (b) φ-E and (c) φ-log(E) formats

39

II-3 (a) P-E, (b) P-E (log E format) and (c) φC-E (log E format) for S. costatum

40

II-4 P-E and φ-E measured for a variety of cultured phytoplankton species using oxygen evolution (a,b) and carbon uptake (c,d)

41

II-5 P-E and φ-E measured for an Arctic field population using oxygen evolution (a,b) and carbon uptake (c,d)

42

II-6 (a) P-E and (b) φC-E measurements with different light colors for S. costatum and D. tertiolecta

43

II-7 Models of P-E with varying amounts of low light φ reduction expressed in (d) P-E, (e) φC-E and (f) φC-log(E) formats

45

II-8 (a) P-E, (b) P-E (log E format), (c) φC-E (log E format) and (d) fraction of maximum slope as a function of number of points used in linear regression for blue-grown and blue-measured S. costatum

46

II-9 Models of P-E with varying amounts of respiration correction error expressed in (d) P-E, (e) φC-E and (f) φC-log(E) formats

47

III-1 Schematic block diagram of the BIG-FRRf

76

III-2 Representative BIG-FRRf output data from WT C. reinhardtii

77

III-3 (a) Chlorophyll a-specific absorption spectra for WT, npq1 and npq2 C. reinhardtii and (b) percent deviation from WT

79

III-4 Photosynthesis-irradiance curves for WT, npq1, and npq2 C. reinhardtii

80

III-5 Representative fluorescence excitation curves from BIG-FRRf

81

ix

III-6 Representative fluorescence decay curves from BIG-FRRf

82

III-7 Fluorescence-derived photosynthetic parameters (Fo, Fm, Fv/Fm, σPSII) as a function of background irradiance for WT, npq1 and npq2 C. reinhardtii

83

III-8 Relative between measured and fluorescence-predicted photosynthetic rates

85

IV-1 Photosynthesis-irradiance relationships for S. costatum

grown at four different irradiance intensities

105

IV-2 φC-E relationships for S. costatum grown at four different irradiance intensities

106

IV-3 Fluorescence-derived photosynthetic parameters (Fo, Fm, Fv/Fm, σPSII, 1/τPSII) as a function of measurement irradiance for (a) very high, (b) high and (c) low light grown S. costatum

107

IV-4 Fv/Fm-irradiance relationships for S. costatum grown at three different irradiance intensities

110

IV-5 σPSII-irradiance relationships for S. costatum grown at three different irradiance intensities

111

IV-6 Relationship between φC and Fv/Fm for all background irradiances

112

IV-7 Relationship between measured and fluorescence-predicted photosynthetic rates

113

V-1 Time course of growth rate properties under N-limitation

140

V-2 Time course of pigmentation properties under N-limitation

141

V-3 Time course of absorption properties under N-limitation

142

V-4 Time course of PSII concentrations under N-limitation

143

V-5 Time course of photosynthetic parameters derived from P-E curves under N-limitation

144

x

V-6 Photosynthesis-irradiance curve comparison for different

levels of N-limitation

145

V-7 Time course of photosynthetic efficiency under N-limitation

146

V-8 Quantum yield-irradiance curve comparison for different levels of N-limitation

147

V-9 Fluorescence-derived photosynthetic parameters (Fo, Fm, Fv/Fm, σPSII) as a function of irradiance for (a) N-replete, (b) moderately N-limited and (c) severely N-limited conditions

148

V-10 Time-course of fluorescence-derived parameters measured in the dark and with background irradiance at 108 µmol quanta m-2 sec-1

151

V-11 Fv/Fm-irradiance curve comparison for different levels of N-limitation

152

V-12 PSII functional cross sectional area-irradiance curve comparison for different levels of N-limitation

153

V-13 Relationships between different measures of photosynthetic efficiencies (Fv/Fm(0), φC,max and α*)

154

V-14 Relationship between Pbmax and its components

155

V-15 Relationship between φC and Fv/Fm for all background irradiances

156

V-16 Relationship between measured and fluorescence-predicted photosynthetic rates

157

V-17 Time course comparison between measured and predicted light saturated photosynthetic rates

158

xi

List of Tables

Table page II-1 Impact of chromatic acclimation on photosynthetic

parameters derived from P-E

36

II-2 Impact of chromatic acclimation on photosystem concentrations and ratios

37

III-1 Parameters of photophysiology for WT, npq1 and npq2 C.

reinhardtii 75

IV-1 Photoacclimation of photosynthetic biomass and

physiological parameters

104

VI-1 Relative success of PSII in describing photophysiological

variability 167

1

Chapter I

Introduction

2

The net primary production of the biosphere amounts to approximately

100 petagrams of carbon per year with the oceans accounting for about 40-50%

of this enormous biogeochemical flux (Antoine et al., 1996; Field et al., 1998).

Although marine primary production is difficult to directly quantify, the importance

of understanding it is two-fold: (1) as the base of the marine food web and (2) as

a major component in global biogeochemical cycles (Sverdrup et al., 1942;

Bougis, 1976; Falkowski, 1994). Photosynthetic rates are regulated by a function

of biomass, irradiance and efficiency. Because biomass and irradiance can be

measured with relative ease, presently the accuracy of the quantification of

global marine photosynthesis is limited by the estimation of photosynthetic

efficiency (Yoder et al., 1993; Behrenfeld and Falkowski, 1997). Understanding

historic or future variations in marine primary production may also require a

working knowledge of the mechanisms by which environmental factors regulate

marine photosynthetic efficiency (Martin, 1990; Sarmiento and Bender, 1994;

Sarmiento and LeQuere, 1996).

Although potentially important, the understanding of the regulation of in

situ marine photosynthetic efficiency is in its infancy (Behrenfeld and Falkowski,

1997). This state largely stems from two classes of difficulties including

inadequate sampling and non-mechanistic characterization of efficiency. The

first area of difficulty is derived from the spatial and temporal variability of the

marine environment, making it logistically difficult to assess the effects of forcing

on efficiency over the necessary space and times scales (Magnuson, 1990;

Dickey, 1991; Powell and Steele, 1995). Because photosynthetic efficiency can

3

vary significantly on time scales of less than 1 hour and over small (<1km) spatial

scales, standard estimates of photosynthetic efficiency made using radiotracers

that are performed with incubation periods of up to one day are not suited for

extensive spatial and temporal coverage (Steemann Nielsen, 1952; Harding et

al., 1981; Falkowski and Raven, 1997). Further, the response of photosynthetic

efficiency to an environmental regulator is often not instantaneous and can lag its

cause significantly (Post et al., 1984; Geider et al., 1993). These coverage and

time-dependent processes complicate the linkage between proximal regulation

and photosynthetic efficiency.

The second general source of difficulty to understanding photosynthetic

efficiency variability stems from most estimates centering on aggregate

responses (ex. Eppley, 1972; Balch et al., 1992; Behrenfeld and Falkowski,

1997). These measures of photosynthetic efficiency focus on broad responses

and use integrated water column averages, maximum water column efficiencies

or photosynthesis-irradiance relationships to provide an integrated photosynthetic

efficiency response to the effect of the environment (Morel, 1991; Karl, 1999;

Johnson and Howd, 2000; Marra et al., 2000). However, these process-

integrated measures of photosynthetic efficiency are composed of multiple steps

(absorption, photosynthetic light utilization, photosystem-specific processes,

photosynthetic electron transport chain efficiency, dark reactions, etc.) that can

vary independently. Thus, the mechanism by which environmental forcing

regulates the variability of aggregate estimates of efficiency remains difficult to

interpret because of imbedded complexity. Without a mechanistic

4

understanding, interpreting present day variability and predicting future variability

is not possible.

In this dissertation I focus on overcoming the inherent limitations

associated with non-mechanistic photosynthetic efficiency measurements. In

particular, I concentrate on the photosynthesis-irradiance (P-E) response curve

as a tool to uncover mechanisms of environmental regulation of marine

photosynthetic efficiency (Jassby and Platt, 1976). This photophysiological

characterization technique has been used extensively in both the laboratory and

field to document the response of phytoplankton photosynthetic efficiency to

diverse environments (Cleveland and Perry, 1987; Cullen, 1990; Sosik and

Mitchell, 1994; Babin et al., 1996; Lindley and Barber, 1998). Nevertheless,

despite these successes, the P-E relationship has limited overall diagnostic and

prognostic capability because of the aforementioned multiple steps between the

incident irradiance intensity and carbon fixation.

Towards addressing this limitation, I concentrate on the light reactions of

photosynthesis as a class of analytic components that in part regulate the

structure and magnitude of the P-E curve. Specifically, in this dissertation I am

interested in the role that photosystem II (PSII)-specific descriptions as measured

by room temperature fluorescence have in determining P-E and efficiency-

irradiance magnitudes and structures.

Previous investigations have provided a strong theoretical background for

the relationship of fluorescence (PSII) to photochemistry (Mauzerall, 1972;

Kitajima and Butler, 1975; Papageorgiou, 1975; Butler, 1978; Lazar, 1999). In

5

particular, fluorescence induction (Kautsy and Hirsch, 1931) and decay (Berens

et al., 1985; LaVergne and Trissl, 1995; Kolber et al., 1998) techniques have

been developed for use in natural marine phytoplankton populations to measure

PSII parameters that have strong correlations with photosynthetic rate and

efficiency estimates under a variety of environmental conditions (Falkowski and

Kiefer, 1985; Kiefer and Reynolds, 1992; Kolber and Falkowski, 1992; Falkowski

and Kolber, 1993). These types of measurements provide a step towards

uncovering the mechanisms that comprise the series of possible rate limiting

steps that regulate photosynthesis rates and efficiency. However, previous

investigations have focused on the properties of PSII in the dark and have not

related the changes in PSII-specific properties, such as PSII cross sectional area

and PSII photochemical conversion efficiency, to changes in photosynthetic rates

and efficiency with irradiance. Because many processes, such as state

transitions and non-photochemical quenching that affect PSII are background

light dependent, dark measurements of PSII may not be indicative of processes

occurring in the presence of light (Bonaventura and Myers, 1969; Demmig-

Adams and Adams, 1992).

The relationship between aggregate photosynthesis-irradiance

relationships and PSII-specific properties can be examined following analytic

descriptions of P-E and associated parameters in combination with similar

descriptions of PSII-specific measures. The P-E function can be mathematically

formulated based on Poisson target theory that is formalized by,

6

( )EP

PPSUPSUτσexp1

max

−= (I-1)

PSU

nP

τ894max = (I-2)

where P/Pmax is relative photosynthesis, Pmax is maximal photosynthetic rate (mol

O2 g Chl a-1 sec-1), σPSU is functional cross sectional area of the photosynthetic

unit (PSU) (m2 µmol quanta), 1/τPSU is PSU turnover rate (sec-1), E is irradiance

(µmol quanta m-2 sec-1), n is mol O2 mol Chl a-1 and 894 converts mol to g Chl a

(Dubinsky et al., 1986; Mauzerall and Greenbaum, 1989). Values of σPSU are

defined by and can be determined directly from oxygen flash yield relationships,

( )fPSU EY

Yσexp1

max

−= (I-3)

where Y/Ymax is the relative oxygen flash yield and Ef is the intensity of each flash

(µmol quanta m-2) (Ley and Mauzerall, 1982).

Equations I-1-3 represent a base series of relationships from which other

properties of photophysiology can be derived. For example, σPSU can be

described as a function of n, quantum yield ( max,2Oφ , mol O2 mol quanta-1) and

mean specific absorption ( *ia m2 mg Chl a-1) (Mauzerall and Greenbaum, 1989)

and can be rearranged to solve for the maximum quantum yield,

n

aO

PSU

*max,2

)894.0( φσ = (I-4a)

7

*max, )894.0(2 a

nPSUO

σφ = (I-4b)

where 0.894 converts mol to mg Chl a and µmol to mols quanta. Similarly,

assuming the initial slope of the P-E curve (α*, mol O2 g Chl a-1 hr-1 (µmol quanta

m-2 sec-1)-1) is linear, α* can be related to max,2Oφ and *ia or to σPSU and n by the

following relationships (Geider and Osborne, 1992)

max,**

26.3 Oa φα = (I-5a)

nPSUσα 03.4* = (I-5b)

where 3.6 converts hours to seconds, g to mg and µmol to mol and 4.03 converts

g to mol Chl a and hours to seconds. These series of equations provide a

mathematical formalism that can be tested using independently measured

parameters and variables. Further, the parameters in these equations, which are

diagnostic of integrated responses, can be compared to components (namely

those of PSII) that make up these integrated responses.

For example, σPSU is the gross photosynthetic unit cross section and is

composed of PSII, PSI, fluorescence and heat cross sections (Dubinsky and

Stambler, 1992). Single turnover (ST) fluorescence induction curves permit the

independent determination of the functional PSII cross section (σPSII) (Kolber et

al., 1998). Thus, σPSU and σPSII can be compared and possible linkages and

8

causalities established. This can be extended to include how variability in σPSII(0)

and σPSII-E influence P-E structures (equation I-1).

Similarly, ST-fluorescence induction curves can also be used to assess

the role of PSII in determining quantum yield of photosynthesis (φmax) variability.

Specifically, fluorescence or more precisely, fluorescence yield, can be

mathematically described as,

ptdf

ff Akkkk

k

+++=φ (I-6a)

where φf is the fluorescence yield and kf, kd, kt and kp are the rate constants for

fluorescence, non-radiative decay, excitation transfer to PSI and photochemistry,

respectively, assuming that other loss processes such as phosphorescence are

minimal (Butler, 1972; Butler and Strasser, 1977). The fraction of open PSII

reaction centers, A, modifies the rate constant of photochemistry such that in the

dark when all reaction centers are open (A=1), fluorescence is minimal (φFo) and

at fully saturating light (A=0) fluorescence is maximal (φFm),

ptdf

fFo kkkk

k

+++=φ (I-6b)

tdf

fFm kkk

k

++=φ (I-6c)

By defining φFv or variable fluorescence as,

9

FoFmFv φφφ −= (I-7)

the ratio of φFv/φFm (or Fv/Fm) is related to the photochemical yield, or

photochemical conversion efficiency for PSII,

pptdf

pFmFv Pkkkk

k

Fm

Fvφφ =

+++==/ (I-8)

Similar to relating variability in σPSU to σPSII(0), variability in photosynthetic

quantum yield (2Oφ , mol O2 mol quanta-1), which is a aggregate property of

photosynthesis, can be related to φp (Fv/Fm), which is a photosystem II-specific

property. Values of Fv/Fm and quantum efficiency can also be compared over a

light gradient.

Thus, using these equations as a theoretical background, I focus on

aggregate photosynthetic rate and efficiency measurements like P-E and

quantum efficiency and PSII-specific measurements like σPSII and Fv/Fm made

over a background light gradient. Recognizing that additional components are

also important in determining the photosynthesis-irradiance response, here I

center on PSII-specific properties because they are easily measured and dark

measured PSII properties such as Fv/Fm and σPSII have been shown to co-vary

with aggregate photosynthesis-irradiance responses under some environmental

conditions (Geider et al., 1993). I use previous aggregate efficiency studies,

10

which have separately demonstrated the influence of environmental variables

such as light and nutrients on analytic components and P-E, as a springboard to

investigate the relationship between these PSII components and P-E. Thus, I

investigate the role that each component individually as well as PSII as a

combined unit play in structuring the P-E curve.

This dissertation is nominally divided into two major sections. In the first

section (chapter II), I focus on processes affecting photosynthetic rates at low

measurement irradiances with emphasis on observed reductions in quantum

efficiency at low irradiance in Skeletonema costatum and Dunaliella tertiolecta.

For these analyses, I use both quantum yield-irradiance and photosynthesis-

irradiance relationships along with aggregate and photosystem-specific biomass

and function properties.

In the second section (chapters III-V), I focus on mechanisms regulating

the structure and magnitude of the photosynthesis-irradiance response curve at

higher irradiances in the context of the photosynthetic electron transport chain.

In chapter III, I use wild type and fluorescence (non-photochemical quenching)

mutants of Chlamydomonas reinhardtii to examine the efficacy of a newly

developed modulated fluorometer to describe photophysiology by examining the

relationship between standard photosynthesis-irradiance relationships and

fluorescence-derived, mechanistic parameters of photosynthesis. In chapters IV

and V, I use this fluorescence induction technique in conjunction with additional

plant photophysiology measurements to uncover the effect of growth irradiance

intensity (chapter IV) and nutrient-limitation (chapter V) on analytical components

11

of photosynthesis-irradiance responses and their relationship to total P-E

responses in S. costatum. Finally, in chapter VI, I use the combined analyses

and conclusions from low light investigations (Chapter II), C. reinhardtii (chapter

III), photoacclimation (chapter IV), and nutrient-limitation (chapter V) chapters to

summarize the contribution of photosystem II-specific processes to the overall

photosynthesis irradiance response in the context of marine variability.

12

Chapter II

On the reduction of photosynthetic quantum efficiency at low irradiances

13

Photosynthesis-irradiance (P-E) curves are widely used to describe

photosynthetic efficiency and potential. In this context, there exist

numerous models of P-E that are helpful in describing data. All

contemporary models assume maximal photosynthetic quantum efficiency

(φφ) at low irradiances. But P-E observations from laboratory and field

studies for mixed populations and monotypic cultures made with both

oxygen evolution and carbon uptake techniques suggest that this is not

always the case. In the context of flashing light, several mechanisms have

been proposed to account for these reductions including S-state decay,

respiration and photosystem I (PSI) limitation. Here I investigate this low

light reduction in φφ using continuous light measurements of P-E with the

diatom Skeletonema costatum (Greville) Cleve and the chlorophyte

Dunaliella tertiolecta Butcher as two model phytoplankton that

photoacclimate using different dominant mechanisms. Under the present

experimental setup, S. costatum is affected by the reduction while D.

tertiolecta does not appear to be affected. Reductions for S. costatum are

relieved when measuring P-E using red-dominated light. From

photosystem-specific biomass estimates and P-E measurements made

with blue-green, white and red light I find that the low-light reductions in φφ

are likely dominated by PSI limitation. Using these measurements along

with modeling exercises, I demonstrate that regardless of the mechanism

responsible, the reductions in φφ at low irradiances are not readily

observable using traditional P-E analyses and are absent from PSII

14

descriptions of photosynthetic efficiency. Yet, the reductions in φφ can

result in significant errors (>50%) in the estimation of the initial slope of the

P-E curve and ultimately the maximum quantum yield of photosynthesis.

The combination of these results is discussed in the context of open ocean

spectral irradiance properties.

Introduction:

Photosynthesis-irradiance (P-E) curves have been used extensively to

probe the efficiency and capacity of photosynthesis with respect to light intensity.

These curves, which are constructed using either oxygen evolution or carbon

uptake, provide a convenient evaluation of photosynthesis in the context of

potential sources of variability. P-E curves have been used broadly in both the

field and laboratory to evaluate nutrient limitation, photoacclimation and

taxonomic effects on photosynthesis (Jassby and Platt, 1976; Perry et al., 1981;

Falkowski et al., 1986; Platt et al., 1987; Cullen et al., 1992; Lindley et al., 1995).

These measured P-E curves generally follow a sigmoidal-type functional

form with photosynthesis increasing with irradiance until it saturates at some

higher light level. Many mathematical models have been formulated to describe

the P-E relationship (Blackman, 1905; Smith, 1936; Jassby and Platt, 1976; Platt

et al., 1980; Leverenz et al., 1990; Geider and Osborne, 1992; Henley, 1995).

Although the specific values of the recovered parameters depends greatly on the

exact model used (Frenette et al., 1993; Henley, 1995), all contemporary models

are generally similar in shape and assume that quantum efficiency is maximal

15

and independent of background light for the low light portion of the quantum

efficiency - irradiance curve. Despite these assumptions, there is evidence that

photosynthetic efficiency is not always maximal at low irradiances (Forbush et al.,

1971; Diner and Mauzerall, 1973; Ley and Mauzerall, 1986). Indeed, there are

many biophysical and biochemical intermediates between initial light absorption

and subsequent carbon incorporation that are background irradiance dependent.

A priori, these multiple, non-linear steps could lead to a non-linear relationship

between excitation energy and photosynthetic rate at low irradiances.

Previous observations of reduced efficiency have been attributed to three

alternative, but not mutually exclusive processes including differential respiration

(the so-called Kok effect), S-state decay (S3 to S2) and imbalances in

photosystem excitation (Kok, 1948; Kok, 1949; Kok, 1956; Forbush et al., 1971;

Diner and Mauzerall, 1973). Each of these mechanisms can potentially reduce

the quantum efficiency at low irradiances such that the maximal quantum yield is

observed at higher irradiance levels. Diner and Mauzerall (1973) critically

examined each of the mechanisms using a variety of techniques including

inhibitors and variable frequency flash yields. They concluded that although all

three mechanisms may be acting to some degree in the presence of flashing

light, it is imbalances in photosystem excitation that dominate the reduction of the

quantum efficiency at low irradiances in Chlorella vulgaris and Phormidium

luridium (Diner and Mauzerall, 1973) (see below).

The observation of a reduction in quantum efficiency at low irradiances

violates the constant and maximal quantum efficiency assumption held by

16

contemporary P-E models. This violation may result in errors when using these

types of models in conjunction with data to evaluate photosynthetic efficiency.

Further, although Diner and Mauzerall (1973) deftly probed this reduction in

quantum efficiency in the presence of flashing light for C. vulgaris and P.

luridium, these responses may not be indicative of all natural populations (Healy

and Myers, 1971) or for continuous light P-E curves measured using 14C

techniques. Significant departures from their original findings could result if

estimates of photosynthesis are made using continuous light (P-E), if different

monotypic or mixed populations are used, or if illuminating wavelength differs.

Thus, the aim of this study is two-fold: (1) to examine the predominance of

the reduction in low light quantum efficiency in the context of different

phytoplankton species, incubation times and colors of continuous light (P-E)

using both oxygen and carbon-14 techniques and (2) to determine the effect of

the reduction on the interpretation of P-E data. My working hypothesis is that

similar to the mechanisms established for flashing light, there can be a reduction

in photosynthetic efficiency as a result of imbalances in the excitation between

photosystems. Because the two photosystems can have different absorption and

action spectra, which are most notable in the far red region, this reduction may

be affected by the species examined as well as spectral characteristics of the

irradiance field (Emerson and Lewis, 1943; Dubinsky et al., 1986). To test these

hypotheses, I evaluate photosystem-specific mechanisms by measuring both

aggregate and photosystem-specific biomass and physiological parameters.

These results are further analyzed in the context of using P-E to estimate

17

quantum efficiency. Finally, results from these experiments are discussed in

relation to open-ocean spectral irradiance properties.

METHODS

Culture conditions

Diatom (S. costatum (CCMP1332) (Greville, 1866; Cleve, 1873)) and chlorophyte

(D. tertiolecta (CCMP1320) (Butcher, 1959)) cultures, originally obtained from

Provasoli - Guillard National Center for Culture of Marine Phytoplankton, were

grown in semi-continuous batch culture at 19oC in sterile (0.2 µm filtered) air

bubbled f/2-amended media made with filtered (GF/F – Whatman) Sargasso Sea

water (Guillard and Ryther, 1962) and were periodically diluted to maintain

exponential growth. Continuous light, which was supplied by fluorescent bulbs,

was attenuated by neutral density and stage screening (Cinemills #019, #141,

#210) to achieve the desired intensity and spectral quality (Figure II-1).

Irradiance intensity averages were as follows: S. costatum 24.3±3.4 and D.

tertiolecta 23.8±2.1 µmol quanta m-2 sec-1. Note that all error bars reported in

this text represent one standard error unless otherwise noted.

Absorption, Pigments, Fluorescence Excitation, Growth Rates, Cell Density

Spectral absorption measurements were made at 2 nm resolution with a 1 sec

integration time on samples in solution using an HP 8452 diode array

spectrophotometer with Labsphere (RSA-HP-84) integrating sphere. Absorption

at 750 nm was assumed to be non-cellular and was subtracted from the

18

absorption curve. Absorption coefficients were calculated following Kirk (1994).

Chlorophyll a was determined on 90% acetone extracts following Parsons et al.

(1984) except without MgCO3 addition, using the trichromatic equations of Jeffrey

and Humphrey (1975). Intrinsic growth rates were calculated from bulk

fluorescence measurements using a Turner Designs 10-005R fluorometer. Cell

concentrations were estimated using a hemacytometer.

Photosystem Quantification

Emerson and Arnold numbers (E&A or Chl a/O2) were measured using a

Hansatech oxygen electrode and a Stroboslave (Type1539A) tunable frequency,

saturating strobe light following Dubinsky et al. (1986). PSII/Chl a was calculated

as four times the initial slope of oxygen evolution rate versus flash frequency

assuming a stoichiometric 4 PSII/O2 relationship, and normalizing to chlorophyll a

concentration. PSI/Chl a were measured on thylakoid membranes using the

chemical oxidation/reduction technique and quantifying the absorption difference

at OD697 normalized to OD725 following Marsho and Kok (1971). Thylakoid

membranes were isolated from phytoplankton cells harvested by centrifugation

for 5 min at 9777×g, gentle sonication (~25 W) of resuspended pellet in 0.02%

Triton X-100 in 50 mM Trizma for 30 secs and then centrifugation at 25000×g for

2 min. Chlorophyll a concentrations of the thylakoid-containing supernatant were

calculated using 60 mM Chl a/OD680 (Thornber et al., 1977). P700

concentrations of thylakoid membranes were calculated using 64 mM

P700/∆OD697/725 where ∆OD697/725 represents the change in optical density from

19

oxidized to reduced spectra at 697 nm relative to 725 nm (Hiyama and Ke, 1972;

Melis and Brown, 1980). All optical density measurements for PSI/Chl a were

made using a split beam Cary 219 spectrophotometer with a 1 cm pathlength.

Photosynthesis vs Irradiance (P-E), Turnover Rates

Photosynthesis-irradiance measurements were made using temperature-

regulated custom-built photosynthetrons. Irradiance, which was supplied by a

250 W ENH projector bulb (Gray Supply), was spectrally modified and attenuated

using a combination of hot and cold mirrors (Optical Coating Laboratory) and

stage screening (Cinemills) (Figure II-1). Incubations of 1 ml samples inoculated

with ~7.4 kBq H14CO3 were terminated after 10 min using 100 µl 37%

formaldehyde and 200 µl HCl and allowed to degas overnight. Carbon uptake

rates were quantified using standard techniques (Barber et al., 1996).

Photosynthetic parameters of the Platt et al. (1980) model were optimized to fit

data using a custom written, non-linear least-squares Levenberg-Marquardt

technique,

( )( ) ( )( )bs

bs

bs

b PEPEPP /exp/exp1 * βα −−−= (II-1)

where Pb is chlorophyll a-normalized photosynthesis (mg C mg Chl a hr-1), E is

irradiance intensity (µmol quanta m-2 sec-1), β is a photoinhibition term (mg C mg

Chl a hr-1(µmol quanta m-2 sec-1)-1), Pbs is the theoretical maximum

photosynthetic rate if β is zero (mg C mg Chl a hr-1), and α* is the maximum light

20

utilization coefficient (mg C mg Chl a hr-1(µmol quanta m-2 sec-1)-1). The light-

saturated chlorophyll a-normalized photosynthetic rate (Pbmax) was calculated

from recovered parameters following Zimmerman et al. (1987). Ek was

calculated as Pbmax/α*. Optimization of model parameters to P-E data was

performed using PAR as the independent variable.

Maximum quantum yield (φC,max) was calculated using two techniques as

the mol C mol quanta-1 absorbed using absorption coefficients that were

spectrally weighted to incident measurement irradiance. In the first (standard)

technique, φC,max was estimated using the initial slope (α*) of the P-E curve

following equation II-2 (Geider and Osborne, 1992):

*

*

max,

0231.0

iC a

αφ = (II-2)

where α* is the maximum light utilization coefficient (mg C mg Chl a-1 hr-1 (µmol

quanta m-2 sec-1)-1), 0.0231 converts grams to moles, and hours to seconds and

**, λλλ aEai

o

= (II-3a)

∫

∫=

nm

nm

nm

nmi

E

aE

a700

400

700

400

*

*

λ

λλ

o

o

(II-3b)

21

where a*λ is the chlorophyll a-specific absorption coefficient at wavelength λ, o

λE

is the relative incident quantum flux at wavelength λ, a*i,λ is the chlorophyll a-

specific absorption coefficient normalized to incident irradiance at wavelength λ,

and *ia (m2 mg Chl a-1) is the mean a*

i,λ over 400-700 nm. In the second

(alternate) technique to calculate φC,max, φC-E curves were constructed from P-E

and normalized absorption (equation II-3) and φC,max was calculated as the

maximum value of the curve ( [ ]EC −φmax ). These values are referred to as

φC,max′.

Irradiance Measurements and Calculations

Spectral power was measured using an Analytical Spectral Devices

spectroradiometer (LabSPEC VNIR 512) with 1o field of view attachment and

converted to relative quantum spectral output using calibrations to a known

source and supplied software. Irradiance intensity, or PAR, was measured using

a Biospherical (QSL-100) 4π scalar irradiance meter. Photosynthetically usable

radiation, PUR, was calculated from PAR, absorption spectra and relative

quantum spectra following Sakshaug et al. (1997):

∫

∫=

nm

nm

nm

nm

i

PARPUR

E

a

a

EE700

400

700

400*

*,

)max(

λ

λ

λ

o

oo

(II-4)

22

where PUREo

is PUR and PAREo

is PAR.

Literature Data

Literature data were obtained from original journals (Perry et al., 1981;

Falkowski et al., 1986; Platt et al., 1987) by scanning in black and white line

mode at 300 dots per inch resolution and digitizing the center of each plotted

point. Relative quantum yield was calculated from recovered P-E data as

photosynthesis divided by incident irradiance following equation II-5:

aE

PX =φ (II-5)

where φX is quantum yield (mol X mol quanta-1), P is photosynthesis (mol X m-3

sec-1), a is absorption (m-1), and E is irradiance intensity (mol quanta m-2 sec-1),

where X is O2 evolved or C uptake. Note that equation II-5 is not the

mathematical first derivative of a P-E curve, but a cumulative derivative.

Results / Discussion:

Contemporary P-E Models

As previously suggested, contemporary models of the P-E relationship

such as the rectangular hyperbola, quadratic, exponential and hyperbolic tangent

functions have the same general shape with photosynthesis increasing with

23

irradiance at low light levels. Photosynthesis saturates to a maximal level at

higher irradiances (Figure II-2a). Similarly, when output from these models is

expressed in quantum yield format, all models have comparable functional forms

with maximal quantum yields at low light levels followed by decreasing values at

higher light levels (Figure II-2b-c).

Inherent in the formulation of these models, it is important to note that the

assumption of maximal and constant quantum yield at low irradiances does not

assume that processes are optimal for photosynthesis. Rather this assumption

simply states that for a given irradiance range comprised of low intensities,

quantum efficiency is maximal and independent of light level. As an example,

PSII and PSI excitation may be out of balance resulting in a reduced maximum

quantum yield, but the models assume that processes that regulate PSII and PSI

distribution are independent of light. Thus, in this example the absolute value of

the maximum quantum yield predicted by the model may be set by PSII / PSI

excitation balance, but not the structure of the φ-E curve at low irradiances.

Another example is nitrogen-limitation, which leads to decreased maximal

quantum yield. Although N-limitation decreases the magnitude of the φ-E curve,

it is assumed not to influence the structure of the curve at low irradiances.

Similar lines of reason can apply to other sources of variability for the φ-E curve

such as photoacclimation, other forms of nutrient limitation, etc.

Observations

24

Contrary to assumptions of constant and maximal efficiency at low light, multiple

observations suggest that quantum yield is variable and reduced at low light.

Multiple investigators have found that for several phytoplankton species O2 flash

yields are not maximal at low background irradiance levels (Forbush et al., 1971;

Diner and Mauzerall, 1973; Ley and Mauzerall, 1986; Falkowski and Raven,

1997). Photosynthesis-irradiance measurements made using continuous light for

10 min with 14C incubations for S. costatum also suggest a reduction in quantum

yield at low irradiances (Figure II-3).

Using both O2 and 14C methodologies, these reductions appear to be

present for a wide range of taxonomies (Figure II-4). However, not all taxa have

similar reductions: some phytoplankton have dramatic reductions (T. fluviatilis)

whereas others may be less affected (D. brightwellii) (but see below). Although

the O2 methodology is less sensitive and both methodologies suffer from reduced

resolution at low background light levels, reductions appear to be present for

both O2 and 14C methodologies for field-observed mixed-populations (Figure II-

5).

In addition to O2 and 14C methodologies for both monotypic cultures and

field populations, these data also encompass other differences. For example,

field data were measured using 4 hr incubations (Platt et al., 1987) (Figure II-5)

whereas laboratory data were collected using either 10 min (Figure II-3) or 2.5 hr

incubations for 14C (Perry et al., 1981) (Figure II-4) and 5-10 min for O2 evolution

(Falkowski et al., 1986) (Figure II-4). Further, multiple investigators performed

each of these experiments. Thus, the reduction in efficiency is not likely to be

25

related to the time of incubation or methodology associated with measuring

photosynthetic rate.

It is important to note that values of quantum yield at low irradiance are

more prone to experimental error than higher irradiance values. This is because

of the decreased relative sensitivity of both photosynthetic rate (both oxygen

evolution and carbon uptake) and irradiance flux. Because quantum yield is the

quotient of photosynthesis and irradiance, small errors in each of the

measurements can lead to large errors in quantum yield that may not necessarily

be observable in standard P-E analyses (example Figure II-4 and II-9). However,

careful observations using different techniques by several investigators

demonstrate the same overall patterns. This suggests that the trends observed

here are 'real' and are not due to methodological difficulties.

Potential Mechanisms

There are multiple potential mechanisms to account for a reduction in quantum

efficiency at low irradiances. Diner and Mauzerall (1973) have explicitly tested

many of these mechanisms in flashing light on C. vulgaris and P. luridium and

found that imbalances in photosystem excitation are largely responsible for

observed reductions. Here I briefly review their findings in the context of my

observations and discuss other potential mechanisms that could account for the

reductions.

Diner and Mauzerall (1973) investigated three basic mechanisms that may

be responsible for the reduction in flash yields at low background irradiances

26

including respiration, S-state decay and photosystem imbalance. Mechanisms

associated with respiration fall into the two basic categories of constant and

variable respiration. A priori, constant respiration corrections do not affect the

overall shape of the P-E curve, thus any sigmoidal behavior (i.e. reduction in

quantum efficiency at low irradiance) is not influenced by changes in constant

respiration. Nevertheless, constant respiration corrections can affect the

estimation of φ-E and ultimately the interpretation of P-E data (see below).

Unlike constant respiration, differential respiration, chlororespiration or the

Kok effect, has the potential to affect the shape of the P-E and φ-E curve (Kok,

1949; Raven and Beardall, 1981; Geider, 1992; Poskuta, 1992). However, both

Diner and Mauzerall (1973) and Healy and Myers (1971) demonstrated that the

reduction in φ still exists under anaerobic conditions where the Kok effect is

negligible (Healy and Myers, 1971; Diner and Mauzerall, 1973). They also show

that the magnitude of respiration signal for both aerobic and anaerobic conditions

is not adequate to explain the reduction in the φ. Further, chlororespiration

results in reduced respiration at low background irradiances that would tend to

increase, not decrease, quantum yield at low irradiances (Kok, 1949). Short-term

gross 14C uptake P-E and φC-E curves, which are largely devoid of direct

extracellular respiration complications, also suggest that respiration is not

responsible for reductions in φ-E (Figure II-3).

In addition to direct effects, respiration may also indirectly influence 14C

uptake estimates of φ-E curves via extracellular dilution of 14C by respired 12C.

However, similar to oxygen respiration, even at low irradiances the respiration

27

rate is low compared to net carbon uptake. For example, analyzing data from

Figure II-3, there is a ~50% reduction in φC relative to φC,max′ at ~25 µmol quanta

m-2 sec-1. A dilution of 14C by respired 12C would require a doubling of the

extracellular 14C pool over the course of the experiment, which in turn would

correspond to a ~2 mM change in ~10 min, or 200 µM/min. This rate exceeds

the light-saturated photosynthetic rate (~1.4 µM/min) by two orders of magnitude

and is therefore unlikely.

At low irradiance levels when extracellular carbon uptake is low, the

internal cellular inorganic carbon pool 14C/12C ratio may be significantly depleted

in 14C relative to the extracellular pool. This could also lead to an apparent drop

in efficiency. However, four lines of evidence suggest that this mechanism is not

dominant. First, the reduction in φ is observed with both continuous and flash

yield oxygen measurements, which are not influenced by carbon equilibration.

Second, the reduction is observed for longer-term (4 hr) measurements, which

should have reduced radiocarbon equilibration problems. Third, a priori

irradiance color should not have an effect on radiocarbon equilibration, but here it

dramatically affects the magnitude of the reduction. And finally, again using data

from Figure II-3, there is an approximately 50% reduction in φC relative to φC,max

at ~25 µmol quanta m-2 sec-1. This irradiance is ~30% of the Ek and well above

the compensation irradiance for S. costatum grown with similar conditions

(Falkowski and Owens, 1978), implying that there is significant carbon transport

across the cell membrane, which in turn would quickly equilibrate the carbon

pool. These lines of evidence do not support a reduction in φ at low irradiance

28

due to cellular inorganic carbon 14C/12C equilibration issues. Thus, evidence

from flash yields and continuous oxygen measurements as well as carbon uptake

measurements strongly suggests that respiration is not responsible for the

reduction in quantum efficiency at low irradiances.

Another potential mechanism to explain the reduction in φ is non-radiative

decay. Under low continuous light or low flash frequencies, the S-states of the

oxygen evolving complex can undergo non-radiative decay that can reduce φ

(Kok et al., 1970; Forbush et al., 1971). However, this deactivation is a relatively

slow process with the most rapid half times for C. vulgaris at 25 oC on the order

of ~1.5 secs (Diner and Mauzerall, 1973). The deactivation rate constant is

independent of background light intensity. Because reductions in φ occur in up to

~10% of the light-saturated value of photosynthesis and the photosynthetic unit

turnover time is less than ~20 msec for Chlorella spp., a ~1.5 secs S-state decay

half time is much too slow to account for reductions in φ even at low light levels

(Myers and Graham, 1971). These results are supported by repetitive double-

flash experiments that show that the deactivation of S3 to S2 on the donor side of

PSII is markedly slower than that necessary to account for the reduction in

φ (Diner and Mauzerall, 1973). S. costatum and D. tertiolecta grown under the

present conditions have similar turnover times (58±3 msec and 63±5 msec,

respectively), which are also much greater than typical S-state decay rates (see

below).

Unlike respiration or S-state mechanisms, several lines of evidence from

Diner and Mauzerall (1973) suggest that low-light reductions in φ are a result of

29

acceptor side limitation of PSII. Experiments with P. luridium using

benzoquinone, which replaces NADP+ as a terminal electron acceptor in the

photosynthetic electron transport chain, demonstrate that the reduction in φ for

flash yields is almost completely attenuated. Conversely, phenazine

methosulfate (PMS), which acts as a PSII acceptor side reducer, significantly

enhances the reduction for flash yields. Similarly, far-red light, which

preferentially excites PSI and ultimately oxidizes the PQ pool, also dramatically

attenuates the reduction in φ (Diner and Mauzerall, 1973). These lines of

evidence all support low light reductions in flash yields being driven by an

imbalance in photosystem excitation.

To examine the influence of this imbalance on continuous light P-E curves

made using short-term 14C incubations, I measured P-E and φC-E response

curves for S. costatum and D. tertiolecta in different colors of continuous light.

These P-E curves are similar in overall structure with identical light-saturated

photosynthetic rates (P>0.15), while the initial slopes are significantly different

among the different measurement colors for S. costatum (P<0.001) and nearly

significantly different for D. tertiolecta (P=0.07) (Table II-1, Figure II-6A). All of

these P-E curves are similar in structure to contemporary P-E models (ex. Figure

II-2).

Although the functional form of the P-E curves are generally consistent

with the models, φC-E curves had a great deal more heterogeneity (Figure II-6B).

The maximum quantum yield of carbon uptake (φC,max) for S. costatum were

slightly different (P=0.043) for the different color measurements with the red-

30

measured value at 0.13 while the white and blue-green values were marginally

reduced at 0.12 mol C/mol quanta. Estimates of φC,max for D. tertiolecta were

statistically indistinguishable (P>0.1) for the different measurement colors (0.12

mol C/mol quanta).

The functional response of φC-E for D. tertiolecta is generally similar to that

predicted by widely-used photosynthesis-irradiance (P-E) empirical models

(Figure II-2); φC,max is observed at the lowest irradiances and φC decreases in an

exponential fashion approximately starting at the photosynthesis saturation

irradiance (Ek). However, functional responses of S. costatum φC are markedly

different with significant reductions in φC at low irradiances – the functional form

of the curve is more log-normal in nature. Decreases in φC at low irradiances for

S. costatum are most pronounced for blue-green- and white-measured curves,

but are significantly attenuated in red-measured populations. This color-

dependent reduction in φC at low irradiance is suggestive of imbalances in

photosystem excitation sensu Emerson and Lewis (1943).

Despite significant reductions for S. costatum, which are measurement

color dependent, the functional form of φC-E for D. tertiolecta remains largely

consistent between different treatments. This increased sensitivity for S.

costatum relative to D. tertiolecta may be due to differences in PSII/PSI ratios: D.

tertiolecta has a PSII/PSI ratio that is near unity while S. costatum's PSII/PSI

ratio is significantly elevated (P<0.01) (Table II-2). D. tertiolecta's more balanced

photosystem ratio may permit mechanisms such as state transitions and non-

31

photochemical quenching, which in part regulate excitation energy distribution

between photosystems, to be more effective at relieving photosystem excitation

imbalance (Bonaventura and Myers, 1969; Demmig-Adams, 1990; Kroon et al.,

1993). For example, state transitions, which favor increased PSI cross sections

under blue light, would tend to mitigate photosystem excitation imbalances.

Conversely, S. costatum, which has a PSII/PSI that is much greater than unity,

may be more prone to excitation imbalance under non-favorable spectra. In spite

of a potentially increased threshold for photosystem excitation imbalance, at

much lower light levels D. tertiolecta may also have reductions in φ. These

results for S. costatum and D. tertiolecta using continuous light are consistent

with oxygen flash yield experiments and proposed PSI limitation mechanism

(Diner and Mauzerall, 1973). Photoacclimation studies, which show that the

reduction in φ is an antennae-dependent process, are also consistent with these

observations (Chapter IV). A reduction in quantum yield measured using

radiocarbon techniques that is possibly due to photosystem excitation imbalance,

does not exclude the role of carbon concentrating mechanisms; carbon

concentrating mechanisms are up-regulated under PSI-favorable light and may

account for decreased reductions for red light (Kaplan and Reinhold, 1999).

A Potential Source of Error in P-E Analysis

Regardless of the mechanism resulting in quantum yield reduction, to

examine the influence of reductions in quantum efficiency at low irradiances on

the structure of P-E curves, I selected the hyperbolic tangent P-E model as a

32

representative contemporary model and incorporated a low-light quantum yield

reduction term (Figure II-7). Four arbitrary levels of low-light quantum yield

reduction ranging from zero to strong affect the φ-E functional form (Figure II-

7b/c). These reductions are most apparent when using a logarithmic abscissa

because it expands the axis in the low light region. However, these effects are

negligible for the associated P-E curves (Figure II-7a). These results suggest

that reductions in quantum efficiency at low irradiance are not easily observable

using traditional P-E analyses.

Although low-light reductions in φ may not significantly modify the overall

structure of the P-E curve, they can affect the interpretation of the P-E curve. For

example, if the initial slope of the P-E curve is determined by linear regression

through the first several points of the P-E curve (Figure II-8a) and these points

are in the low-light reduced-quantum yield region (Figure II-8c), the estimated

initial slope may be significantly reduced compared to the maximum slope

(Figure II-8d). This type of error may be significant because this initial slope of

the P-E curve (α*) is used in conjunction with absorption measurements to

estimate the maximum quantum yield of photosynthesis (Geider and Osborne,

1992; Lindley et al., 1995; Falkowski and Raven, 1997). For the present case

(Figure II-8), φC,max could be under-estimated by greater than 50% unless the

appropriate (exact) number of points is used to construct the linear regression.

While the linear regression technique under-estimated the true maximum

quantum yield, other techniques such as fitting P-E model parameters to data

can over-estimate the maximum quantum efficiency. For example, using 51 P-E

33

curves that have a three-fold range in the maximum light utilization coefficient

(α*), the Platt et al. (1980) model over-estimates the true maximum quantum

yield by an average of 40% (data not shown). This problem is not unique to the

Platt et al. (1980) model: other P-E model formulations recover significantly

different parameter values that depart from the true values (Frenette et al., 1993;

Henley, 1995). Thus, because model structures are not consistent with observed

data patterns (i.e. low light reductions in φ), there are difficulties associated with

both linear and non-linear contemporary model regressions on P-E data.

Over- or under-estimates of the true maximum quantum efficiency is

highly data- and model-specific: large or small numbers of data points in the

reduced quantum yield region of the P-E curve significantly affects the degree of

error associated with the linear regression and Platt et al. (1980) techniques. To

avoid these potential errors, estimates of the maximum quantum yield of carbon

uptake should be performed using φ-E analyses.

In addition to errors associated with low-light φ reduction and curve fitting

analyses, there also exist additional errors in respiration corrections that can

affect the low-light region of the φ-E structure. Although, light-independent

(constant) respiration per se is not responsible for the sigmoidal structure of the

P-E curve, respiration or respiration-like corrections can affect the structure of the

φ-E curve. For example, using a generalized reduced quantum efficiency model

at low irradiances as a control case, various respiration errors are added to the

P-E and associated φ-E curves (Figure II-9). Similar to the previous conceptual

model graphs, this family of curves demonstrates that respiration errors are not

34

readily observed using traditional P-E analyses. However, when φ-E is analyzed

on a semi-log plot, there can be significant errors associated with small

respiration inaccuracies. For example, an error of +0.1% (under-estimation of

respiration by 0.1% of the light saturated photosynthetic rate), induces large

changes in the φ-E structure. Similarly, -0.1% leads to anomalous negative

values for φ. In the +0.2% case, maximum φ values become dramatically

elevated over the true value. These types of errors can be associated with

corrections made for oxygen respiration and for so-called dark uptake or

adsorption of 14C (Williams and Lefevre, 1996; Markager, 1998). This type of

error is most common when signal-to-noise levels are reduced and the

subtracted value (oxygen respiration or 14C adsorption/dark uptake) represents a

significant fraction of the signal level, such as is typically the case in the low light

region of the curve. Errors in low light φ linked to respiration (or 14C uptake)

corrections are commonly observed for both oxygen evolution and carbon uptake

field and laboratory experiments (Figures II-3-5) and proximally account for the

larger variability in estimated quantum yields at low background irradiances.

Environmental Significance

Observations of spectrally dependent photosynthetic efficiencies at low

irradiances, which are enhanced by red-wavelength light, suggest a mechanism

involving PSI limitation by which Emerson enhancement can take place. This

finding may be particularly important for oceanic photosynthesis; because low

irradiance intensities in the open ocean are associated with blue-dominated

35

irradiance spectra, low-light depressions in φ may significantly reduce light-limited

primary production (Jerlov, 1976; Kirk, 1994). Alternatively, phytoplankton that

dominate at low irradiance intensities such as Prochlorococcus spp. (Olson et al.,

1990) may have photosynthetic pigments or machinery that are optimized for

equal distribution of excitation energy between the two photosystems (Goericke

and Repeta, 1992; Garczarek et al., 1998; Vanderstaay et al., 1998; Johnson et

al., 1999). Indeed, photosynthetic bacteria that are enriched in PSI particles tend

to dominate at lower light levels, deep in the water column. Nevertheless,

reductions in quantum efficiencies at low irradiance have been observed for both

nutrient-replete and -limited surface phytoplankton populations. Regardless of

mechanism, these types of reductions are only apparent when data are analyzed

in a φ-E fashion and are not present for PSII-inferred descriptions of P-E and φC-

E because the reductions in φC are mediated by processes downstream of PSII

(Falkowski et al., 1986; Falkowski and Raven, 1997; Chapters III-V).

36

Table II-1 Recovered P-E parameters using the model of Platt et al. (1980) with data from Figure II-6A. (mean ± SE)

Properties Units Species Color Value

Pbmax mg C

mg Chl a-1 hr-1 S. costatum blue-green 1.54±0.01

white 1.42±0.03

red 1.53±0.08

D. tertiolecta blue-green 1.09±0.04

white 1.06±0.01

red 0.98±0.04

α* (mg C

mg Chl a-1 hr-1) S. costatum blue-green 0.0609±0.0011

(µmol quanta m-2 sec-1)-1

white 0.0341±0.0001

red 0.0333±0.0004

D. tertiolecta blue-green 0.0234±0.0013

white 0.0202±0.0010

red 0.0245±0.0009

φC,max mol C

mol quanta-1 S. costatum blue-green 0.123±0.002

white 0.120±0.000

red 0.128±0.002

D. tertiolecta blue-green 0.107±0.006

white 0.121±0.006

red 0.128±0.005

37

Table II-2 Photosystem Specific Biomass Properties (mean ± SE)

Property Units S. costatum D. tertiolecta

PSII/Chl a mmol mol-1 2.41±0.11 1.84±0.08

PSI/Chl a mmol mol-1 0.63±0.05 1.48±0.15

PSII/PSI mol mol-1 3.83±0.49 1.25±0.18

38

Photosynthetron and Growth Spectra

Wavelength (nm)

400 450 500 550 600 650 700 750

Rel

ativ

e Q

uant

um O

utpu

t

0.0

0.2

0.4

0.6

0.8

1.0

Blue-green

White Red

Figure II-1: Relative quantum output for growth and blue-green,white and red measurement spectra.

Growth

39

Irradiance (µmol quanta m-2 sec-1)

0.1 1 10 100 1000

Qua

ntum

Yie

ld

Irradiance (µmol quanta m-2 sec-1)

0 75 150 225 300

Pho

tosy

nthe

tic R

ate

Rectangular HyperbolaQuadraticExponentialHyperbolic Tangent

Irradiance (µmol quanta m-2 sec-1)

0 75 150 225 300Q

uant

um Y

ield

A B C

Figure II-2: (a) Modeled photosynthesis-irradiance (P-E) curves constructed using identical initial slopes (α*) and maximalphotosynthetic rates (Pbmax) using four contemporary P-E models. (b) quantum yield-irradiance (φ-E) curves associated withthe P-E models. (c) as (b) except with common log abscissa scale. Note that none of these models used here includes aphotoinhibition term.

40

Irradiance (µmol quanta m-2 sec-1)0 75 150 225 300

φ C (

mol

C m

ol q

uant

a-1)

-0.02

0.00

0.02

0.04

0.06

Irradiance (µmol quanta m-2 sec-1)0 75 150 225 300

Pb (

mg

C m

g C

hl a

-1 h

r-1)

0.0

0.5

1.0

1.5

2.0

Irradiance (µmol quanta m-2 sec-1)1 10 100 1000

φ C (

mol

C m

ol q

uant

a-1)

-0.02

0.00

0.02

0.04

0.06

A B C

Figure II-3: (a) Measured P-E curves for nutrient-replete S. costatum grown in continuous moderate intensity (108 µmol quanta m-2 sec-1)white light, (b) φC-E curves associated with panel (a) and (c) as (b) except common log abscissa scale.

41

PAR (µmol quanta m-2 sec-1)

0 500 1000 1500 2000

Rel

ativ

e O

2 E

volu

tion

S. costatum

C. vulgaris

T. weisflogii

I. galbana

D. tertiolecta

PAR (µmol quanta m-2 sec-1)

1 10 100 1000

Rel

ativ

e Q

uant

um Y

ield

PAR (µmol quanta m-2 sec-1)

0 200 400 600

g C

g C

hl a

-1 h

r-1

0

2

4

6

8

D. brightwellii

D. euchlora

T. fluviatilis

I. galbana

C. sanicus

T. pseudonana

PAR (µmol quanta m-2 sec-1)

10 100 1000

Rel

ativ

e Q

uant

um Y

ield

A B

C D

Figure II-4: P-E (a) and associated relative φO2-E (b) calculated from oxygen evolution

for Skeletonema costatum, Chlorella vulgaris, Thalassiosira weisfloggi, Isochrysis galbana,and Dunaliella tertiolecta grown in continuous 67 µmol quanta m-2 sec-1 light. These data are

replotted from Falkowski et al. 1986. P-E (c) and associated relative φC-E (d) calculated fromcarbon uptake for Ditylum brightwellii, Dunaliella euchlora, Thalassiosira fluviatilis, Isochrysisgalbana, Chaetoceros danicus and Thalassiosira pseudonana grown in continuous300 µmol quanta m-2 sec-1 light. These data are replotted from Perry et al. 1981. Note thatreductions at higher irradiances for carbon measured curves likely occur because cultures were grown at higher irradiances compared to cultures measured using oxygen techniques.

42

PAR (W m-2)

0 100 200 300 400

Rel

ativ

e O

2 E

volu

tion

PAR (W m-2 )

1 10 100 1000

Rel

ativ

e Q

uant

um Y

ield

PAR (W m-2)

0 100 200 300 400

Rel

ativ

e C

arbo

n U

ptak

e

PAR (W m-2)

1 10 100 1000

Rel

ativ

e Q

uant

um Y

ield

A B

C D

Figure II-5: P-E (a) and associated relative φO2-E (b) calculated from oxygen evolution

and P-E (c) and associated relative φC-E (d) calculated from carbon uptake for identicalsamples for a mixed field population of phytoplankton collected in the Canadian high arctic.All data are replotted from Platt et al. 1987.

43

blue-green white redS

. cos

tatu

mD

. ter

tiole

cta

Photosynthetically Usable Radiation (PUR)

0 50 100 150 200

0.0

0.5

1.0

1.5

2.0

0 50 100 150 200

0.0

0.5

1.0

1.5

2.0

0 50 100 150 200

0.0

0.5

1.0

1.5

2.0

0 50 100 150 2000.0

0.5

1.0

1.5

2.0

0 50 100 150 2000.0

0.5

1.0

1.5

2.0

0 50 100 150 2000.0

0.5

1.0

1.5

2.0

Photosynthesis-Irradiance Curves

Figure II-6A: Photosynthesis-irradiance relationships using photosynthetically usableradiation (µmol quanta m-2 sec-1) measured with blue-green-, white- and red-dominatedwavelengths (columns) for S. costatum and D. tertiolecta (rows). Circles, squares andtriangles represent data from independent triplicate measurements. Note that bothS. costatum and D. tertiolecta were grown in blue-green light (see figure II-1).

44

blue-green white red

S. c

osta

tum

D. t

ertio

lect

a

Photosynthetically Usable Radiation (PUR)

0.1 1 10 1000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.1 1 10 1000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.1 1 10 1000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.1 1 10 1000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.1 1 10 1000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.1 1 10 1000.00

0.02

0.04

0.06

0.08

0.10

0.12

Quantum Yield Irradiance Curves

Figure II-6B: Quantum yield of carbon uptake (mol C mol quanta-1) as a function ofphotosynthetically usable radiation (µmol quanta m-2 sec-1) measured using blue-green-,white- and red-dominated wavelengths (columns) for S. costatum and D. tertiolecta (rows).Circles, squares and triangles represent data from independent triplicate measurements.Note that both S. costatum and D. tertiolecta were grown in blue-green light.

45

Irradiance (µmol quanta m-2 sec-1)0 75 150 225 300

Qua

ntum

Yie

ld

Irradiance (µmol quanta m-2 sec-1)0 75 150 225 300

Pho

tosy

nthe

tic R

ate

abcd

Irradiance (µmol quanta m-2 sec-1)0.1 1 10 100 1000

Qua

ntum

Yie

ld

A B C

Figure II-7: (a) modeled P-E curves (tanh model used) with increasing levels of photosystem excitation imbalance (red=most balanced,black=least balanced), (b) φ-E curves with varying levels of photosystem excitation imbalance, (c) as (b) except common log abscissascale. Note that none of these models used here includes a photoinhibition term. The choice of model used in panels a-c does notsignificantly affect the outcome. Also note that in panel A, all curves are plotted and lie under curve d.

46

PAR (µmol quanta m-2 sec-1)

0 50 100 150

Pb (

mg

C m

g C

hl a

-1 h

r-1)

0.0

0.5

1.0

1.5

# points used in slope determination

0 20 40 60 80

Fra

ctio

n of

Max

imum

Slo

pe (

alph

a)

0.0

0.2

0.4

0.6

0.8

1.0

PAR (µmol quanta m-2 sec-1)

1 10 100

φ C (

mol

C m

ol q

uant

a-1)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

A

C D

PAR (µmol quanta m-2 sec-1)

0.1 1 10 100

Pb (

mg

C m

g C

hl a

-1 h

r-1)

0.0

0.5

1.0

1.5

B

Figure II-8: (a) P-E for blue-green-grown S. costatum and measured with whiteirradiance. (b) as (a) except on common log abscissa. (c) φC-E curve associated withpanels (a) and (b). (d) Fraction of maximum slope estimated by linear regression as afunction of the number of points used in the regression. Note that the photosynthesissaturating irradiance intensity (Ek) occurs at approximately data point number fifteen.

Irradiance (µmol quanta m-2 sec-1)0 75 150 225 300

Qua

ntum

Yie

ld

Irradiance (µmol quanta m-2 sec-1)0 75 150 225 300

Pho

tosy

nthe

tic R

ate

-0.1%control+0.1%+0.2%

Irradiance (µmol quanta m-2 sec-1)0.1 1 10 100 1000

Qua

ntum

Yie

ld

A B C

Figure II-9: (a) modeled P-E curves (tanh model used) with variable levels of respiration (or dark uptake) correction error (+0.1% is anunder estimation of respiration by 0.1% of the light saturated photosynthetic rate), (b) φ-E curves associated with (a), and (c) as (b)except common log abscissa scale. The choice of model used in panels a-c does not significantly affect the outcome. Also note thatin panels A & B, all curves are plotted and either lie under the 0.2% curve or the y-axis.

47

48

Chapter III

Photophysiological characterization of wild type and non-photochemical quenching mutants of Chlamydomonas reinhardtii (WT, npq1, npq2) using

standard and novel modulated fluorescence techniques

49

Non-photochemical quenching (NPQ) via the xanthophyll cycle is a

significant pathway in the regulation of photosynthetic excitation energy

distribution. Although the mechanism of NPQ is not fully understood and

may vary among different classes of plants, the basic pathway in part

involves the epoxidation and de-epoxidation of zeaxanthin, antheraxanthin

and violaxanthin pigments and specifically affects photosystem II (PSII).

Here using Chlamydomonas reinhardtii mutants with different epoxidase

and de-epoxidase genetic lesions (npq1 and npq2), I investigate the role of

NPQ on (1) photophysiology and (2) the relationship between PSII

parameters and photosynthetic rates and efficiency. npq1, which lacks

violaxanthin de-epoxidase activity and has a reduced NPQ capacity, has

increased chlorophyll b/a and reduced PSII/cell, light utilization,

photosynthetic capacity and quantum efficiency all relative to WT. npq2,

which lacks zeaxanthin epoxidase and has increased NPQ induction

kinetics (but also has reduced NPQ capacity), has reduced PSI/cell,

photosynthetic unit cross section, light utilization and quantum efficiency

relative to WT. Although both mutants are different than WT, npq1 is more

severely affected than npq2. On dark relaxed cells, measures of PSII cross

sectional area in the dark and in the presence of background light are

consistent with standard measurements and support NPQ acting as a

photosynthetic antenna excitation energy trap. The combination of

standard and fluorescence-derived results suggests that the xanthophyll

cycle directly and indirectly affects thylakoid composition and excitation

50

energy processing. In the context of NPQ, single turnover fluorescence

(PSII) measurements describe well the general patterns of measured

photosynthetic rates, but not absolute rates or efficiencies because of

indirect effects of the xanthophyll cycle.

INTRODUCTION

Oxygenic photoautotrophs must maintain a balance between having adequate

excitation energy to drive photosynthesis and limiting the effect of potentially

damaging over-excitation (Barber and Andersson, 1992). A primary mechanism

towards achieving this goal is the dissipation of excess excitation energy via the

xanthophyll cycle (Demmig-Adams and Adams, 1992; Niyogi, 1999). Although

complex and not fully characterized, this mechanism is based on the

interconversion of xanthophyll pigments by specific enzymes: under normal

conditions xanthophyll pigments absorb light and transfer excitation energy to

photochemistry, whereas under high-light, excess-excitation energy conditions

xanthophylls are converted to a form that dissipates the energy as heat (Demmig

et al., 1987; Demmig-Adams, 1990; Demmig-Adams and Adams, 1996; Niyogi et

al., 1997; Havaux and Niyogi, 1999; Lohr and Wilhelm, 1999). This pigment

interconversion is triggered by an elevated pH gradient across the thylakoid

membrane generated by reduction of the plastoquinone pool (Demmig-Adams

and Adams, 1992). These xanthophyll pigments are fully interconvertible and

provide a flexible mechanism to aid in the acclimation to naturally fluctuating light

conditions (Casper-Lindley and Bjorkman, 1998; Moisan et al., 1998).

51

Using Chlamydomonas reinhardtii as a model organism, Niyogi et al.

(1997) have created several algae mutant in specific pathways of xanthophyll

pigment interconversion to study this process. npq1 mutants lack violaxanthin

de-epoxidase activity and are unable to convert violaxanthin to zeaxanthin in high

light. Consequently they have a reduced, but non-zero capacity for non-

photochemical quenching (NPQ) of fluorescence (Niyogi et al., 1997).

Nonphotochemical quenching of fluorescence, which is highly light intensity and

duration dependent, can be defined mathematically as (Fm(0)-Fm(x))/Fm(x) where

Fm(0) is maximal fluorescence in the dark and Fm(x) is the maximal fluorescence

in the presence of background irradiance (x) (see below) (Niyogi et al., 1997).

Conversely, npq2 mutants lack zeaxanthin epoxidase activity and are unable to

convert zeaxanthin back to violaxanthin under normal excitation energy

conditions. Despite this lesion, npq2 NPQ returns to pre-illumination values in

the presence of low light because its maintenance requires both pigment

conversion and a pH gradient. Unexpectedly, npq2 NPQ is also lower than wild

type (WT), but is more quickly induced in the presence of high light (Niyogi et al.,

1997). Although the pathways lesioned in npq1 and npq2 are not responsible for

all NPQ, together with WT C. reinhardtii these mutants provide a window into the

photophysiological effects of NPQ (Niyogi, 1999).

Because npq1 and npq2 were originally isolated and characterized as

fluorescence mutants (Niyogi et al., 1997), they also provide a unique opportunity

to examine the effect of NPQ on fluorescence induction properties as they relate

to photophysiology. Fluorescence measures provide a rapid, non-destructive

52

assessment of the structure and function of photosystem II (Papageorgiou, 1975;

Schreiber et al., 1986; Dau, 1994; Kolber et al., 1998). From the magnitude and

kinetics of fluorescence induction and decay curves, specific measures of

photosystem II functional size, conversion efficiency, connectivity, turnover and

NPQ can be assessed (Ley and Mauzerall, 1982; Trissl et al., 1993; LaVergne

and Trissl, 1995; Lazar, 1999). In particular, single turnover type-fluorescence

measures of PSII functional cross sectional area provide a rapid assessment of

the effect of NPQ on the functional antennae size. Also, measures of PSII

conversion efficiency and cross sectional area as a function of background

irradiance intensity may provide insight into the effect of NPQ on the

photosynthesis-irradiance response curve.

Here I use measures of photosynthetic biomass properties such as

pigment concentrations, photosystem concentrations and absorption properties

and conventional measures of photosynthetic functionality such as

photosynthesis-irradiance relationships, flash yields and turnover time in

combination with a novel single turnover (ST)-type fluorometer to

photophysiologically characterize WT, npq1 and npq2 C. reinhardtii. Using these

data, my primary goal is to assess the influence of the xanthophyll cycle on

photophysiology. In the context of these findings, my secondary goal is to

investigate the role that PSII-specific processes (here the xanthophyll cycle and

NPQ), as probed by fluorescence measurements, have in regulating the overall

photosynthesis-irradiance (P-E) curve structure.

53

My working hypothesis is that the xanthophyll cycle through NPQ

proximally acts as an alternate non-photosynthetically active trap in the

photosynthetic antenna, effectively reducing the cross sectional area of the

photosynthetic unit. Thus, under NPQ favorable conditions, the first order result

of a lesioned xanthophyll cycle should be that the functional cross sectional area

is elevated for npq1 and reduced for npq2 relative to WT because of the relative

loss and addition, respectively, of traps to the antennae. Indirectly, NPQ lesions

may lead to altered pigmentation, cross sections, quantum efficiency and growth

rates relative to WT because of second order changes in pigmentation, damage

to reaction centers and alterations to PSII/PSI ratios. Since NPQ, by definition, is

a photophysiological process that affects fluorescence, fluorescence-based

estimates of P-E should reveal similar patterns as conventional methods in the

context of direct NPQ variability.

NPQ mutants are significantly different from WT in biomass and

physiological properties due to direct and indirect effects from xanthophyll cycle

lesions. Both standard and fluorescence induction analyses suggest that the

direct effects of these lesions are centered in antennae pigment-bed composition

and functionality with indirect manifestations including altered photosynthetic

conversion efficiencies, turnover times and photosystem ratios. Finally, in the

context of NPQ variability, ST-type fluorescence measurements do well in

describing P-E structure, but not in describing the magnitudes of P-E and max2Oφ .

METHODS

54

Culture conditions

Chlamydomonas reinhardtii (CC125 - WT), (CC3682 - npq1), (CC3683 - npq2)

obtained from Duke University Chlamydomonas Genetics Center were grown in

aqueous HS media (Sueoka, 1960). Semi-continuous batch cultures, grown in

continuous light at 200 µmol quanta m-2 sec-1 at 19oC and bubbled with 5% CO2,

were periodically diluted with fresh media to maintain exponential growth.

Absorption, Pigments, Growth Rates, Cell Density

Spectral absorption measurements were made at 2 nm resolution with a 1 sec

integration time on samples in solution using an HP 8452 diode array

spectrophotometer with Labsphere (RSA-HP-84) integrating sphere. Absorption

at 750 nm was assumed to be non-cellular and was subtracted from the

absorption curve. Absorption coefficients were calculated following Kirk (1994).

Chlorophyll a and b were determined on 90% acetone extracts following Parsons

et al. (1984), except without MgCO3 addition, using the trichromatic equations of

Jeffrey and Humphrey (1975). Intrinsic growth rates were calculated from bulk

fluorescence measurements made with a Turner Designs 10-005R fluorometer.

Cell concentrations were estimated using a hemacytometer.

Photosystem Quantification

Emerson-Arnold (E&A) numbers (Chl a/O2) were measured using a Hansatech

oxygen electrode and a Stroboslave (Type1539A) tunable frequency, saturating

strobe light following Dubinsky et al. (1986). PSII/Chl a was calculated as four

55

times the initial slope of oxygen evolution rate versus flash frequency, normalized

to chlorophyll concentration. Chl a/PSI were measured on thylakoid membranes

using the chemical oxidation/reduction technique and quantifying the absorption

difference at OD697 normalized to OD725 following Marsho and Kok (1971) and

Melis and Brown (1980). Thylakoid membranes were isolated from

phytoplankton cells harvested by centrifugation for 5 min at 9777×g, sonication

(25 W) of the resuspended pellet in 0.02% Triton X-100 in 50 mM Trizma for 30

seconds and then centrifugation at 25000×g for 2 min. Chlorophyll concentration

of the thylakoid-containing supernatant was calculated using 60 mM Chla/OD680

(Thornber et al., 1977). P700 (PSI) concentrations of thylakoid membranes were

calculated using 64 mM P700/∆OD697/725 where ∆OD697/725 represents the change

in optical density from oxidized to reduced spectra at 697 nm relative to 725 nm

(Hiyama and Ke, 1972; Melis and Brown, 1980). All optical density

measurements for Chl a/PS I were made using a split beam Cary 219

spectrophotometer with a 1 cm pathlength.

Photosynthesis vs Irradiance (P-E), Turnover Rates

Oxygen-based photosynthesis-irradiance measurements were made using an

automated Clark-type oxygen electrode with computer-controlled red LED light

source (Hansatech). Oxygen concentrations were sampled digitally and oxygen

evolution rates calculated using custom written software. Irradiance intensity

(PAR) was measured using a Biospherical Instruments (QSL-100) 4π scalar

irradiance meter. Photosynthetic parameters of the Platt et al. (1980) model (α*,

56

Ps, β) were optimized to fit the data using a custom written, non-linear least-

squares Levenberg-Marquardt technique. Optimization of model parameters to

P-E data was performed using PAR as the independent variable. Secondary

parameters (Pbmax and Ek) were calculated following Zimmerman et al. (1987).

Maximum quantum yield ( max2Oφ ) was calculated as the mol O2 evolved to mol

quanta absorbed using absorption coefficients that were spectrally weighted to

incident measurement irradiance (Sakshaug et al., 1997) following:

*

*

max, 2782

i

Oa

αφ = (III-1)

where α* is the maximum light utilization coefficient (mol O2 g Chl a-1 hr-1 (µmol

quanta m-2 sec-1)-1) and *ia is the spectrally-weighted mean chlorophyll a-specific

absorption coefficient (m2 g Chl a-1) (Chapter II) and 278 converts hours to

seconds and µmol to mol. Photosynthetic unit turnover rate constant (sec-1),

1/τPSU, was calculated as:

n

P b

PSU

max248.01 =τ (III-2)

where Pbmax is light-saturated photosynthesis (µmol O2 µg Chl a-1 hr-1), n is the

concentration of PSII (mol O2 mol Chl a-1) and 0.248 converts the units from

57

hours to seconds and grams to moles. The photosynthetic unit cross section

(σPSU) was calculated as α* multiplied by E&A and correcting for units.

Modulated Fluorescence

Modulated fluorescence measurements were performed with a custom-built fast

repetition rate type fluorometer (Kolber et al., 1998) with computer-controlled

background irradiance gradient (BIG-FRRf) (Figure III-1). This instrument is

centered around a cuff of super bright blue LEDs (Nichia NSPB500S) that

illuminate an aqueous sample of phytoplankton producing fluorescence. One

bank of LEDs (84) is operated in AC mode (1MHz) and is used to excite and

probe photosystem II. A second bank of LEDs (28) is operated in DC mode and

is used to provide a variable intensity, continuous background irradiance source

that is calibrated using a Biospherical Instruments (QSL-100) 4π scalar irradiance

meter. The fluorescence signal generated is isolated through a long pass

dichroic (Melles-Griot #RG665) and interference (Oriel #53970) filter set and

quantified by means of a photomultiplier tube (Hamamatsu #R2066). A fraction

of the excitation energy is quantified with a PIN photodiode (Hamamatsu

#S1722-02). Excitation and emission signals are synchronously sampled at 10

MHz and passed through an analog to digital converter (Gage Compuscope

1012PCI).

The measurement protocol is computer-controlled by custom-written

software via a graphical user interface (GUI). Four basic functions are supported

by the GUI including: excitation protocol, decay protocol, background irradiance

58

protocol and PMT gain settings. The software allows a great deal of flexibility in

experimental protocol and permits modification of intensity, duration and

frequency of both excitation and fluorescence decay protocols. The background

irradiance can be regulated both in intensity and duration. For this study, the

excitation protocol was run for 350 µsec at high intensity with a 100% duty cycle

followed by the decay protocol that was run for 7.5 msec at low intensity with a

1% duty cycle. The pre-illumination background irradiance duration was 500

msec. Samples were dark adapted for >15 min for each background irradiance

setting.

Raw data are internally processed by the same GUI to isolate excitation

and fluorescence signals from background noise. The frequency resolution of

the reduced data is 1 MHz. Reduced data are displayed graphically on the GUI.

Reduced data were analyzed with custom-written software (MATLAB 5.3v11).

Single turnover type fluorescence induction curves were fit using a custom

written, non-linear least-squares Levenberg-Marquardt technique following

Kolber et al. (1998) to determine Fo, Fm, σPSII and p where Fo is initial

fluorescence, Fm is the maximal fluorescence, σPSII is the functional cross-

sectional area, p is a connectivity parameter. Values of σPSII for Fv (Fm-Fo) <

0.075 were excluded from the analysis because of poor model fits to data.

Representative data from the BIG-FRRf is presented in Figure III-2. For

the experimental protocol used in this study, raw data output contains a 350 µsec

excitation region followed by a 7.5 msec decay region (Figure III-2a). Raw data

are in 0.1 µsec intervals and have not been corrected for background signals.

59

Raw fluorescence excitation data (Figure III-2b) contain little white noise, but are

strongly affected by background signals. Reduced fluorescence excitation data

(Figure III-2c) is in 1 µsec intervals and individual excitation points have been

binned and averaged over the 1 µsec blocks thus averaging out a great deal of

white noise. Fluorescence excitation has also been corrected for background

excitation (non-signal, DC voltage). Similarly, emission points have been binned

and averaged into 1 µsec blocks and have been corrected for background

fluorescence (non-signal, DC voltage) and have been normalized to the relative

excitation signal thus giving fluorescence yield. It is this fluorescence yield curve

in combination with a cumulative excitation curve, which is subsequently

analyzed.

Raw fluorescence decay curves are similar to fluorescence excitation

curves except that because the duty cycle is 1%, there are many short duration

peaks in the raw data (Figure III-2d/e). Both excitation and emission peaks have

a half-peak height of approximately 1 µsec and are easily isolated from the

background (non-signal, DC voltage) using simple signal extraction techniques.

Again, fluorescence is normalized to excitation to produce fluorescence yields

and this time-dependent curve can be analyzed (Figure III-2f).

For each background irradiance intensity, both fluorescence excitation and

decay curves are generated as in Figure III-2a. Hence, for each background

irradiance there is generated a suite of fluorescence-derived parameters. These

parameters are referred to with the subscript indicating the background

irradiance intensity. Thus, Fv/Fm(0) is photosystem II photochemical conversion

60

efficiency in the dark and σPSII(521) is the PSII functional cross sectional area at

521 µmol quanta m-2 sec-1.

RESULTS

Photosynthetic Biomass

Results from photosynthetic biomass measurements are summarized in Table III-

1. Chlorophyll a and absorption cross section per cell (σcell) were relatively

constant between WT and NPQ mutants. However, chlorophyll b/a pigment

ratios were significantly elevated for npq1 relative to WT. WT and npq1

chlorophyll a specific absorption coefficients (σChla) were similar while npq2

values were elevated. These increases were dominated by increases in the blue

and red regions of absorption (Figure III-3). Nevertheless, there were large

percentage increases in npq2 specific-absorption in the 500-600 nm region.

Similarly, photosynthetic unit cross sections (σPSU) were identical between WT

and npq1, but were reduced for npq2.

Photosystem concentrations were also dramatically different between WT

and NPQ mutants (Table III-1). Chlorophyll a/PSI sizes were significantly

reduced for npq1 and increased for npq2 relative to WT. Emerson-Arnold

numbers were similar between WT and npq2, but were significantly elevated

~50% for npq1. These differences in PSI and PSII concentrations also translated

into differences in PSII/PSI ratios. WT PSII/PSI molar ratios were near unity,

whereas npq1 were reduced ~50% and npq2 were elevated ~50%.

61

Photophysiology

Photosynthesis-irradiance curves (P-E) were dramatically different between WT

and NPQ mutants both in shape and magnitude (Figure III-4). WT and npq2 had

similar light-saturated photosynthesis, but npq1 Pbmax was significantly reduced.

The initial slope of the P-E curve, or maximum light utilization coefficient (α*),

was highest for WT and reduced ~30% for npq2 and ~45% for npq1 (Table III-1).

The maximum quantum yield of oxygen evolution ( max2Oφ ) largely followed

the patterns of α* with the highest values for WT and the lowest values for npq1.

Although σChla were somewhat different between WT and NPQ mutants, the

absolute changes were relatively small when compared to differences in α*.

Similarly, intrinsic growth rates of WT and NPQ mutants followed the same

general patterns as α* (r2=0.87) and max2Oφ (r2=0.85) with WT having the highest

growth rates followed by npq2 and npq1.

Because Pbmax and Emerson-Arnold numbers were similar for WT and

npq2, both also had similar photosystem turnover rates (1/τPSU). npq1 had

dramatically reduced Pbmax and despite an increase in E&A, its 1/τPSU was also

significantly reduced.

Fluorescence Induction

Characteristic fluorescence excitation and decay data are presented in Figures

III-5 and -6. These fluorescence excitation and decay curves are representative

of WT and NPQ mutant C. reinhardtii. Fluorescence excitation curves increased

62

in a saturating exponential fashion. Both Fo and Fm increased with increasing

background irradiance (Figure III-5, -7a,b). The increase in fluorescence as a

function of cumulative excitation energy, which is described by σPSII, was

influenced by background irradiance (see below). Fluorescence decay was also

significantly influenced by background irradiance (Figure III-6). Here, the

variable fluorescence, or relative amount of fluorescence recovery, declined

dramatically between low and high background irradiance intensities.

Differences in Fo and Fm structure and magnitude translated into

significant differences in Fv/Fm-irradiance curves (Fv/Fm-E) for WT, npq1 and

npq2 (Figure III-7c). All Fv/Fm-E curves decreased rapidly with increasing

irradiance such that at background irradiances above ~300 µmol quanta m-2 sec-1

Fv/Fm was less than 0.1. This decrease was nearly exponential in structure.

The overall magnitude of the WT Fv/Fm curve was greatest.

Photosystem II functional cross sectional area-irradiance curves (σPSII-E)

were less effected by background light and had only minor variations (Figure III-

7d). Values of σPSII(0) were reduced for npq2 relative to WT and npq1. These

small differences in σPSII(0) were also present at higher irradiance values on the

σPSII-E curve.

Overall, all modulated fluorescence photosynthetic parameters, with the

exception of σPSII, were strongly dependent on background irradiance intensity

(Figure III-7). The structure of this irradiance-dependence was also different

between WT and NPQ mutants. Nevertheless, all parameter curves were

63

generally consistent with those generated for other species under different

nutritional and irradiance regimes (Chapter IV & V).

DISCUSSION

Photosynthetic Biomass

Although some properties remain the same between WT and NPQ mutants,

there remain significant differences in the photosynthetic biomass between WT,

npq1 and npq2. For example, although cellular chlorophyll a concentrations and

σcell are similar between WT and NPQ mutants, which is likely due to a general

compensatory balance between photosystem-specific processes and

photosynthetically active and non-active accessory pigments, respectively, σChla

is significantly elevated for npq2 relative to WT. This increase may be due to an

accumulation of xanthophyll pigments relative to WT (Niyogi et al., 1997); since

npq2 lacks zeaxanthin epoxidase, any excess production of zeaxanthin cannot

be recycled and potentially leads to a surplus of xanthophyll pigments. An

increase in xanthophyll pigments is also consistent with the dramatic proportional

increase in chlorophyll a-normalized absorption in the 500-600 nm absorption

band relative to WT and the large absorption increase in the 400-500 nm

absorption band (Figure III-3) as well as the decrease in σPSU and σPSII(0) despite

increases in σChla.

In addition to bulk pigment concentrations, photosystem-specific

properties are also affected by modifications to the xanthophyll cycle. The

PSII/PSI molar ratio for WT is near unity suggesting that photosystem excitation

64

energy balancing mechanisms such as pigment complementation, state

transitions and NPQ are responsible for photosystem excitation balance

(Bonaventura and Myers, 1969; Murakami et al., 1997). The relative decrease in

functional cellular PSII concentration for npq1 and the decrease in cellular PSI

concentration for npq2 suggests that xanthophyll cycling significantly affects the

distribution of excitation energy through photosystem I and II. For example,

when the xanthophyll cycle has been abated and NPQ is reduced such as for

npq1, (Niyogi et al., 1997), there may be an excess of excitation energy through

PSII (Melis et al., 1989). Despite this potential increase, npq1 has an enhanced

E&A relative to WT. This increase, which is in the opposite direction one would

expect to decrease the probability of photon interception for PSII, is probably due

to the decreases in PSII/cell while PSI/cell remains constant. This is supported

by WT and npq1 having similar σPSII(0).

For npq2, the opposite is true: because of the accumulation of

photoprotective xanthophyll pigments and subsequent reduction in excitation

energy flux, an increase in the relative PSII concentrations may be expected

(Niyogi et al., 1997). A reduced flux through PSII leads to a reduced requirement

for PSI and may account for a ~40% reduction in npq2 PSI cellular

concentrations relative to WT. The overall effect of modification of PSI and PSII

concentrations as affected by the xanthophyll cycle is that npq1 has a reduced

PSII/PSI ratio whereas npq2 has an increased PSII/PSI. These modifications in

pigmentation and photosystem concentrations suggest that the xanthophyll cycle

can significantly alter the distribution of excitation energy between the two

65

photosystems. Therefore, it is likely a critical component in the acclimation to

both irradiance intensity and color (Kroon et al., 1993; Murakami et al., 1997;

Chapter II).

Photophysiology

In addition to differences in the pigmentation and photosystem complement

between WT, npq1 and npq2 there are significant differences in photophysiology.

Both α* and max2Oφ are reduced for npq1 and npq2 relative to WT. Evidence from

npq1 Fv/Fm(0) suggests that the decreases in α* and max2Oφ may be due to a

relative increase in damaged reaction centers. Because NPQ represents a

significant protection mechanism from over-excitation, npq1 PSII reaction centers

likely experience increased stress from oxygen radicals associated with over-

excitation (Aro et al., 1993; Anderson et al., 1998). This reaction center damage

may also in part lead to the observed ~50% reduction in Pbmax relative to WT.

This is consistent with similar σChla, σPSU and σPSII(0) between npq1 and WT, but

decreased max2Oφ . Relative to WT, npq2 also has reduced α* and max2Oφ , but here

reductions in efficiency are likely due to the over-accumulation of non-

photosynthetically active xanthophylls and not due to a net increase in PSII

reaction center damage (see below). This is supported by similar Pbmax values

for WT and npq2. A reduced σPSU and σPSII(0) for npq2 is consistent with excess

xanthophyll pigments, which acting as a competing trap, lead to smaller effective

absorption cross sections and α*.

66

The net result of these insults on the photophysiology of npq1 and npq2 is

a reduction in both of the growth rates relative to WT. Of the two mutants, the

growth rate of npq1, which is reduced by ~50%, is most significantly affected.

The reduction in growth rate is likely the direct result of photodamage to PSII, but

autotrophic growth is still possible because the lesion does not fully eliminate

NPQ (Niyogi et al., 1997). Further, additional pathways such as state transitions

and alteration of pigmentation help to avoid some damage (Demmig-Adams and

Adams, 1992 ; Long et al., 1994). It is also likely that protein turnover, especially

those proteins associated with the reaction center core such as D1/D2/CP43, is

increased relative to WT (Aro et al., 1993).

The growth rate for npq2 is also reduced relative to WT, but by only ~20%.

Here the reduction is likely due to the over-accumulation of non-photosynthetic

xanthophyll pigments (Niyogi et al., 1997). The over-accumulation of xanthophyll

pigments, which in turn is associated with de-excitation, likely reduces the flux of

excitation energy available to photochemistry and ultimately reduces the growth

rate.

Fluorescence Induction

Modulated fluorescence measurements provide further evidence of the direct and

indirect effects of npq1 and npq2 lesions on photophysiology. Photosynthetic

conversion efficiency of PSII (Fv/Fm(0)) was maximal for WT, but reduced for both

npq1 and npq2. The reduction in Fv/Fm(0) in npq1 is likely due to damage to PSII

67

reaction centers and is consistent with reductions in max2Oφ , α* and PSII/cell and

increases in E&A.

The reduction in the Fv/Fm(0) signal of npq2 is more complicated and may

not be due to reductions in PSII conversion efficiency per se: two lines of

evidence suggest that the apparent decrease in Fv/Fm(0) may be due to an

increase in antennae pigment fluorescence. The first line of evidence is that

other properties linked to PSII efficiency are not consistent with the magnitude of

reduction in Fv/Fm(0). For example, α* and max2Oφ are ~20% higher in npq2

relative to npq1, while Fv/Fm(0) is ~10% reduced. In addition, WT and npq2 have

similar E&A and PSII/cell, but different Fv/Fm(0). These data suggest that Fv/Fm(0)

may not be exclusively linked to true PSII efficiency estimates in npq2. The

second line of evidence is that the NPQ of npq2 is significantly reduced in spite

of an accumulation of NPQ-favorable zeaxanthin (Niyogi et al., 1997). An

increase in antennae pigment-based fluorescence, which could lead to an

increased dark Fm (and Fo), would reduce the measured NPQ and Fv/Fm(0).

Further, an increase in antennae-based fluorescence, which would provide an

additional de-excitation path not measured by NPQ, would also account for

discrepancies between reduced NPQ, but also reduced σPSU and σPSII(0) relative

to WT. This induction of antennae-based fluorescence may be the result of

abnormally high levels of zeaxanthin that in turn induces an alternate de-

excitation pathway. Regardless of the mechanism, efficiency estimates along

68

with NPQ evidence suggest that low npq2 Fv/Fm(0) may not be due to a true

reduction in PSII efficiency.

Values of σPSII(0) are also generally consistent with standard

photophysiological measurements. Similar to σChla, σcell and σPSU, σPSII(0) is

similar between WT and npq1. This suggests that in spite of a reduction in the

number of excitation traps that could lead to increases in the apparent cross

section, WT and npq1 cross sections remain similar because of small

compensatory changes in pigmentation and photosystem balance. Conversely,

the σPSII(0) of npq2 is reduced by ~10% relative to WT. This reduction is

consistent with increases in σChla due to elevated xanthophyll concentrations and

decreases in σPSU due to increased proportion of non-photosynthetically active

(i.e. xanthophyll) traps.

The irradiance structure for fluorescence-based parameters provides

further insight into WT, npq1 and npq2 photophysiology (Figure III-7). Initial (Fo)

and maximal (Fm) fluorescence both increase as a function of irradiance. Both

Fo and Fm likely continue to increase to some saturating value at very high

irradiance values. Generally, increases in Fo are largely due to a progressive

decrease in the fraction of open reaction centers: as the background irradiance

increases, the probability of a given reaction center processing excitation energy

increases. By definition, at a fully saturating background irradiance intensity, the

probability that any reaction center is closed is 1.0.

The general pattern of Fm as a function of background irradiance is largely

mediated by two general processes: NPQ and multiple turnovers (MT) of PSII.

69

Previous work has demonstrated a significant increase in Fm when it is

measured with MT flashes compared to a single turnover (ST) flash system

(Schreiber et al., 1986; Schreiber et al., 1989; Kolber et al., 1998). Differences in

Fm between ST and MT measurements in turn lead to significant differences in

reported Fv/Fm(0), which has generated controversy regarding the true saturation

of the PSII (Falkowski et al., 1994; Schreiber and Krieger, 1996; Kolber et al.,

1998). Similar to previously described fast repetition rate fluorometers that have

no background irradiance (i.e. Fm(0)), the BIG-FRRf measurement of Fm is most

consistent with a ST flash (Kolber and Falkowski, 1992; Kolber et al., 1998).

However, as the background irradiance increases, the plastoquinone pool

becomes progressively reduced and the Fm value becomes more similar to a MT

measured Fm(0), despite always being measured with a ST-like flash protocol.

Superimposed on this increase in Fm due to progressive reduction of the

plastoquinone pool is the effect of NPQ. Although each measurement of Fo and

Fm as a function of background irradiance was performed on a dark-adapted

cellular suspension, NPQ may have had an effect on fluorescence because of

the duration of the background irradiance. NPQ is strongly dependent on the

duration of the background irradiance and can increase several fold over the

course of just a few minutes (Niyogi et al., 1997; Casper-Lindley and Bjorkman,

1998). Although here the background irradiance duration was only 500 ms, this

may be sufficient to initiate a reduction in Fm from dark to low irradiance values

(Figure III-7). At greater irradiances, the increase in Fm due to convergence on a

MT-like Fm measurement overwhelms the reduction in Fm due to NPQ.

70

Although the overall patterns are similar, there remain differences

between WT, npq1, and npq2 Fo- and Fm-E curves that are the direct and

indirect result of modifications in xanthophyll cycling. For example, while the

structure of the WT and npq1 Fo-E curves are very similar, the magnitude of the

npq1 is generally higher and only converges with WT at very high irradiances.

This pattern is consistent with an overall reduction in the number of functional

reaction centers for npq1 relative to WT that was postulated from photosynthetic

biomass and photophysiology measurements. Further, although WT and npq1

P-E curves are quite different in magnitude, their normalized structure is similar

including comparable saturating irradiance intensities (Ek). This similarity is also

present for WT and npq1 Fo-E curves suggesting that the processes regulating

the structure of P-E curves may be similar to those regulating Fo-E. (see below).

Unlike npq1, the npq2 Fo-E structure is different in both magnitude and structure

when compared to WT suggesting that different regulatory processes may be

acting. From P-E relationships, which are also significantly different between

npq2 and WT, as well as σChla, σPSII(0) and σPSU, it is likely that there is an over-

accumulation of non-photosynthetic pigments. This over-accumulation increases

the irradiance intensity required to reduced PSII reaction centers and thus leads

to a reduced saturation rate. The magnitude of Fo may be elevated due to an

increase in antennae-based fluorescence (see above).

Fm-E curves show similar grouping between WT, npq1 and npq2: WT and

npq1 show similar patterns as Fo-E curves, but different magnitudes, whereas

npq2 curves have a somewhat different structure. Decreases in Fm at very low,

71

non-zero irradiances, which may be indicative of NPQ, are most pronounced for

WT and reduced for both npq1 and npq2. Predicted reductions in NPQ for both

npq1 and npq2 relative to WT are consistent with previous estimates (Niyogi et

al., 1997).

The net result of the structure and magnitude of the Fo- and Fm-E curves

is Fv/Fm-E curves that are similar to previously reported curves using ST flash

techniques such as pump and probe modulated fluorescence (Falkowski et al.,

1986). The general structure of the Fv/Fm-E curves is an exponential decay with

the maximal Fv/Fm observed in the dark (Fv/Fm(0)).

All Fv/Fm-E curves are generally in agreement with other measures of

photophysiology. Similar to P-E and Fo curves, WT and npq1 Fv/Fm-E curves

have nearly identical structures. Like Fo curves, these similarities are rooted in

the same photophysiological processes affecting both P-E and Fv/Fm-E curves.

The magnitude of the npq1 Fv/Fm-E curve is reduced relative to WT due to

increases in the magnitude of the Fo-E curve, which in turn are the result of a

reduction in the fraction of functional reaction centers. Similarly, the npq2 Fv/Fm-

E curve has an altered structure compared to WT and npq1 that is again due to

its Fo-E curve. In particular, the decrease in the rate of npq2 Fv/Fm-E decline as

a function of irradiance intensity is consistent with P-E curves and the increase in

non-photosynthetically active pigments in the PSII antennae leading to a

reduction in σPSII(0).

Unlike Fo, Fm and Fv/Fm-E curves, σPSII-E curves have relatively little

structure for WT, npq1 and npq2; σPSII-E curves decrease slightly at moderate

72

irradiance intensities, perhaps due to minor induction of NPQ from the brief

duration background light or from state 2 to state 1 transitions (Bonaventura and

Myers, 1969). This background irradiance-independent nature of σPSII is

consistent with previous oxygen flash yield observations that have also

demonstrated a relatively constant σPSII (Myers and Graham, 1971). Thus,

although the xanthophyll cycle does affect the magnitude of σPSII-E curves, it

does not dramatically modify the structure or shape of the σPSII-E curves for C.

reinhardtii.

Relationship between fluorescence and oxygen

There are many similarities between the patterns of standard physiology

measurements such as σPSU and P-E and fluorescence-derived measurements

such as σPSII(0) and Fo-E. Because both of these methodologies probe similar

(though not the same) portions of the photosynthetic apparatus (i.e. PSII), there

is good reason to expect a strong relationship between oxygen and fluorescence

(Butler and Kitajima, 1975; Papageorgiou, 1975; Butler and Strasser, 1977;

Genty et al., 1989; Lazar, 1999). Nevertheless, NPQ, which in part is regulated

by the xanthophyll cycle, is strictly a fluorescence phenomenon. Thus, changes

to the xanthophyll cycle (and NPQ) could differentially affect fluorescence and

oxygen measurements.

Despite this potential for divergence, WT, npq1 and npq2 fluorescence-

derived photosynthetic rates are strongly (r2=0.96) related to measured oxygen

evolution rates when using a known Pbmax (Figure III-8). This relationship is

73

most robust at light levels above the compensation irradiance (~0.2 eq. on Figure

III-8). This deviation may be due to respiration complications or photosystem

excitation balance considerations (Chapter II). Regardless, the fluorescence

measurements from WT, npq1 and npq2 all have similarly strong relationships

with oxygen P-E, thus changes in the xanthophyll cycle do not appear to

significantly modify the relative fluorescence-oxygen relationship.

Although the structure of fluorescence-determined P-E curves are

consistent with measured P-E, absolute rates are not. For the three samples

measured here, there is no significant relationship between oxygen and

fluorescence maximum Pbmax (r2<0.1). This lack of correlation is due to

processes that are downstream of PSII, such as RuBisCO concentrations and

turnover, regulating maximum photosynthetic rates (Sukenik et al., 1987; Stitt

and Schulze, 1994). Similarly, the correlation between Fv/Fm(0) and φC,max is

marginal (r2=0.51), suggesting that processes other than PSII conversion

efficiency, such as non-photosynthetic pigment concentrations, are regulating

efficiency. Thus, while PSII-specific processes may be regulating the general

shape and structure of the P-E curve via modifications of cross section, they are

not regulating its magnitude or efficiency.

Conclusions

There are significant differences in the photosynthetic biomass and physiology

between WT and xanthophyll mutants of Chlamydomonas reinhardtii (npq1 and

npq2). Loss of violaxanthin de-epoxidase (npq1), which leads to a reduced NPQ,

74

does not significantly affect biomass and photosynthetic cross sections, likely

due to compensatory changes in pigment type and distribution between

photosystems. But, npq1 does have a decreased PSII/PSI and reduced α*,

Pbmax, max2Oφ and Fv/Fm(0) relative to WT probably due to damage to PSII. Loss

of zeaxanthin epoxidase (npq2) results in increased xanthophyll pigments and

decreased photosystem cross sections along with increased PSII/PSI and

reduced α* and max2Oφ . Unexpectedly, both Fv/Fm(0) and NPQ are also reduced,

but this may be the result of induction or increase of an antenna pigment-based

fluorescence. Taken together, biomass and physiology measurements for npq1

and npq2 in relation to WT support the notion of the xanthophyll cycle and NPQ

acting as an antenna quencher of excitation energy, thus affecting the functional

photosynthetic cross section. Modification of the xanthophyll cycle (npq1 and

npq2) does not affect the robust structural (relative) relationships between

fluorescence and oxygen derived photosynthetic rates, but does influence the

correlation among absolute rates and efficiency.

75

Table III-1: Photophysiological parameters of WT, npq1, npq2 C. reinhardtii (mean ± SE, * P<0.05, ** P<0.01 different from WT)

Property Units WT npq1 npq2

Chlorophyll a pg Chla cell-1 1.15±0.12 0.93±0.06 1.02±0.09 σcell µm2 cell-1 8.10±0.93 7.03±0.33 8.69±0.67 σChla m2 g Chl a-1 7.0±0.1 7.6±0.2 8.6±0.1**

σPSII(0) a.u. 1.00±0.05 1.08±0.03 0.88±0.03* σPSU m2 (µmol quanta)-1 1.11±0.11 0.94±0.24 0.77±0.07*

Chlorophyll b/a g g-1 0.305±0.005 0.361±0.011** 0.319±0.007 Chla/P700 mol Chl a mol PSI-1 663±23 526±27** 996±77**

E&A mol Chl a mol O2-1 2537±99 3814±269** 2555±138

PSI / cell amol cell-1 1.94±0.27 1.98±0.23 1.15±0.19* PSII / cell amol cell-1 2.03±0.29 1.09±0.15* 1.79±0.25 PSII / PSI PSII PSI-1 1.05±0.08 0.55±0.07** 1.56±0.20*

α* mol O2 g Chl a-1 hr-1

(µmol quanta m-2 sec-1)-1 0.00176

±0.00011 0.00100

±0.00037* 0.00121

±0.00004**

Pbmax mol O2

g Chla-1 hr-1 0.301±0.005 0.122±0.045** 0.310±0.016

Ek µmol quanta m-2 sec-1 194±12 174±50 286±16** 1/τPSU sec-1 213±12 138±35* 219±23

max2Oφ mol O2 mol quanta-1 0.052±0.003 0.030±0.011* 0.035±0.001**

Fv/Fm(0) unitless 0.68±0.01 0.62±0.01* 0.55±0.01* growth rate day-1 1.04 0.46 0.81

76

77

78

79

m2 m

g C

hl a

-1

0.000

0.005

0.010

0.015

0.020 WTnpq1npq2

Wavelength (nm)

400 500 600 700

% D

evia

tion

from

WT

-50

0

50

100

A

B

Figure III-3: (a) Mean chlorophyll a-specific absorption spectra for WT, npq1 and npq2C. reinhardtii. (b) Percentage deviation from WT chlorophyll a-specific absorptionspectra for npq1 and npq2.

80

Photosynthesis-Irradiance Response Curves

PAR (µmol quanta m-2 sec-1)

0 250 500 750 1000

mol

O2

gChl

a h

r-1

-0.1

0.0

0.1

0.2

0.3

WTnpq1npq2

Figure III-4: Photosynthesis irradiance response curves for WT, npq1 and npq2 C. reinhardtii.Curves represent the mean P-E model fit to data.

81

Fluorescence Excitation

Cumulative Excitation Energy (a.u.)

0 100 200 300 400

Flu

ores

cenc

e (a

.u.)

0.0

0.5

1.0

1.5

2.0

increasing background irradiance0

47

108

433

Figure III-5: Representative fluorescence excitation curves for four continuous backgroundirradiances. Data from one of the WT replicates. Number next to curve is intensity ofbackground irradiance in µmol quanta m-2 sec-1. Note that all curves were generated withthe same PMT voltage setting and biomass concentration.

82

Fluorescence Decay

Seconds

0.000 0.002 0.004 0.006 0.008

Flu

ores

cenc

e (a

.u.)

0.8

1.0

1.2

1.4

1.6

1.8

0

47

108

433

increasing background irradiance

Figure III-6: Fluorescence decay curves (normalized to fluorescence at 7 msec) for fourcontinuous background irradiances. Numbers as figure III-5.

83

Flu

ores

cenc

e (a

.u.)

0.0

0.2

0.4

0.6

0.8

1.0Fo (WT)Fo (npq1)Fo (npq2)

PAR (µmol quanta m-2 sec-1)

0 100 200 300 400 500

Flu

ores

cenc

e (a

.u.)

0.0

0.2

0.4

0.6

0.8

1.0

Fm (WT)Fm (npq1)Fm (npq2)

0 25 50 75 1000.0

0.2

0.4

0.6

0.8

0 25 50 75 1000.4

0.6

0.8

1.0

A

B

Figure III-7: Parameters (a) Fo, (b) Fm, (c) Fv/Fm and (d) σPSII derived from fluorescenceexcitation and decay curves as a function of irradiance for WT, npq1 and npq2C. reinhardtii. Fo and Fm curves have been normalized to Fm at 433 µmol quanta m-2 sec-1

for each respective population. Inset panels are close-up views of low irradiance values.

84

PAR (µmol quanta m-2 sec-1)

0 100 200 300 400 500

σ PS

II (a

.u.)

0.00

0.01

0.02

0.03

σPSII (WT)

σPSII (npq1)

σPSII (npq2)

0 25 50 75 100

0.010

0.015

D

Fv/

Fm

0.0

0.2

0.4

0.6Fv/Fm (WT)Fv/Fm (npq1)Fv/Fm (npq2)

C

0 25 50 75 1000.0

0.2

0.4

0.6

85

relative Fv/Fm-determined oxygen flux

0.0 0.2 0.4 0.6 0.8 1.0

rela

tive

mea

sure

d O

2 flu

x

0.0

0.2

0.4

0.6

0.8

1.0

WTnpq1npq2

Figure III-8: Relationship between measured oxygen evolution andfluorescence-derived oxygen flux. Fluorescence-derived measurementswere calculated using ((Fo-Fo(0))/Fo(0))-E curves that were normalized to

measured oxygen evolution Pbmaxs. The slope of the linear regressionis not significantly different from 1.0 with an r2=0.96.

86

Chapter IV

Mechanisms and consequences of photoacclimation to photosynthetic efficiency in Skeletonema costatum

87

Open ocean environments are complex light environments for

phytoplankton photosynthesis with irradiance intensities varying several

orders of magnitude. In this context, phytoplankton have multiple

mechanisms to acclimate, which in turn can affect both photosynthetic

rates and efficiency. Here using Skeletonema costatum as a model

organism, I expand on previous photoacclimation studies (1) to determine

the response of photosynthetic quantum yield-irradiance (φφC-E) curves to

photoacclimation and (2) to quantify the role that photosystem II (PSII)

plays in structuring φφC-E curves. High growth irradiance induces

significant increases in chlorophyll a-specific absorption, light saturated

photosynthesis, photosynthesis saturating irradiance and significant

decreases in cellular chlorophyll a, photosynthetic unit cross sectional

area, PSII cross sectional area, light utilization, maximum quantum yield

and Emerson and Arnold numbers. The net result of these changes in

photosynthetic biomass and photophysiology is high light φφC-E curves that

are reduced in magnitude and have increased irradiance at which φφC

decreases from φφC,max. Changes in relative P-E and φφC-E curves due to

photoacclimation are well-described by PSII specific measurements

including Fv/Fm-E and σσPSII-E. However, in the context of photoacclimation,

Pbmax and φφC,max are not well-described by fluorescence measures because

they are regulated by non-PSII processes such as non-photosynthetic

pigment absorption and processes downstream of PSII.

INTRODUCTION

88

In the open ocean, light is quickly attenuated with depth due to strong absorption

by water and biogenous materials such that only the upper skin of the open

ocean is light replete with respect to photosynthesis (Jerlov, 1976; Morel, 1988;

Kirk, 1994). Vertical mixing of phytoplankton within the euphotic zone from light-

saturated to light-limited regions can impose additional limitations on

photosynthesis (Sverdup, 1953; Marra, 1980; Johnson and Howd, 2000). Far

from statically responding to different light levels, phytoplankton have multiple

mechanisms of acclimation, which in turn can affect photosynthetic rates and

efficiencies.

There is a significant body of research devoted to understanding the role

that irradiance plays in regulating photosynthetic responses. For steady-state

light, photoacclimation is rooted as the effect of light intensity on gene expression

and protein production regulation via redox poise (Levings III and Siedow, 1995;

Durnford and Falkowski, 1997). This regulation occurs for both chloroplast and

nuclear encoded genes through sensing of the redox state of the plastoquinone

pool in the chloroplast or via production of reducing equivalents from PSI that

regulate thioredoxin (Danon and Mayfield, 1994; Escoubas et al., 1995; Durnford

and Falkowski, 1997; Dai et al., 2000). Although these mechanisms are not fully

characterized, they do point to irradiance-mediated gene-regulation and

ultimately protein production as the partial mechanistic basis for steady-state

photoacclimation.

Following this redox poise, light-mediated genetic basis of regulation,

previous studies have demonstrated that there are significant differences in

89

specific photosynthetic biomass properties in relation to photoacclimation. For

example, compared to low light grown cells, high light grown S. costatum have

reduced cellular chlorophyll a, cellular PSII and PSI concentrations and

photosynthetic accessory pigments (chlorophyll c and fucoxanthin) (Falkowski

and Owens, 1980; Falkowski et al., 1981; Perry et al., 1981; Gallagher et al.,

1984; Caron et al., 1988). Other proteins that are not directly involved in the light

harvesting process such as RuBisCO and D1 have also been shown in

phytoplankton to be influenced by light intensity (Laroche et al., 1991; Sciandra

et al., 1997; Shapira et al., 1997). It is these light harvesting and excitation

processing related proteins that are affected by photoacclimation that in part lead

to the observed changes in the photosynthetic rates and efficiency.

For example, photoacclimation to high light in S. costatum can increase

photosynthetic capacity (Pbmax) and decrease PSII functional cross sectional

area (σPSII(0)) and Emerson & Arnold numbers (E&A) (Falkowski et al., 1981;

Cosper, 1982; Gallagher et al., 1984; Kolber et al., 1988). High growth irradiance

has also been shown to dramatically reduce light utilization and quantum

efficiency relative to low light grown populations while not significantly effecting

the photochemical conversion efficiency of PSII (Fv/Fm(0)) (Kolber et al., 1988;

Herzig and Dubinsky, 1992; Lindley et al., 1995; Fisher et al., 1996; Marra et al.,

2000). Photosynthetic unit turnover rates can also be influenced by growth

irradiance (Falkowski et al., 1981; Dubinsky et al., 1986; Behrenfeld et al., 1998).

This body of evidence suggests that photoacclimation is fairly consistent between

90

species and embodies a series of standard biomass and photophysiological

responses.

Taken together, previous research has demonstrated a photoacclimation

response that affects photosynthetic efficiency, rates and biomass that is based

at the gene-regulation level. Although the effect of photoacclimation on biomass

and process components has been characterized for many phytoplankton

(Falkowski et al., 1981), there remain some gaps in the mechanistic linkage

between analytic components and processes. Specifically, there is no analytic

relationship for the role of PSII in regulating photosynthetic rates (P-E) and

efficiency (φC-E) as a function of background irradiance in the context of

photoacclimation.

To aid in closing these gaps, the goals of this study are (1) to characterize

the quantum efficiency-irradiance (φC-E) curve in the context of photoacclimation

and (2) to quantify the role of irradiance-dependent PSII-specific processes such

as σPSII-E and Fv/Fm-E in determining φC-E curves. For this investigation I use

standard photophysiological measures such as flash yields, radiocarbon uptake,

spectral absorption and pigment concentrations in combination with a newly

developed active fluorescence induction technique that measures PSII

photosynthetic parameters as a function of background irradiance (Chapter III).

Results from this study show (1) a decrease in the magnitude of φC-E

(φC,max) with increasing growth irradiance and (2) a lateral (irradiance) shift of the

φC-E curve that is consistent with growth irradiance and photosynthesis

saturating irradiances (Ek). This shift is also present for Fv/Fm-E curves and is

91

supported by altered σPSII(0), σPSU and E&A These data show that PSII-specific

measures describe well the relative φC-E and P-E structures, but that the

magnitudes of these curves are not well established by PSII-specific

measurements; in the context of photoacclimation, φC,max and Pbmax are in part

regulated by non-photosynthetic pigments and processes downstream of PSII,

respectively.

METHODS

Culture conditions

Skeletonema costatum (SKEL) cultures were grown in semi-continuous batch

culture at 19oC in sterile (0.2 µm filtered) air bubbled f/2-amended media made

with filtered (GF/F – Whatman) Sargasso Sea water (Guillard and Ryther 1962)

and were periodically diluted to maintain exponential growth. Continuous light (

211±8, 111±8, 20±0 and 8±1 µmol quanta m-2 sec-1), which was supplied by

fluorescent bulbs, was attenuated by neutral density and stage screening

(Cinemills) to achieve the desired intensity and spectral quality.

Absorption, Pigments, Growth Rates, Cell Density

Spectral absorption measurements were made at 2 nm resolution with a 1 sec

integration time on samples in solution using an HP 8452 diode array

spectrophotometer with Labsphere (RSA-HP-84) integrating sphere. Absorption

at 750 nm was assumed to be non-cellular and was subtracted from the

absorption curve. Absorption coefficients were calculated following Kirk (1994).

92

Chlorophyll a and b and carotenoids were determined on 90% acetone extracts

following Parsons et al. (1984), except without MgCO3 addition, using the

trichromatic equations of Jeffrey and Humphrey (1975). Intrinsic growth rates

were calculated from chlorophyll a concentrations. Cell concentrations were

estimated using a hemacytometer.

Photosystem Quantification

Emerson-Arnold numbers (Chl a/O2) were measured using a Hansatech oxygen

electrode and a Stroboslave (Type1539A) tunable frequency, saturating strobe

light following Dubinsky et al. (1986). PSII/Chl a was calculated as four times the

initial slope of oxygen evolution rate versus flash frequency, normalized to

chlorophyll concentration.

Photosynthesis vs Irradiance (P-E), Turnover Rates

Carbon-based photosynthesis-irradiance measurements were made using

custom-built temperature-regulated photosynthetrons. Irradiance, which was

supplied by a 250 W ENH projector bulb (Gray Supply), was spectrally modified

and attenuated using a combination of hot and cold mirrors (Optical Coating

Laboratory) and stage screening (Cinemills). Irradiance intensity (PAR) was

measured using a Biospherical Instruments (QSL-100) 4π scalar irradiance

meter. Incubations of 1 ml samples inoculated with ~7.4 kBq H14CO3 were

terminated after 10 min using 100 µl 37% formaldehyde and 200 µl HCl and

allowed to degas overnight. Carbon uptake rates were quantified using standard

93

techniques (Barber et al., 1996). Photosynthetic parameters of the Platt et al.

(1980) model (α*, Ps, β) were optimized to fit data using a custom written, non-

linear least-squares Levenberg-Marquardt technique (Frenette et al., 1993).

Optimization of model parameters to P-E data was performed using PAR as the

independent variable. Secondary parameters (Pbmax and Ek) were calculated

following Zimmerman et al. (1987). Maximum quantum yield (φC,max) was

calculated from model predicted α* as the mol C mol quanta-1 absorbed using

absorption coefficients that were spectrally weighted to incident measurement

irradiance (Chapter II). Maximum quantum yield (φC,max') calculated using the

[ ]EC −φmax method (Chapter II) yielded similar patterns, but ~25% reduced

values.

Photosynthetic unit turnover rate constant (sec-1), 1/τPSU, was calculated

using two methods. In the first (standard) method 1/τPSU was calculated as:

n

P b

PSU

max0269.01 =τ (IV-1a)

where Pbmax is light-saturated photosynthesis (mg C mg Chl a-1 hr-1), n is the

concentration of PSII (mol O2 mol Chl a-1) and 0.0269 converts the units from

hours to seconds and grams to moles and carbon to oxygen using a

photosynthetic quotient (PQ) of 1.3 (Laws, 1991). Because a constant PQ is

assumed, estimates of 1/τPSU should be interpreted with caution. In the second

94

(alternate) method relative 1/τPSU was calculated following Dubinsky et al. (1986)

as:

[ ]kPSIIPSU

E)0('1 στ = (IV-1b)

where σPSII(0) is the relative PSII functional cross sectional area and Ek is the

photosynthesis saturating irradiance. These estimates of relative 1/τPSU are

referred to as 1/τPSU'.

Modulated Fluorescence

Modulated fluorescence measurements were performed with a previously-

described custom-built fast repetition rate type fluorometer with computer-

controlled background irradiance gradient (BIG-FRRf) (Chapter III) (Kolber et al.,

1998). The background light is produced by super-bright blue LEDs and has a

maximum output wavelength of ~550 nm (Chapter III). For this study, the

excitation protocol was run for 350 µsec at high intensity with a 100% duty cycle

followed by the decay protocol that was run for 7.5 msec at low intensity with a

1% duty cycle. The pre-illumination background irradiance duration was 500

msec. Samples were dark adapted for >15 min for each background irradiance

setting. Data were analyzed using custom-written software based in MATLAB

5.3v11 following Kolber et al. (1998) to determine Fo, Fm, σPSII and p where Fo is

initial fluorescence, Fm is the maximal fluorescence, σPSII is the functional cross-

sectional area, p is a connectivity parameter.

95

For each background irradiance there is generated a suite of

fluorescence-derived parameters. These parameters are referred to with the

subscript indicating the background irradiance intensity. For example, Fv/Fm(0) is

photosystem II photochemical conversion efficiency in the dark and σPSII(521) is

the PSII functional cross sectional area at 521 µmol quanta m-2 sec-1.

RESULTS / DISCUSSION

Photosynthetic Biomass

Cellular chlorophyll a concentrations and chlorophyll a-specific absorption cross

sections (σChl a) were significantly affected by growth irradiance (Table IV-1).

These changes in pigmentation and absorption properties in response to different

growth irradiances likely involve the balance between two processes:

photosynthetic and non-photosynthetic protective pigment (NPP) optimization

with the balance shifting depending on whether there is limiting or excess light,

respectively. Increases in σChla with growth irradiance support the relative

proportion of NPP increasing with growth irradiance and suggest σChla being

dominated by NPP. Conversely, σcell remains constant with growth irradiance,

suggesting that there are compensatory changes in photosynthetic active

accessory pigments. Curiously, neither chlorophyll c/a nor carotenoid/chlorophyll

a mass ratios have significantly different values between growth irradiances

(Gallagher et al., 1984). However, the increase in the mean chlorophyll c/a that

is not statistically significant is consistent with previously measured increases in

chlorophyll c/a with decreased growth irradiance (Kolber et al., 1988). Similarly,

96

although relative measured carotenoid concentrations remain constant for

different growth irradiances, this stability may be influenced by imprecise

spectrophotometric carotenoid measurement; spectrophotometer estimates of

carotenoids can be significantly affected by other accessory pigments such as

fucoxanthin (Jeffrey et al., 1997). Although there are some inconsistencies,

taken together these data support a compensatory increase in NPP and

decrease in photosynthetically active pigments with increasing growth irradiance

that leads to a relatively constant cellular absorption cross section.

Commensurate with changes in aggregate biomass properties were

changes in photosystem biomass properties. Both the Emerson and Arnold

number (E&A) and photosynthetic unit cross sectional area (σPSU) increased

significantly with decreasing irradiance (Table IV-1) and are consistent with

previous observations of increased photosynthetic cross sections for decreased

light (Falkowski et al., 1981; Dubinsky and Stambler, 1992). Increases in the

photosynthetic cross section with decreased irradiance are also supported by

elevated σPSII(0) (see below).

Photophysiology

Photoacclimation, partially through changes in photosynthetic biomass

properties, dramatically affected the structure and magnitude of photosynthesis-

irradiance (P-E) curves as well as associated photophysiological parameters

(Table 1, Figure IV-1). Light saturated photosynthesis (Pbmax) and the maximum

light utilization coefficient (α*) generally followed opposite patterns with Pbmax

97

increasing and α* decreasing with growth irradiance. However, α* and Pbmax

patterns were uncorrelated (r2=0.04) suggesting that they were regulated by

different processes. Increases in Pbmax were related (r2=0.67) to reductions in

E&A and were uncorrelated with turnover time, which did not change significantly

with growth irradiance. Nevertheless, although overall variability in Pbmax is

most closely related to E&A, at higher irradiances Pbmax may in part be

regulated by 1/τPSU (see below).

Increases in α* with decreased growth irradiance were correlated with

increases in σPSU (r2=0.76), σPSII(0) (r2=0.80) and E&A (r2=0.50) and decreases in

σChla (r2=0.54) pointing to differences in cross section mediating the initial slope.

The negative correlation with σChla is likely due to an inverse correlation between

photosynthetically active accessory pigments and the NPP that are responsible

for the majority of variability in σChla. The saturating irradiance intensity (Ek) also

increased with growth irradiance and was correlated with decreases in cross

section (σPSU r2=0.84) as well as E&A (r2=0.94).

Similar to α*, φC,max was inversely correlated with growth irradiance.

Because photosynthetic conversion efficiency (Fv/Fm(0)) remains relatively

constant among growth irradiances, the reductions in φC,max are likely due to

changes in pigmentation and absorption. Increases in σChla and decreases in α*,

both due to the relative increase in NPP, support a dominant role for absorption.

In addition to changes in magnitude, there are also changes in the φC-E

structure with growth irradiance (Figure IV-2). Most notably, high growth

98

irradiance increases the irradiance at which φC begins to depart from φC,max. For

this photoacclimation study, this irradiance has similar patterns to Ek. Because

the photosynthetic turnover time (1/τPSU) is not significantly affected by the

growth irradiance, this irradiance shift is largely regulated by changes in cross

sectional area (σPSU and σPSII(0)). These patterns are similar to those of Ek.

Although there are significant increases in Pbmax with growth irradiance,

1/τPSU (and 1/τPSU') remains relatively constant because E&A decreases with

increasing irradiance. This pattern suggests that for S. costatum grown in the

present conditions, photoacclimation does not significantly affect photosynthetic

unit functionality via 1/τPSU (see below). This may not be true for all

phytoplankton taxa: previous investigations have found dramatic alterations in

1/τPSU as a function of growth irradiance for other phytoplankton (Dubinsky et al.,

1986; Behrenfeld et al., 1998).

Fluorescence-derived Parameters

All estimates of Fo-, Fm-, Fv/Fm- and σPSII-E (Figure IV-3) were generally

consistent with previous measurements of curve structure using other

phytoplankton taxa under nutrient-replete conditions (Chapter III). Nevertheless,

similar to standard photophysiology measurements, fluorescence-derived

estimates of PSII properties were also significantly affected by growth irradiance.

Consistent with previous studies, growth irradiance did not significantly

affect Fv/Fm(0) with all values ~0.60. However, σPSII(0) increased significantly at

99

lower growth irradiances (Table IV-1) (Kolber et al., 1988). This increase in

σPSII(0) at low irradiances is consistent with increases in the σPSU (r2=0.98) and

E&A (r2=0.99) and suggests that either σPSU are dominated by changes in σPSII(0)

or that photosystem I cross sections follow similar trends (i.e. are larger at lower

growth irradiances) as σPSII(0). These patterns are also consistent with φC-E

curves.

To examine the effect of photoacclimation on Fv/Fm and σPSII in more

detail, I constructed Fv/Fm-E and σPSII-E curves (Figures IV-4 and IV-5).

Although Fv/Fm(0) was not significantly affected by growth irradiance, the

structure of Fv/Fm-E curves was. Curves from low light grown populations

decrease more rapidly than do high light grown populations. Because 1/τPSU

remains relatively constant, differences in structure between high and low light

grown populations are likely due to σPSII(0); more rapid reduction in Fv/Fm-E

curves for low light grown populations is consistent with larger σPSII(0).

Similar to Fv/Fm-E curves, σPSII-E curves are also significantly different in

magnitude and structure for different growth irradiances. The magnitude of σPSII-

E curves are dominated by σPSII(0), thus low growth irradiance σPSII-E curves are

consistently elevated above high growth irradiance σPSII-E curves.

In addition to changes in the magnitude, relative σPSII-E curves also differ

in structure in two major fashions. Firstly, high light σPSII-E curves have an

elevated maximum to minimum σPSII ratio. Decreases in σPSII at moderate light

levels are likely in part due to nonphotochemical quenching (NPQ). Thus, an

100

increase in the maximum to minimum σPSII ratio suggests that high light grown

populations have elevated NPQ related to low light grown populations. This is

consistent with increases in relative NPP concentrations suggested by increases

in σChla at high light and with previous studies suggesting an increased NPQ

potential for high light plants (Demmig-Adams and Adams, 1992; Casper-Lindley

and Bjorkman, 1998).

The second way photoacclimation influences σPSII-E structure is that high

light σPSII-E curves have a minor local peak at very low background light that is

not present for low light σPSII-E curves (Figure IV-5 bottom inset). Because of the

limited number of samples at low background light, using the present data it is

not possible to tell if the localized peak is present for all growth irradiances and

simply shifted laterally to lower irradiances or if the peak is simply not present for

low growth irradiance σPSII-E. Regardless, the very high light (211 µmol quanta

m-2 sec-1) localized peak is approximately 10% above measured σPSII(0). One

potential mechanism for this minor peak is that the increase is approximately the

same order as state transition effects (Bonaventura and Myers, 1969; Falkowski

and Raven, 1997). Populations at high growth intensities are likely in state 1 and

may require a low (non-zero) background light, potentially as a source of ATP, to

induce dephosphorylation of the pigment bed to initiate a change to state 2. This

process could lead to an effective increase in σPSII. This increase would not be

present for low growth irradiances because they are likely already in state 2. The

decrease in σPSII in all populations at moderate background light levels are due to

101

NPQ, thus only high growth irradiances would have a minor peak at low

background lights because low growth irradiances do not have an initial increase.

A second potential mechanism for this minor peak is the minor population

of PSIIβ (Melis and Anderson, 1983; Melis, 1984). Because these reaction

centers have reduced cross section and decreased turnover time, a very dim

background light could close a significant number PSIIβ, but not PSIIα and may

result in an increased apparent PSII photosynthetic cross section. Previous work

has demonstrated an increase in the PSIIα/PSIIβ ratio for low compared to high

light grown plants, which is consistent with observed σPSII-E trends (Hodges and

Barber, 1983). Irrespective of the mechanism, the localized peak, which is

photoacclimation-dependent, does affect the shape, albeit minimally, of σPSII-E

curves.

Fluorescence-Photosynthesis Relationship

Photoacclimation to different growth irradiances induces significant changes in

photosynthetic biomass and physiology parameters. Fluorescence measures of

properties of PSII also document significant changes in photophysiology

properties. How well do changes in σPSII-E and Fv/Fm-E document changes in P-

E and φC-E? Or, in the context of photoacclimation, how well does PSII variability

describe the variability in photosynthetic rates and efficiency?

Photoacclimation significantly affects photosynthetic efficiency (φC,max).

This variability is not due to reaction center damage, but rather changes in

102

absorption. Because Fv/Fm is absorption independent, Fv/Fm(0) remains

relatively constant despite significant decreases in φC,max with increasing growth

light (Figure IV-4). Nevertheless, Fv/Fm-E and φC-E have similar curve

structures, such that paired comparisons of Fv/Fm and φC over a background light

gradient are significant (r2=0.66) (Figure IV-6). However, this relationship is

driven almost exclusively by changes in the relative structure of Fv/Fm-E and φC-

E curves that are highly correlated (r2=0.93). Thus, factors that regulate the

shape and structure of efficiency curves (Fv/Fm-E and φC-E) such as

photosynthetic cross sections and turnover time appear to equally affect both

Fv/Fm-E and φC-E in the context of photoacclimation, but factors that affect

maximal efficiency such as NPP differentially affect Fv/Fm-E and φC-E.

From strong Fv/Fm and φC correlations, a priori one would expect equally

robust photosynthetic rate predictions, even though photoacclimation embodies

changes in cross section and concentration of photosynthetic units, that in turn

affect both the structure and magnitude of P-E related to photoacclimation.

Indeed, when normalizing to known Pbmax values, PSII rate variability describes

nearly all of the variability in both absolute (r2=0.97) and relative (r2=0.97)

photosynthetic rates (Figure IV-7). This finding is consistent with the robust

relationship between σPSU and σPSII(0) (r2=0.98) that describe the structure of the

P-E and fluorescence-predicted P-E curves, respectively.

However, Pbmax is not described well by fluorescence measurements

(r2=0.37). This is somewhat surprising given the small variability in 1/τPSU and a

103

robust σPSII(0) - E&A relationship (r2=0.99). The deviation between predicted and

observed Pbmax is mainly due to a fluorescence predicted increase (that is not

observed) in Pbmax for very high (211 µmol quanta m-2 sec-1) compared to high

(111 µmol quanta m-2 sec-1) grown populations. The lack of strong correlation

between predicted and observed Pbmax suggests that processes downstream of

PSII, such as RuBisCO concentration and turnover, are controlling maximum

photosynthetic rates (Sukenik et al., 1987; Stitt and Schulze, 1994). A corollary

of downstream regulation is that there is excess capacity available through PSII

such that if functional PSII reaction centers are removed, Pbmax is not

significantly affected (Behrenfeld et al., 1998). In the context of variable light

fields, this excess capacity may provide "insurance" against future reaction

center damage from potentially increased light levels (Neale, 1987; Aro et al.,

1993). At lower growth irradiances, the chance of incurring damage to PSII is

reduced and photosynthetic electron transport chain and downstream

components, such as RuBisCO concentrations and turnover, may be more

balanced (Genty and Harbinson, 1996). Regardless, this excess PSII capacity

leads to a divergence in the relationship between fluorescence-predicted and

observed Pbmax, limiting accurate descriptions of photosynthetic rate and

efficiency by fluorescence techniques to relative descriptions.

104

Table IV-1: Photoacclimation of photosynthetic biomass and physiological parameters (Means ± SE) Superscripts a, b, c and d signify that the sample is significantly different at P<0.05 from very high, high, low and very low growth irradiance sample, respectively.

Property Units Very High High Low Very Low growth PAR µmol quanta m-2 sec-1 211±8 111±8 20±0 8±1

Chl a fg Chl a cell-1 408±7 c,d 420±8 c,d 583±42 a,b 547±29 a,b

Chl c/a g g-1 0.159±0.012 0.169±0.009 0.176±0.005 0.217±0.018

carot/Chla g g-1 0.382±0.017 0.336±0.015 0.378±0.008 0.374±0.012

σcell µm2 cell-1 4.55±0.37 4.30±0.20 5.23±0.66 4.60±0.23

σChla m2 g Chl a-1 11.1±0.8 d 10.2±0.5 d 9.2±0.4 8.4±0.1 a,b

σPSU m2 µmol quanta-1 0.77±0.21 c 1.04±0.12 c 1.86±0.28 a,b 1.70±0.47

σPSII(0) a.u. 0.030±0.001 c 0.032±0.001 c 0.044±0.001 a,b n/a

α mg C mg Chl a-1 hr-1

(µmol quanta m-2 sec-1)-1 0.0190±0.0013 b,c 0.0241±0.0007 a 0.0283±0.0015 a 0.0244±0.0016

Pbmax mg C mg Chl a hr-1 1.30±0.04 d 1.43±0.02 c,d 1.19±0.01 b,d 0.72±0.02 a,b,c

Ek µmol quanta m-2 sec-1 69±6 c,d 59±3 c,d 42±2 a,b,d 30±2 a,b,c

φC,max mol C mol quanta-1 0.039±0.003 b,c,d 0.055±0.002 a,c 0.071±0.004 a,b 0.067±0.004 a

φC,max' mol C mol quanta-1 0.030 0.044 0.051 0.056

E&A mol Chl a mol O2-1 1514±239 c 1606±88 c 2455±242 a,b 2613±484

1/τPSU sec-1 53.1±10.0 61.8±4.4 78.8±8.2 50.9±10.6

1/τPSU' relative 1.12±0.12 1.02±0.06 1.00±0.07 n/a

µ d-1 1.64 1.44 0.26 0.16

105

S. costatum Photosynthesis - Irradiance

Pb

(mg

C m

g C

hl a

-1 h

r-1)

0

1

2

3

211111208

0.1 1 10 100

PAR (µmol quanta m-2 sec-1)

0 100 200 300 400 500 600

Pb

(rel

ativ

e)

0.00

0.25

0.50

0.75

1.00

0.1 1 10 100

Figure IV-1: Absolute (top) and relative (bottom) photosynthesis-irradiance response curvesfor S. costatum grown at four irradiance intensities (211, 111, 20 and 8 µmol quanta m-2 sec-1) 211 - open circles, dotted lines; 111 - light gray circles, light gray line; 20 - dark gray circles,dark gray line; 8 - closed circles, black line. Inset panels same data except with common logabscissa.

106

-0.02

0.00

0.02

0.04

0.06

0.08

211 µmol quanta m-2 sec-1

111 µmol quanta m-2 sec-1

20 µmol quanta m-2 sec-1

8 µmol quanta m-2 sec-1

φ C (

mol

C m

ol q

uant

a-1)

-0.02

0.00

0.02

0.04

0.06

-0.02

0.00

0.02

0.04

0.06

PAR (µmol quanta m-2 sec-1)1 10 100 1000

-0.02

0.00

0.02

0.04

0.06

Figure IV-2: φC-E curves for S. costatum grown at four light levels.Dotted vertical lines are for reference only.

107

S. costatum 211 µmol quanta m-2 sec-1

Fo

or F

m (

a.u.

)

0.0

0.5

1.0

1.5

Fv/

Fm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FoFmFv/Fm

PAR (µmol quanta m-2 sec-1)

0 200 400 600

σ PS

II (a

.u.)

0.00

0.02

0.04σPSII

Figure IV-3a: Fluorescence-derived parameters as a function of irradiance for S. costatumgrown in continuous 211 µmol quanta m-2 sec-1 light. (top) initial fluorescence (Fo, openred circles), saturated fluorescence (Fm, closed red circles), and PSII photochemicalconversion efficiency (Fv/Fm, blue squares), (middle) PSII functional cross-sectional area(σPSII, green triangles). Note that the background light used here and for all fluorescence datawas blue-dominated as compared to the "white" spectra used for growth and P-Eand φC-E curves.

108

S. costatum 111 µmol quanta m-2 sec-1

Fo

or F

m (

a.u.

)

0.0

0.5

1.0

1.5

Fv/

Fm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FoFmFv/Fm

PAR (µmol quanta m-2 sec-1)0 200 400 600

σ PS

II (a

.u.)

0.00

0.02

0.04

σPSII

Figure IV-3b: As Figure IV-3a except for grown in 111 µmol quanta m-2 sec-1.

109

S. costatum 20 µmol quanta m-2 sec-1

Fo

or F

m (

a.u.

)

0.0

0.5

1.0

1.5

Fv/

Fm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FoFmFv/Fm

PAR (µmol quanta m-2 sec-1)

0 200 400 600

σ PS

II (a

.u.)

0.00

0.02

0.04

σPSII

Figure IV-3c: As Figure IV-3a except for 20 µmol quanta m-2 sec-1

110

PAR (µmol quanta m-2 sec-1)

0 200 400 600 800

rela

tive

Fv/

Fm

0.0

0.5

1.0

0 25 50 75 100

Figure IV-4: Relative Fv/Fm-irradiance (Fv/Fm-E) curve comparison for differentgrowth irradiances. Inset panel is same data except with an expanded abscissa.Symbols as in figure IV-1.

S. costatum Fv/Fm-E Comparison

111

S. costatum PSII functional cross section

σ PS

II (a

.u.)

0.02

0.04

0.06

PAR (µmol quanta m-2 sec-1)

0 200 400 600 800

rela

tive

σ PS

II

0.0

0.5

1.0

1.5

0 25 50 75 100

0 25 50 75 100

Figure IV-5: PSII functional cross sectional area-irradiance (σPSII-E) curve comparison for

different growth irradiances. (top) Absolute σPSII-E curves (a.u.) and (bottom) relative σPSII-Ecurves. Inset panels are same data except with an expanded abscissa. Symbols as figure IV-1.

112

Measured

Fv/Fm(x) (all irradiances)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

φ C(x

) (al

l irr

adia

nces

)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Relative

Normalized Fv/Fm(x) (all irradiances)

0.0 0.2 0.4 0.6 0.8 1.0

Nor

mal

ized

φC

(x) (

all i

rrad

ianc

es)

0.0

0.2

0.4

0.6

0.8

1.0

Figure IV-6: (top) Measured and (bottom) relative (to maximum observed infor Fv/Fm-E or φC-E curve, respectively) relationship between PSII conversion

efficiency (Fv/Fm) and photosynthetic quantum efficiency (φC) for all background

light intensities. For the purpose of interpolation, φC was assumed to approach

φC,max as the background light approached zero. Absolute r2=0.66 and relative r2=0.93.

113

Measured

Fls-derived Pb (all irradiances)

0.0 0.5 1.0 1.5 2.0

Pb (

all i

rrad

ianc

es)

0.0

0.5

1.0

1.5

2.0

Relative

Normalized Fls-derived Pb (all irradiances)

0.0 0.2 0.4 0.6 0.8 1.0

Nor

mal

ized

Pb (

all i

rrad

ianc

es)

0.0

0.2

0.4

0.6

0.8

1.0

Figure IV-7: (top) absolute and (bottom) relative (to maximum observed infor P-E curve) relationship between fluorescence-derived photosynthetic ratesand measured photosynthetic rates. Fluorescence-derived rates were calculatedusing E*(Fo-Fo(0))/Fo(0) and normalizing the resultant curve to measured Pbmax

values. Measured r2=0.97 and relative r2=0.97.

114

Chapter V

Mechanisms of reduction in photosynthetic efficiency under nitrogen-limitation in Skeletonema costatum

115

Nutrient-limitation of phytoplankton, which can significantly affect

photosynthetic rates and efficiency, may be typical in open-ocean

ecosystems. Of the different types of nutrient-limitation, insufficient levels

of nitrogen may be dominant in the present-day ocean. Previous

investigations have shown that nitrogen-limitation can significantly

influence protein turnover and in turn photosynthetic biomass, rates and

efficiency. Here I expand on these studies to include fluorescence-based

measures of photosystem II (PSII) as a function of background irradiance

to investigate the regulatory role of PSII on photosynthetic efficiency in the

context of N-limitation. Reductions in the initial slope (αα*) of

photosynthesis-irradiance (P-E) curves and in quantum efficiency (φφC)

under nitrogen-limitation are mechanistically related to changes in the

magnitude and structure of PSII photochemical conversion efficiency

(Fv/Fm)- and PSII functional cross sectional area (σσPSII)- irradiance curves.

Specifically, these findings demonstrate that nitrogen-limitation (1)

decreases the magnitude of the quantum efficiency -irradiance (φφC-E) curve

via reductions in dark measured Fv/Fm, (2) decreases the magnitude of P-E

by decreasing the turnover time and (3) reduces the irradiance at which φφC

starts to decrease from φφC,max via increases in the magnitude of σσPSII-E. This

final reduction is not predicted from standard P-E curve-analyses (Ek), but

can be clearly observed in Fv/Fm-E and φφC-E curve architecture. These

results demonstrate that changes in photosynthetic unit cross section as

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well as both the magnitude and structure of φφC-E, Fv/Fm-E and σσPSII-E

curves are important in describing the photosynthesis-irradiance response

to nutrient limitation. These results show that in the context of non-steady

state N-limitation, PSII-specific processes account for the majority of

variability in terms of relative photosynthetic efficiency and P-E and φφC-E

structure, but do not describe well absolute P-E curves because processes

unlinked to PSII such as RuBisCO concentration and turnover time are

likely regulating light saturated photosynthesis.

INTRODUCTION

Because major plant nutrients are typically low compared to half-saturation

constants, nutrient-limitation of photosynthesis may be standard in many

oceanographic regions (Eppley et al., 1969; Levitus et al., 1993; Falkowski et al.,

1998). Of the various forms of nutrient-limitation, nitrogen may play a

disproportionately important role in regulating historic and present-day marine

photosynthesis (Codispoti, 1989; Barber, 1992; Falkowski, 1997).

In this capacity, the effect of nitrogen-limitation on photosynthesis has

historically been well-studied in both the field and laboratory (Cleveland and

Perry, 1987; Cleveland et al., 1989; Herzig and Falkowski, 1989; Henley et al.,

1991; Babin et al., 1996). These studies have revealed a suite of photosynthetic

biomass and functional responses to N-limitation that ultimately are rooted in a

phytoplankter’s inability to construct functional chloroplast proteins (Coleman et

al., 1988; Turpin, 1991). In part, chloroplast proteins are more susceptible to N-

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limitation because they turn over rapidly in response to the particularly large

swings in redox potential (Crofts and Wraight, 1983). However, both proteins

with normally high levels of turnover such as reaction center associated proteins

D1, D2 and CP43 as well as slower turnover, non-reaction center proteins such

as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) are sensitive to

N-stress (Falkowski et al., 1989; Beardall et al., 1991; Aro et al., 1993; Garcia-

Ferris and Moreno, 1994; Vasilikiotis and Melis, 1994).

Because it alters the production of necessary proteins, N-limitation leads

to altered photosynthetic functionality. For example, reaction center proteins,

which are crucial to the efficient use of excitation energy, can become damaged

under N-limitation that in turn can lead to marked reductions in photosystem II

(PSII) photosynthetic conversion efficiencies. These decreases also lead to an

overall increase in the functional size of PSII antennae (Kolber et al., 1988;

Geider et al., 1998). Similarly, N-limitation can lead to significant reductions in

both the concentration and activity of RuBisCO (Beardall et al., 1991; Garcia-

Ferris and Moreno, 1994). Other chloroplast and mitochondrial proteins, which

affect the photosynthesis process, are also specifically influenced by N-limitation

and lead to reduced functionality (La Roche et al., 1993).

In addition to these direct effects on excitation energy processing, other

components and processes of photosynthesis are affected by N-limitation. For

example, pigment concentrations are dramatically altered under N-limitation

including chlorosis as well as the relative increase in non-photosynthetically

active pigment concentrations (Sosik and Mitchell, 1991; Collier and Grossman,

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1992; Latasa and Berdalet, 1994). In turn, these changes in pigmentation can

affect absorption properties (Sosik and Mitchell, 1991; Geider et al., 1993).

Aggregate pigment indices also suggest that pigment-dependent processes such

as non-photochemical quenching of excitation energy may be altered under N-

limitation (Demmig-Adams and Adams, 1992; Verhoeven et al., 1997).

From studies of the effects of N-limitation on protein turnover and

expression, it is then not surprising that photosynthetic rates and efficiencies are

significantly reduced under N-limitation (Cleveland and Perry, 1987; Kolber et al.,

1988; Sosik and Mitchell, 1991; Geider et al., 1998). These reductions are

characterized by decreases in the magnitude and altered structure of

photosynthesis-irradiance response curves and specifically result in decreased

light-saturated photosynthesis (Pbmax) and quantum efficiencies. However, the

effect of nutrient-limitation in general and nitrogen-limitation in particular on the

mechanisms that determine photosynthesis-irradiance curves has been difficult

to unravel (Cullen et al., 1992).

In this study, my goal is to uncover the effect of N-limitation on the

quantum efficiency-irradiance (φC-E) relationship by focusing on PSII, to

analytically link previously documented changes in mechanistic properties such

as photochemical conversion efficiencies (Fv/Fm(0)) and PSII functional cross

sectional area (σPSII(0)) to observed changes in photosynthesis-irradiance (P-E)

and quantum efficiency-irradiance response curves.

My working hypothesis is that impaired reaction center protein turnover

will lead to decreased Fv/Fm(0), and because of partial photosynthetic unit (PSU)

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connectivity, associated increases in σPSII(0). In addition, because they are

indicative of the flux of excitation energy through PSII, Fv/Fm-E and σPSII-E

curves will be linked to the general structure of P-E and φC-E curves. However,

N-limitation alterations to RuBisCO and other non-PSII proteins, which alter the

photosynthetic electron transport chain downstream of PSII, could result in some

disparities between PSII predicted and measured P-E.

For this study, I use standard photophysiological measures such as flash

yields, carbon uptake, spectral absorption and pigment concentrations in

combination with a newly developed active fluorescence induction technique

(Chapter III). This technique measures several mechanistic properties of PSII

over a light gradient, providing an analytical connection to P-E response curves.

These findings demonstrate that N-limitation can affect both the magnitude and

structure of P-E, φC-E, Fv/Fm-E and σPSII-E relationships and that the

fluorescence-based (PSII) measures of the mechanistic parameters of

photosynthesis describe well the analytic processes affecting the overall relative

P-E and absolute φC-E response. However, in the context of N-limitation, these

PSII descriptions do not adequately describe absolute P-E because non-PSII

specific processes such as RuBisCO concentration and turnover time appear to

be regulating Pbmax.

METHODS

Culture conditions

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Replicate Skeletonema costatum (SKEL) cultures (Exp#1 and Exp #2) were

grown in batch culture in 5 l glass containers at 19oC in air bubbled, modified f/2

media (N/20 and 1Si, with major plant nutrient atom ratios of N:P:Si 2.4:1:5.8)

(Guillard and Ryther, 1962). Assuming balanced Redfield growth, (N:P:Si

16:1:10.4-15.0), nitrogen is predicted to limit growth first (Redfield, 1958;

Brzezinski, 1985). Nominal nitrogen limitation was relieved by addition of f/2

concentrations of nitrogen stock solution (0.88 mM N, final concentration).

Continuous light (~100 µmol quanta m-2 sec-1) was supplied by cool white

fluorescent bulbs. Cultures were started and remained optically thin throughout

the duration of the experiment. Replicate experiments were created by adding a

known volume and cell density of culture to a known volume of fresh media.

However, due to slight errors in estimating the volumes and cell densities, there

remain minor "timing" discrepancies between the replicates.

Absorption, Pigments, Growth Rates, Cell Density

Spectral absorption measurements were made at 2 nm resolution with a 1 sec

integration time on samples in solution using an HP 8452 diode array

spectrophotometer with Labsphere (RSA-HP-84) integrating sphere. Absorption

at 750 nm was assumed to be non-cellular and was subtracted from the

absorption curve. Absorption coefficients were calculated following Kirk (1994).

Chlorophyll a and b and carotenoids were determined on 90% acetone extracts

following Parsons et al. (1984), except without MgCO3 addition, using the

trichromatic equations of Jeffrey and Humphrey (1975). Intrinsic growth rates

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were calculated from bulk fluorescence measurements. Cell concentrations were

estimated using a hemacytometer.

Photosystem Quantification

Emerson-Arnold numbers (Chl a/O2) were measured using a Hansatech oxygen

electrode and a Stroboslave (Type1539A) tunable frequency, saturating strobe

light following Dubinsky et al. (1986). PSII/Chl a was calculated as four times the

initial slope of oxygen evolution rate versus flash frequency, normalized to

chlorophyll concentration.

Photosynthesis vs Irradiance (P-E), Turnover Rates

Carbon-based photosynthesis-irradiance measurements were made using

custom-built temperature-regulated photosynthetrons. Irradiance, which was

supplied by a 250 W ENH projector bulb (Gray Supply), was spectrally modified

and attenuated using a combination of hot and cold mirrors (Optical Coating

Laboratory) and stage screening (Cinemills). Irradiance intensity (PAR) was

measured using a Biospherical Instruments (QSL-100) 4π scalar irradiance

meter. Incubations of 1 ml samples inoculated with ~7.4 kBq H14CO3 were

terminated after 10 mins using 100 µl 37% formaldehyde and 200 µl HCl and

allowed to degas overnight. Carbon uptake rates were quantified using standard

techniques (Barber et al., 1996). Photosynthetic parameters of the Platt et al.

(1980) model (α*, Ps, β) were optimized to fit data using a custom written, non-

linear least-squares Levenberg-Marquardt technique (Frenette et al., 1993).

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Optimization of model parameters to P-E data was performed using PAR as the

independent variable. Secondary parameters (Pbmax and Ek) were calculated

following Zimmerman et al. (1987). Maximum quantum yield (φC,max) was

calculated from model predicted α* as the mol C mol quanta-1 absorbed using

absorption coefficients that were spectrally weighted to incident measurement

irradiance (Chapter II). Maximum quantum yield (φC,max') calculated using the

[ ]EC −φmax method (Chapter II) yielded similar patterns, but ~50% reduced

values (compare Figures V-7 and -8). Photosynthetic unit turnover rate constant

(sec-1), 1/τPSU, was calculated as:

n

P b

PSU

max0269.01=

τ (V-1)

where Pbmax is light-saturated photosynthesis (mg C mg Chl a-1 hr-1), n is the

concentration of PSII (mol O2 mol Chl a-1) and 0.0269 converts the units from

hours to seconds and grams to moles and carbon to oxygen using a constant

photosynthetic quotient of 1.3 (Laws, 1991). Patterns of 1/τPSU, calculated using

σPSII(0) and Ek following Dubinsky et al. (1986) that are referred to as 1/τPSU',

yielded similar results.

Modulated Fluorescence

Modulated fluorescence measurements were performed with a previously-

described custom-built fast repetition rate type fluorometer with computer-

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controlled background irradiance gradient (BIG-FRRf) (Chapter III; Kolber et al.,

1998). The background light is produced by super-bright blue LEDs and has a

maximum output wavelength of ~550 nm (Chapter III). For this study, the

excitation protocol was run for 350 µsec at high intensity with a 100% duty cycle

followed by the decay protocol that was run for 7.5 msec at low intensity with a

1% duty cycle. The pre-illumination background irradiance duration was 500

msec. Samples were dark adapted for >15 mins for each background irradiance

setting. Data were analyzed using custom-written software based in MATLAB

5.3v11 following Kolber et al. (1998) to determine Fo, Fm, σPSII and p where Fo is

initial fluorescence, Fm is the maximal fluorescence, σPSII is the functional cross-

sectional area, p is a connectivity parameter.

For each background irradiance there is generated a suite of

fluorescence-derived parameters. These parameters are referred to with the

subscript indicating the background irradiance intensity. For example, Fv/Fm(0) is

photosystem II photochemical conversion efficiency in the dark and σPSII(521) is

the PSII functional cross sectional area at 521 µmol quanta m-2 sec-1.

RESULTS / DISCUSSION

Photosynthetic Biomass

S. costatum growth curves are exponential for approximately 1.5 days, at which

point growth rates decrease significantly and populations become stationary

(Figure V-1). Nitrogen addition at ~5.5 days re-initiated population growth. The

reversal of stationary to exponential growth phase with the addition of nitrogen

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supports nitrogen-limited growth, as predicted from growth media atom ratios.

That the ratio of fluorescence to chlorophyll, which can be used as a crude index

of physiological status, increased nearly two-fold from N-replete to -limited

conditions and returned to pre-limitation values after N-addition also suggests a

N-limited culture.

N-limitation caused cellular chlorophyll a to decrease nearly four-fold, but

the levels of chlorophyll c relative to chlorophyll a remained fairly constant

(Figure V-2). As fluorescence to chlorophyll a increased and cellular chlorophyll

a decreased, relative carotenoid concentrations increased approximately two-

fold. These decreases in photosynthetically active pigments along with relative

increases in the non-photosynthetically active protective pigments (NPP) are

consistent with previous investigations into the effect of nitrogen limitation on

photosynthetic biomass (Geider et al., 1993; Latasa and Berdalet, 1994). They

suggest that under N-limitation, S. costatum becomes chlorotic, but maintains

NPP content to avoid photodamage (Henley et al., 1991; Verhoeven et al., 1997).

Differences in pigmentation under N-limitation lead to some significant

changes in absorption properties (Figure V-3). The cellular absorption cross

section (σcell) decreased significantly under nitrogen limitation. However,

chlorophyll a-specific absorption cross section (σChla) displayed a more complex

behavior. For both replicate experiments, σChla initially increased and then

decreased. Although the general pattern for the two experiments is similar, the

timing of the pattern is not. These differences in timing, which are also apparent

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for other photophysiological parameters (σPSU, α*, Pbmax, etc.), may be the result

of slight differences in the initial conditions of the “replicates.”

Timing aside, initial increases in σChla following the onset of N-limitation

are consistent with a relative increase in NPP and subsequent decreases in

absorption are generally consistent with the likely decreases in the relative

concentrations of photosynthetic accessory pigments not measured here (ex.

fucoxanthin). Despite the minor changes in σChla that are linked to the pigment

complementation, the overall magnitude of change is quite low when compared

to dramatic shifts in cellular chlorophyll a concentrations. Thus, the combination

of photosynthetically active accessory pigment degradation and maintenance of

NPP with decreasing chlorophyll a concentration leads to a relatively constant

σChla.

Similar to σcell patterns, functional photosynthetic cross sections (σPSU)

decrease precipitously under N-limitation (Figure V-3). These decreases are

consistent with reduced chlorophyll concentrations and the relative increase in

protective carotenoids. The decreases in σPSU are also likely significantly

affected by the dramatic decreases in PSU turnover time (1/τPSU) (see below).

In addition to changes in aggregate pigment and absorption properties

under N-limitation, there are also changes in PSII concentrations (Figure V-4).

Cellular concentrations of functional PSII decrease significantly under N-limitation

from N-replete levels of ~1.0 amol PSII cell-1 to ~0.1 amol PSII cell-1 under

extreme N-limitation. Emerson and Arnold (E&A) number trends are less

conclusive suggesting a decrease in one replicate, but no general trend in the

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second replicate. Nevertheless, it is clear that individual S. costatum cells have

reduced active PSII levels under N-limitation (see below).

Photophysiology

Similarly to photosynthetic biomass properties, photophysiology as interpreted

from P-E curve analysis was significantly compromised under N-limitation (Figure

V-5). The maximum light utilization coefficient (α*) decreased from ~0.04 mg C

mg Chl a-1 hr-1 (µmol quanta m-2 sec-1)-1 to nearly zero at maximum N-limitation.

This decrease was paralleled by similar decreases in the light-saturated

photosynthetic rate (Pbmax) from ~2.0 mg C mg Chl a-1 hr-1 to near zero. Despite

these dramatic decreases in α* and Pbmax, the general structure of the P-E

curves remained remarkably constant from N-replete to severely N-limited, with

the light saturation level (Ek) resting at ~50 µmol quanta m-2 sec-1 throughout the

experiment (but see below).

Although Ek remained relatively constant, N-limitation induced more subtle

changes in P-E architecture. These changes are not readily observed when

using absolute P-E curves, but are apparent when examining relative P-E

structure (Figure V-6). Using this type of analysis, N-limited, relative P-E curves

are shifted to lower irradiances relative to N-replete curves. This property implies

that relative to N-replete, N-limited functional cross sections are elevated and

that it takes less light to saturate photosynthesis under N-limited conditions. A

corollary suggested by this observation is that under N-replete conditions, S.

costatum is able to utilize higher irradiance intensities compared to N-limited

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conditions. Despite these properties, it is important to note that under N-replete

conditions, more photosynthate is produced per photon because of the increased

magnitude of the P-E curve. The Ek value, which is calculated as Pbmax / α*

(Talling, 1966), does not adequately document this change in photophysiology

because it is not a sensitive measure of the structure of the P-E curve. The

calculation of Ek implicitly assumes P-E structure to be bi-linear and disregards

convexity (Blackman, 1905; Henley, 1995). These changes in the magnitude

and relative structure of P-E are supported by additional photosystem biomass

and physiological characterizations (see below).

As implied from P-E curves and absorption properties, φC,max decreases

significantly under N-limitation (Figure V-7). Values of φC,max are near maximal

(~0.125 mol C mol quanta-1) under N-replete conditions and decrease to near

zero as nitrogen becomes more limiting. These decreases are consistent with

previous findings of N-sensitive φC,max (Cleveland and Perry, 1987; Babin et al.,

1996). Decreases in φC,max are almost exclusively due to reductions in α* and are

largely independent of absorption (σChla) properties (Figures V-3 and -5).

Estimates of photosystem turnover (1/τPSU) derived from oxygen flash

yields and light saturated photosynthesis values also decreased significantly

under nitrogen limitation (~80 to ~20 sec-1). This decrease in turnover is almost

entirely driven by decreases in Pbmax (Equation V-1); even in experiment #1

when E&A was somewhat reduced under N-limitation, turnover time decreases

because of dramatic reductions in Pbmax. Nevertheless, these estimates of

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1/τPSU should be interpreted with caution because they were calculated using a

constant photosynthetic quotient (PQ) of 1.3 (mol O2 mol C-1). However, a ~6-

fold reduction in growth rate due to N-limitation lead to only a minor (<~20%)

increase in PQ (Bidigare et al., 1989; Genty et al., 1989; Geider et al., 1998)

suggesting that the dramatic reductions in turnover measured here are probably

not driven exclusively by oxygen evolution / carbon uptake ratios. Independently

measured 1/τPSU (1/τPSU'), which are not dependent on PQ, show similar trends.

Similarly to P-E analyses, φC-E structure is significantly affected by N-

limitation (Figure V-8). Contrary to P-E curves, the magnitude of the φC-E curve

decreases only slightly under moderate N-limitation. However, under severe N-

limitation, the magnitude of the φC-E curve decreases in a similar manner to P-E

curves. This delayed onset of decrease in φC,max relative to photosynthetic rates

could be driven by changes in pigmentation or turnover rates. However, the

structure and magnitude of chlorophyll a-specific absorption spectra are

remarkably constant from N-replete to N-limited cells (data not shown),

suggesting that changes in the fate of absorbed light and not absorption (σChla)

are driving φC-E. In addition, Fv/Fm-E, which are absorption (σChla) independent,

are also consistent with φC-E suggesting that changes in turnover times are

mediating the drops in P-E magnitude and do not affect the magnitude of φC-E or

Fv/Fm-E (see below).

While the magnitude of the φC-E curve remains high under moderate N-

limitation, the structure of the curve is significantly different under N-limitation

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with the φC,max occurring at a lower irradiance (Figure V-8). Similar to P-E curves,

this reduction in the irradiance at which φC,max occurs in effect shifts the relative

φC-E curve to lower irradiances. Both moderately and severely N-limited φC-E

curves are shifted to lower irradiances. For S. costatum grown at ~100 µmol

quanta m-2 sec-1, this shift results in a dramatic reduction in realized quantum

efficiency. For example, φC at 100 µmol quanta m-2 sec-1 (the growth irradiance)

is reduced ~50% for moderately N-limited relative to N-replete, despite only

minor (~10%) reductions in φC,max. Thus, although φC,max is at first not affected by

N-limitation, the shift in irradiance structure significantly reduces the operational

quantum efficiency and in part leads to reduced P-E magnitudes. Similar to P-E

and Fv/Fm-E , this shift is due to changes in cross section (see below).

Fluorescence-derived Measurements

N-replete estimates of Fo-, Fm-, Fv/Fm- and σPSII-E (Figure V-9a) were generally

consistent with previous observations using other phytoplankton taxa under

nutrient-replete conditions (Chapter III). N-limited estimates of these parameters

were also consistent with previous fluorescence parameter versus irradiance

patterns, but here the level of N-limitation significantly affected curve magnitudes

(Figure V-9b,c).

Similar to previous studies, Fv/Fm(0) and σPSII(0) were greatly affected by N-

limitation (Figure V-10) (Kolber et al., 1988; Geider et al., 1993). Like φC,max,

Fv/Fm(0) started to decrease approximately one day after growth rates began to

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decline. Fv/Fm(0) decreased about 3-4 fold to ~0.15 after five days of N-limitation,

but quickly recovered (1-2 days) to pre-limitation values after the addition of

nitrate. Commensurate with decreases in Fv/Fm(0) were increases in σPSII(0).

From N-replete to severely N-limited conditions, σPSII(0) increased from ~0.02 to

0.05 (a.u.). These increases also quickly recovered upon addition of nitrate.

Values of σPSII(0) were inversely correlated with functional PSII

concentrations (r2=0.70). Because photosynthetic units are partially energetically

connected and in-between independent and connected-units antennae

arrangements, decreases in functional PSII concentrations can lead to increases

in the functional size of PSII (Trissl et al., 1993; LaVergne and Trissl, 1995; Trissl

and LaVergne, 1995).

Clearly N-limitation affects the magnitude of Fv/Fm(0) and σPSII(0).

However, responses of dark measured parameters may not be typical of Fv/Fm-E

or σPSII-E responses. To gauge the possible differential response to N-limitation

along a Fv/Fm-E or σPSII-E curve, I selected a background irradiance of 108 µmol

quanta m-2 sec-1 as a representative middle irradiance value that is close to the

growth irradiance to measure the effects of differential N-limitation on Fv/Fm-E

and σPSII-E. Similar to dark values, Fv/Fm(108) and σPSII(108) were also dramatically

affected by N-limitation (Figure V-10) with Fv/Fm(108) decreasing and σPSII(108)

increasing upon N-limitation. However, although these parameters had the same

general patterns with respect to N-limitation when measured in the dark or in the

presence of light (Figure V-10), N-limitation had relatively less influence on

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σPSII(108) when compared to σPSII(0); σPSII(0) increased ~150% while σPSII(108)

increased only ~50% under N-limitation. Meanwhile, the relative reduction in

Fv/Fm(108) with N-limitation was nearly identical to that of Fv/Fm(0).

To examine these differential reductions in more detail, I compared the

irradiance structure of Fv/Fm and σPSII for three different levels of N-limitation

(Figure V-11-12). Fv/Fm-E curves are very similar in generalized structure and

relative magnitude to φC-E curves. Moderately N-limited Fv/Fm-E curves are

somewhat reduced in magnitude compared to N-replete curves, while severely

N-limited Fv/Fm-E curves are dramatically reduced in magnitude. However,

similar to normalized φC-E curves, moderate and severe N-limitation leads to an

overall shift of the normalized Fv/Fm(max) plateau from higher to lower irradiances

(Figure V-11). This shift is indicative of changes in photosynthetic cross sections

(see below).

Using the integrated area under the Fv/Fm-E curve as a measure of

summed potential photosynthetic capacity, I calculated the relative influence of

(1) the changes in the magnitude of the curves and (2) the shift from high to low

irradiances under N-limitation. The integrated area (0-500 µmol quanta m-2 sec-

1) for N-replete Fv/Fm-E was 75% and 167% greater than for moderately (2.9

days) or severely (4.9 days) N-limited Fv/Fm-E, respectively. Of this increase,

87% and 47% was due to the shift of N-replete Fv/Fm-E over moderately or

severely-limited Fv/Fm-E, respectively. Although crude, this type of analysis

highlights the importance of the irradiance shift in determining the light response

curve of photosynthetic conversion efficiency.

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N-replete and -limited σPSII-E curves also differ in magnitude and structure

(Figure V-12) with increases in the magnitude of the σPSII-E curves even under

moderate N-limitation. This increase in magnitude is present throughout the

σPSII-E curve. Thus, unlike Fv/Fm-E and φC-E curves, changes in magnitude

dominate the overall shape of the curve.

Although the magnitude component of the curve dominates, there remain

some notable differences between the relative structures. Specifically, at middle

irradiances σPSII decreases more under N-limitation than for N-replete (Figure V-

12). As previously described, decreases in σPSII at moderate irradiance

intensities are likely a result of non-photochemical quenching (NPQ), thus this

data suggest that N-limitation induced elevated NPQ (Chapter III). To the degree

that NPQ-regulation may be a critical component in the regulation of

photosynthetic light-harvesting, especially under non-favorable nutrient regimes

(Niyogi, 1999; Demmig-Adams and Adams, 2000; Li et al., 2000), increases in

NPQ under N-limitation are consistent with S. costatum that have with reduced

photosynthetic yield (Bungard et al., 1997; Verhoeven et al., 1997). This

increase in relative NPQ capacity is also supported by the increase in the

carotenoid to chlorophyll a pigment ratio under N-limitation (Figure V-2).

Integration

It is clear from this and previous studies that N-limitation significantly affects

photosynthetic biomass, rates and efficiency. To step beyond simply

documenting changes, an emergent question is: Can changes in the analytic

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components of photosynthesis be related to the total photosynthetic response?

Or for modeling purposes, can the measured mechanistic properties of PSII such

as Fv/Fm and σPSII be used to predict observed photosynthetic rates (P-E) and

efficiency (φC-E) in the context of N-limitation? Nominally this task can be broken

into three major descriptions: α*, Pbmax and the structure or convexity of the P-E

relationship (Frenette et al., 1993; Henley, 1995).

The maximum light utilization coefficient (α*) is composed of two terms

including chlorophyll a-specific absorption (σChla) and quantum efficiency (φC,max) :

max,*

CChlaφσα = (V-2)

From this relationship, one can examine the differential effect of absorption and

efficiency on α*. Because of the dramatic changes in α* (Figure V-5) with only

minor changes associated with σChla (Figure V-3), a priori variability in α* in the

context of N-limitation should be dominated by changes in quantum efficiency.

Indeed, greater than 98% of the change in α* is due to changes in φC,max (Figure

V-13).

Numerous processes including non-photosynthetic pigment content,

reaction center functionality, or RuBisCO concentrations and activity can

potentially influence quantum efficiency (Bidigare et al., 1989; Genty et al., 1989;

Geider et al., 1998). In turn, each of these processes can be influenced by

nitrogen nutrition and therefore has the potential to affect the realized quantum

efficiency (Herzig and Falkowski, 1989; Beardall et al., 1991; Latasa and

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Berdalet, 1994). However, in S. costatum grown under the present conditions,

variability in φC,max is highly correlated (r2=0.93) with PSII photosynthetic

conversion efficiency (Fv/Fm(0)) (Figure V-13). Even though there are definitive

differences under N-limitation in carotenoid content (Figure V-2) and are likely

changes in RuBisCO activity (Geider et al., 1998), overall these factors may be

less important in regulating φC,max under N-limitation for S. costatum. The specific

vulnerability of PSII to N-limitation is consistent with previous observations

(Kolber et al., 1988; Berges et al., 1996). By association, because absorption

(σChla) remains relatively constant, greater than 90% of the variability in α* is

explained by Fv/Fm(0) variability.

Similar to α* analyses, Pbmax can be described as the product of the

concentration of photosynthetic units (1/E&A) and the turnover rate of the units

(1/τPSU) (see methods). In the present study, 1/τPSU dominates Pbmax variability

with only minor contributions from changes in E&A (Figure V-14). Although

1/τPSU and Pbmax are not truly independent variables, the lack of a strong

correlation (r2=0.27) between E&A and Pbmax, which are independent, suggests

that it is the functionality of the units and not the relative concentrations of the

units per se that dominantly determines light-saturated photosynthesis under N-

limitation for S. costatum. This is consistent with observations under nutrient-

replete conditions that suggest that Pbmax may be limited by RuBisCO, which in

turn affects 1/τPSU (Sukenik et al., 1987; Stitt and Schulze, 1994).

The lack of a strong relationship between E&A and Pbmax is intriguing in

light of dramatic reductions in Fv/Fm(0). The reduction in Fv/Fm(0), but no

135

significant effect in E&A, may be due to the balance of two complementary

processes: although the number of functional reaction centers decreases

dramatically under N-limitation (Figure V-4), chlorosis also occurs (Figure V-2).

These simultaneous reductions may lead to the overall stability in E&A despite

precipitous reductions in Pbmax and may ultimately account for 1/τPSU describing

most of the variability in Pbmax.

The final major component of the P-E curve, convexity, also appears to be

influenced by N-limitation (Figure V-6). These changes in structure are also

present for φC-E and Fv/Fm-E curves (Figure V-8, V-11). This consistency

between P-E, φC-E and Fv/Fm-E suggests that the processes mediating convexity

may be acting through PSII antennae and excitation energy processing

mechanisms and may be documented by dynamic PSII cross sections (σPSII-E).

A priori changes in the convexity and shape of P-E and φC-E curves are

not due to alterations in σPSU or σPSII(0) alone, but due to changes in their

irradiance structure. Examples of σPSII-E curves (Figure V-9 and V-12) do

suggest that N-limitation induces alterations to the relative σPSII-E structure. For

example, in addition to the dramatic changes in the magnitude of σPSII-E curves,

N-limited σPSII-E curves also have significantly reduced relative σPSII at moderate

light levels (~200 µmol quanta m-2 sec-1) when compared to N-replete curves.

This reduction is suggestive of increased NPQ. These changes in σPSII-E could

be the proximal mechanism for altered structure: a simple exponential P-E model

using only E and σPSII(E) as inputs to estimate P/Pbmax reproduces with

136

considerable fidelity the differences between N-replete and N-limited relative P-E

curves (data not shown). These results also imply that PSII-specific processes

are largely responsible for changes in the shape of the relative P-E curve under

N-limitation.

Related to α*, Pbmax and convexity is the N-limitation induced lateral shift

in P-E, φC-E and Fv/Fm-E curves and associated alterations to σPSII-E curves.

From the above data and previous studies, in the context of N-limitation there are

two major processes acting to modify the shape and magnitude of the P-E

relationship including (1) reductions in efficiency from both reductions in

functional reaction centers and turnover time and (2) increases in σPSII(0).

However, only turnover time and σPSII(0) are potentially responsible for the lateral

shift; reaction center functionality does not directly affect the shape of P-E

curves. When P-E curves are normalized, which removes the contribution of

turnover time to curve variability, there is still a lateral shift in P-E curves. This

suggests that the shifts in normalized P-E curves and Fv/Fm-E and φC-E curves

from high to low light under N-limitation is a result of changes to cross section.

Indeed, σPSII(0) increases significantly from N-replete to N-limited conditions.

Taken together, comparisons between α*, Pbmax and convexity and PSII

analytic components implies that N-limitation affects PSII and that variability in

PSII-specific processes like Fv/Fm and σPSII describe well the efficiency and

relative structure of P-E curves as a whole. Direct comparisons between Fv/Fm

and φC over a wide-range of background light levels support this for

137

photosynthetic efficiency (Figure V-15). However, it is important to note that this

relationship does partially breakdown at low efficiency values associated with

high irradiance levels (positive Fv/Fm(x) intercept in Figure V-15). This may be

due to non-linear electron throughput of PSII from cyclic electron flow around

PSII (Falkowski et al., 1986; Prasil et al., 1996) or other processes (Whitmarsh et

al., 1994). Nevertheless, in spite of slight deviations, the overall Fv/Fm(x)-φC(x)

relationship is strong.

Consistent with strong Fv/Fm - φC correlations, fluorescence-derived

photosynthetic rates are also strongly correlated with measured rates when using

a known Pbmax (Figure V-16). Further, fluorescence-derived maximum

photosynthetic rates have similar patterns to observed Pbmax (Figure V-17).

However, the uncoupling of these two measurements at the initiation of and

recovery from N-limitation suggests that this correlation may be due to the

simultaneous reduction in PSII electron transport and turnover time and not due

to Pbmax limitation by PSII electron transport per se (Genty and Harbinson,

1996). The uncoupling of maximum PSII and photosynthetic rates may also be

indicative of small changes in the convexity of the P-E curve.

Fluorescence measures of photosynthetic rates and efficiency are focused

on variability in the function and functional structure of PSII. Several components

downstream of PSII in the photosynthetic electron transport chain, notably

RuBisCO concentrations and turnover, are severely influenced by N-limitation.

Thus, while PSII measures of φC-E and P-E structure are robust because PSII

regulates their structure under N-replete and -limited conditions, the relationship

138

between fluorescence-measured parameters and σPSU, 1/τPSU and Pbmax

(absolute P-E structure) are not robust because they are not solely regulated by

PSII-specific processes.

CONCLUSIONS

N-limitation induces dramatic changes in the photosynthetic biomass and

physiology of Skeletonema costatum. Reductions in maximal quantum efficiency

(φC,max) are largely driven by reductions in photosystem II conversion efficiency

(Fv/Fm(0)). Reductions in Fv/Fm(0) also lead to increases in PSII cross sectional

area (σPSII(0)) because of the partial interconnectedness of photosynthetic units.

Further, both Fv/Fm-E and φC-E efficiency curves, which are shifted to lower

irradiances under N-limitation, are consistent with altered σPSII(0) and σPSII-E

curves. This suggests that photosynthetic efficiency is dominated by PSII-

specific processes. Relative P-E curves (and associated α*) are also consistent

with σPSII-E. However, unlike σPSII(0) and inconsistent with φC-E, Fv/Fm-E and

relative P-E, photosynthetic unit cross sections (σPSU) are reduced under N-

limitation. Values of σPSU deviate from the other parameters because they are

significantly affected by decreased photosynthetic unit turnover rates (1/τPSU).

These reductions in 1/τPSU are likely due to previously described changes in

RuBisCO content and function under N-limitation and are not robustly quantified

by PSII measurements. Combined, these results support a general regulatory

role of PSII in photosynthetic efficiency under N-limitation. In the context of non-

139

steady state N-limitation, PSII-specific processes describe well φC-E and relative

P-E curves, but do not describe well absolute P-E curves because processes not

linked to PSII such as RuBisCO concentration and turnover appear to be

regulating Pbmax.

140

ln (

fluor

esce

nce)

-7

-6

-5

-4

Exp#1 Exp#2

Gro

wth

Rat

e (d

ays-1

)

0

1

Days0 2 4 6 8 10

Flu

ores

cenc

e/C

hlor

ophy

ll a

(a.u

.)

1

2

3

4

5

S. costatum N-limitation - general growth characteristics

NO3 addition

Figure V-1: Growth rate properties. (top) Bulk fluorescence (a.u.), (middle) intrinsicgrowth rates calculated using bulk fluorescence (d-1), and (bottom) bulk fluorescenceto chlorophyll a ratio as an indicator of physiologic status.

141

S. costatum N-limitation - Pigments

NO3 addition

g C

hlor

ophy

ll a

/ cel

l

0

1e-13

2e-13

3e-13

4e-13

Exp#1Exp#2

Chl

orop

hyll

c/a

(g/g

)

0.10

0.12

0.14

0.16

0.18

0.20

Days0 2 4 6 8 10

Car

oten

oids

/ C

hlor

ophy

ll a

(g/g

)

0.3

0.4

0.5

0.6

0.7

Figure V-2: Pigmentation properties. (top) Cellular chlorophyll a concentration (g cell-1),(middle) relative chlorophyll c concentrations (g Chl c g Chl a-1) and (bottom) relativecarotenoid concentrations (g Car. g Chl a-1).

142

σ Chl

a (m

2 mg

Chl

a-1

)

0.008

0.010

0.012

0.014

0.016

Exp#1Exp#2

σ cell (

m2 c

ell-1

)

0

1e-12

2e-12

3e-12

4e-12

S. costatum N-limitation - Absorption Cross Sections

NO3 addition

Figure V-3: Absorption properties. (top) Cellular absorption cross-section (m2 cell-1),(middle) chlorophyll a specific absorption cross section (m2 mg Chl a-1) and (bottom)photosynthetic unit cross section (m2 quanta-1).

Days0 2 4 6 8 10

σ PS

U (

m2 µ

mol

qua

nta-1

)

0.0

0.5

1.0

1.5

143

Days

0 2 4 6 8 10

Em

erso

n an

d A

rnol

d N

umbe

r(m

ol C

hl a

mol

O2-1

)

0

500

1000

1500

2000

2500

Exp#1Exp#2

S. costatum N-limitation - PSII Concentrations

NO3 addition

activ

e P

SII

(am

ol P

SII

cell-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Figure V-4: PSII Concentrations. (top) Active (functional) PSII (amol PSII cell-1)and (bottom) Emerson and Arnold number (mol Chl a mol O2

-1).

144

α*

(mg

C m

g C

hl a

-1 h

r-1

(µm

ol q

uant

a m

-2 s

ec-1

)-1)

-0.01

0.00

0.01

0.02

0.03

0.04

0.05

Exp#1Exp#2

Pb m

ax(m

g C

mg

Chl

a h

r-1)

0.0

0.5

1.0

1.5

2.0

Days0 2 4 6 8 10

Ek

(µm

ol q

uant

a m

-2 se

c-1)

0

50

100

150

S. costatum N-limitation - P-E

NO3 addition

Figure V-5: Photosynthetic Parameters. (top) maximum light utilization coefficient (α*)(mg C mg Chl a-1 hr-1 (µmol quanta m-2 sec-1)-1), (middle) light-saturated photosynthetic rate(Pbmax) (mg C mg Chl a-1 hr-1) and (bottom) saturation intensity (Ek) (µmol quanta m-2 sec-1).

145

S. costatum N-limitation - P-E Comparison

Pb (

mg

C m

g C

hl a

-1 h

r-1)

0

1

2

3

4

RepleteTime 2.9 daysTime 4.9 days

PAR (µmol quanta m-2 sec-1)

0 100 200 300 400 500

Rel

ativ

e P

b

0.0

0.2

0.4

0.6

0.8

1.0

1 10 100 1000

1 10 100 1000

Figure V-6: Photosynthesis-Irradiance (P-E) curve comparison for different levels ofN-limitation. (top) Absolute P-E curves (mg C mg Chl a-1 hr-1) and (bottom) relative (a.u.)P-E curves. Inset panels are same data except on common-log abscissa. Circles andblack line (time 0.4 days) are nominally N-replete, squares and dark gray line (time 2.9days) are moderately N-limited, and triangles and light gray line (time 4.9 days) areseverely N-limited. (See figure V-1)

146

φ C,m

ax (

mol

C m

ol q

uant

a-1)

0.00

0.04

0.08

0.12

0.16

Exp#1Exp#2

Days

0 2 4 6 8 10

1/τ P

SU (

sec-1

)

0

20

40

60

80

S. costatum N-limitation - Photosynthetic Efficiency

NO3 addition

Figure V-7: Efficiency of Photosynthesis. (top) maximum quantum yield of carbon uptake(φC,max) (mol C mol quanta-1) and (bottom) photosynthetic unit turnover rate (1/τPSU) (sec-1).

Note that φC,max were calculated using P-E model fit α* in conjunction with a*.

147

S. costatum N-limitation φC-E comparisons

φ C (

mol

C m

ol q

uant

a-1)

0.000

0.025

0.050

0.075

0.100

0.125

PAR (µmol quanta m-2 sec-1)

0 100 200 300 400 500

Rel

ativ

e φ C

0.0

0.2

0.4

0.6

0.8

1.0

1 10 100 1000

1 10 100 1000

Figure V-8: Quantum Yield-Irradiance (φC-E) curve comparison for different levels of

N-limitation. (top) Absolute φC-E curves (mol C mol quanta-1) and (bottom) relative

φC-E curves. Symbols and inset panels as in figure V-6.

148

S. costatum N-limitation - 0.43 days (N-replete)

Fo

or F

m (

a.u.

)

0.0

0.5

1.0

1.5

Fv/

Fm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FoFmFv/Fm

PAR (µmol quanta m-2 sec-1)0 200 400 600

σ PS

II (a

.u.)

0.000

0.025

0.050

0.075

σPSII

Figure V-9a: Fluorescence-derived parameters as a function of irradiance for N-repletepopulation (0.4 days). (top) initial fluorescence (Fo, open red circles), saturated fluorescence(Fm, closed red circles), and PSII photochemical conversion efficiency (Fv/Fm, blue squares),and (bottom) PSII functional cross-sectional area (σPSII, green triangles).

149

S. costatum N-limitation - 2.93 days (moderate N-limitation)

Fo

or F

m (

a.u.

)

0.0

0.5

1.0

1.5

Fv/

Fm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FoFmFv/Fm

PAR (µmol quanta m-2 sec-1)0 200 400 600

σ PS

II (a

.u.)

0.000

0.025

0.050

0.075

σPSII

Figure V-9b: Fluorescence-derived parameters as a function of irradiance for moderatelyN-limited population (2.9 days). Panels and symbols as in Figure V-9a.

150

S. costatum N-limitation - 4.93 days (severe N-limitation)

Fo

or F

m (

a.u.

)

0.0

0.5

1.0

1.5

Fv/

Fm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

FoFmFv/Fm

PAR (µmol quanta m-2 sec-1)0 200 400 600

σ PS

II (a

.u.)

0.000

0.025

0.050

0.075

σPSII

Figure V-9c: Fluorescence-derived parameters as a function of irradiance for severelyN-limited population (4.9 days). Panels and symbols as in Figure V-9a.

151

S. costatum N-limitation - fluorescence parameter time course

Fv/

Fm

(0)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

σP

SII(0) (a.u.)

0.00

0.02

0.04

0.06

0.08

Fv/FmσPSII

Figure V-10: Time-course of fluorescence-derived parameters measured in the dark (top)and with moderate intensity background irradiance (108 µmol quanta m-2 sec-1) (bottom).PSII photochemical conversion efficiency (Fv/Fm, squares) and PSII functionalcross-sectional area (σPSII, circles).

Days

0 2 4 6 8 10

Fv/

Fm

(108

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

σP

SII(108) (a.u.)

0.00

0.02

0.04

0.06

Dark

108 µmol quanta m-2 sec-1

152

S. costatum N-limitation Fv/Fm Comparisons

Fv/

Fm

0.0

0.1

0.2

0.3

0.4

0.5

0.6

RepleteTime 2.9 daysTime 4.9 days

PAR (µmol quanta m-2 sec-1)

0 200 400 600 800

Rel

ativ

e F

v/F

m

0.0

0.2

0.4

0.6

0.8

1.0

1 10 100 1000

1 10 100 1000

Figure V-11: Fv/Fm-Irradiance (Fv/Fm-E) curve comparison for different levels ofN-limitation. (top) Absolute Fv/Fm-E curves and (bottom) relative Fv/Fm-E curves.Symbols and inset panels as in figure V-6.

153

S. costatum N-limitation σPSII comparisons

σ PS

II (a

.u.)

0.00

0.02

0.04

0.06

0.08

PAR (µmol quanta m-2 sec-1)

0 200 400 600 800

Rel

ativ

e σ P

SII

0.0

0.5

1.0

1.5

2.0

2.5

1 10 100 1000

1 10 100 1000

Figure V-12: PSII Functional Cross Sectional Area-Irradiance (σPSII-E) curve comparison

for different levels of N-limitation. (top) Absolute σPSII-E curves (a.u.) and (bottom) relative

σPSII-E curves. Symbols and inset panels as in figure V-6.

154

φC,max (mol C mol quanta-1)

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

α*

(mg

C m

g C

hl a

hr-1

(µ m

ol q

uant

a m

-2 s

ec-1

)-1)

0.00

0.01

0.02

0.03

0.04

0.05

S. costatum N-limitation φC,max correlations

Fv/Fm(0)

0.1 0.2 0.3 0.4 0.5 0.6

φ C,m

ax (

mol

C m

ol q

uant

a-1

)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

Figure V-13: Relationship between different measures of photosynthetic efficiencies for thecombined N-limited data set. (top) α* vs. φC,max and (bottom) φC,max vs. Fv/Fm(0).

Note that φC,max were calculated using P-E model fit α* in conjunction with a*.

155

S. costatum N-limitation Pbmax correlations

Emerson and Arnold Number (mol Chl a mol O2-1)

0 1000 2000 3000

Pb m

ax (

mg

C m

g C

hl a

-1 h

r-1)

0

1

2

1/τPSU (sec-1)0 20 40 60 80

Pb m

ax (

mg

C m

g C

hl a

-1 h

r-1)

0

1

2

3

Figure V-14: Relationship between Pbmax and its components. (top) Pbmax vs. 1/τPSU and

(bottom) Pbmax vs. Emerson and Arnold number. Red circles are from Exp#1 and bluesquares are from Exp #2.

156

Measured

Fv/Fm(x) (all irradiances)

0.0 0.1 0.2 0.3 0.4 0.5 0.6

φ C(x

) (al

l irr

adia

nces

)

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Relative

Normalized Fv/Fm(x) (all irradiances)

0.0 0.2 0.4 0.6 0.8 1.0

Nor

mal

ized

φC

(x) (

all i

rrad

ianc

es)

0.0

0.2

0.4

0.6

0.8

1.0

Figure V-15: (top) Measured and (bottom) relative (to maximum observed infor Fv/Fm-E or φC-E curve, respectively) relationship between PSII conversion

efficiency (Fv/Fm) and photosynthetic quantum efficiency (φC) for all background

light intensities. For these graphs, φC was assumed to approach φC,max as the

background light approached zero. Absolute r2=0.92 and relative r2=0.95.

157

Measured

Fls-derived Pb (all irradiances)

0.0 0.5 1.0 1.5 2.0

Pb (

all i

rrad

ianc

es)

0.0

0.5

1.0

1.5

2.0

Relative

Normalized Fls-derived Pb (all irradiances)

0.0 0.2 0.4 0.6 0.8 1.0

Nor

mal

ized

Pb (

all i

rrad

ianc

es)

0.0

0.2

0.4

0.6

0.8

1.0

Figure V-16: (top) absolute and (bottom) relative (to maximum observed infor P-E curve) relationship between fluorescence-derived photosynthetic ratesand measured photosynthetic rates. Fluorescence-derived rates were calculatedusing E*(Fo-Fo(0))/Fo(0) and normalizing the resultant curve to measured Pbmax

values. Measured r2=0.97 and relative r2=0.96.

158

Days

0 2 4 6 8

Pb m

ax (

mg

C m

g C

hl a

-1 h

r-1)

0

1

2

3

Relative F

luorescence-derived Maxim

um R

atePbmaxFls

Figure V-17: Time course comparison between maximum measured photosynthetic rateand maximum fluorescence-derived rate. Fluorescence rates calculated as Figure V-16.Both rates are correlated at r2=0.69.

159

Chapter VI

Conclusions

160

INTRODUCTION

Excitation energy from light absorption by marine phytoplankton has three fates

including non-radiative dissipation (heat), photochemistry and fluorescence. The

distribution of excitation energy to each of these processes can be modeled

using one of several models of exciton flow including the bipartite model, tripartite

model and the reversible radical pair model, among others (Butler and Kitajima,

1975; Butler and Strasser, 1977; Schatz et al., 1988; LaVergne and Trissl, 1995;

Lazar, 1999). Using these types of relationships, it can be demonstrated that the

ability of the PSII reaction center to send electrons through the primary quinone

regulates both fluorescence and photochemistry, and since these pathways are

mutually exclusive, photochemical and fluorescence yields are approximately

inversely related to each other (Duysens and Sweers, 1963; Malkin and Kok,

1966; Bonaventura and Myers, 1969; Butler, 1972).

Taking advantage of this property, several equations have been

formulated that use parameters derived from fluorescence induction curves

measured in the presence and absence of light to formalize this fluorescence -

photochemistry relationship (Weis and Berry, 1987; Genty et al., 1989; Seaton

and Walker, 1990; Falkowski and Kolber, 1993). Each of these equations has

demonstrated a great deal of homology with observations (Seaton and Walker,

1992). From the success of the equations in combination with the fact that room

temperature fluorescence is almost exclusively associated with photosystem II

(Geacintov and Breton, 1987) variability in photochemistry has largely been

ascribed to PSII-dependent processes (Fasham and Platt, 1983; Geider and

161

Osborne, 1992; Falkowski and Raven, 1997). The dependence of photosynthetic

rates and efficiency on PSII processes may be heightened in the context of

marine photosynthesis since typical sources of variability such as N-limitation,

Fe-limitation and photoinhibition / photodamage may differentially impact PSII

(Aro et al., 1993; Geider et al., 1993; Vassiliev et al., 1995).

Nevertheless, direct assignment of PSII-specific processes to marine

photosynthetic rate and efficiency variability is limited (Falkowski and Kolber,

1993; Babin et al., 1996; Boyd et al., 1997) and there is evidence that PSII-

specific processes may not be regulating photosynthetic rates and efficiency at

higher light levels (Sukenik et al., 1987; Behrenfeld et al., 1998). In particular,

there is little evidence relating photosynthetic rate and efficiency in the presence

of background light to PSII-specific processes. To aid in closing this gap, in this

dissertation I have specifically examined the role PSII, as measured by

fluorescence induction (σPSII, Fv/Fm), has in regulating P-E and φC-E curves in the

context of marine environmental variability.

SOURCES OF VARIABILITY

In the open ocean there are many sources of variability that can potentially

influence phytoplankton photosynthetic rates and efficiency. In this dissertation I

have selected four physiological conditions as representative of some of the

major processes affecting marine photophysiology. These conditions include:

low light (Chapter II), xanthophyll cycling (Chapter III), photoacclimation (Chapter

IV) and nitrogen limitation (Chapter V). These conditions do not exhaustively

162

represent all sources of marine photophysiological variability and other sources

may prove to be equally or more important in regulating photophysiological

responses. However, the conditions characterized here do encompass light- and

nutrient-limitation, which are probably the dominant limitations to marine

phytoplankton photosynthesis and growth (Sverdrup et al., 1942; Bougis, 1976).

Here, focusing on the role of PSII, I summarize my findings of how these four

environmental factors related to light- and nutrient-limitation impact

photophysiology (Table VI-1).

Low Light

Only the upper few meters of the open ocean are light-saturated with respect to

photosynthesis, thus processes that impact low light photophysiology may be

disproportionately responsible for observed photosynthetic rates and efficiencies

(Kirk, 1994). In Chapter II I investigate the nature and potential mechanisms of

the low light reduction in quantum efficiency. For the experimental setup used S.

costatum, but not D. tertiolecta, has reduced quantum efficiency at low irradiance

that is relieved in the presence of red-dominated light. This reduction, which is

likely the result of photosystem excitation imbalance via PSI limitation, does not

significantly impact the overall shape of P-E curves on linear-linear plots because

the reductions occur in the portion of the curve that does not greatly influence the

magnitude or structure of the P-E curve.

None of the potential mechanisms to explain the reduction in quantum

efficiency at low irradiances (respiration, S-state decay, photosystem excitation

163

imbalance), including the most probable: photosystem excitation imbalance, is a

PSII-limitation process that is quantified by standard fluorescence induction

techniques. Measures of Fv/Fm-E are always maximal at low background

irradiances and subsequently decrease at higher background irradiances

(Falkowski et al., 1986; Chapter III, IV, V). Thus, when quantum efficiency is

significantly reduced at low background irradiances such as for S. costatum,

fluorescence-based measures that show maximum PSII efficiency at low

irradiances do not adequately represent irradiance structure of photosynthetic

efficiency. However, when there is not a significant reduction in quantum

efficiency at low irradiance such as observed with D. tertiolecta, Fv/Fm-E

relationships do adequately represent the curve structure (Chapter II). A priori,

there is no way to distinguish between these two cases using standard

fluorescence induction techniques.

Xanthophyll Cycling

There are significant changes in the photosynthetic biomass and physiology

between WT and xanthophyll mutants of C. reinhardtii (npq1 and npq2), many of

which are related to alterations in PSII-specific processes. Loss of violaxanthin

de-epoxidase (npq1) results in a reduced non-photochemical quenching (NPQ)

capacity due to loss of non-photosynthetically active antenna traps. Conversely,

loss of zeaxanthin epoxidase (npq2) results in an increase in non-

photosynthetically active antenna traps (Chapter III). These modifications lead to

measurable changes in photosynthetic unit and PSII cross sections. Neglecting

164

low light reductions in quantum efficiency, it is modifications to antenna

properties as a function of irradiance that dominantly determine both φ-E and P-E

relative structure. Hence, in the context of xanthophyll cycle variability,

fluorescence measures of PSII describe well the relative irradiance structure of

both rates and efficiency (Chapter III). However, modifications to the xanthophyll

cycle and NPQ induce additional secondary alterations to photophysiology such

as carotenoid content and photosynthetic unit turnover time. These properties

are not directly assessed by fluorescence. These changes do not impact the

relative structures of φ-E and P-E, but do impact the respective magnitudes of

these curves. Thus, PSII conversion efficiency and maximum electron

throughput rate are uncorrelated with quantum efficiency or Pbmax, respectively

(Chapter III). In short, in the context of xanthophyll cycling and NPQ,

fluorescence-derived parameters describe well the relative structures, but not the

magnitudes of φ-E and P-E.

Photoacclimation

Photoacclimation affects a myriad of photosynthetic biomass and physiology

properties (Johnsen and Sakshaug, 1996; Schanz et al., 1997). Focusing on P-E

and φ-E, major results from this study show (1) an independent increase in

Pbmax and decrease in α* with increasing growth irradiance, (2) a decrease in

the magnitude of φC-E (φC,max) with increasing growth irradiance and (3) a lateral

(irradiance) shift of P-E and φC-E curves (Chapter IV). These data show that

165

PSII-specific measures (fluorescence) describe well the relative φC-E and P-E

structures. For example, Fv/Fm-E curves are shifted in a manner similar to P-E

and φC-E and are consistent with measured σPSII-E. However, the magnitudes of

P-E and φC-E curves are not well-established by PSII; in the context of

photoacclimation, φC,max and Pbmax are largely regulated by non-photosynthetic

pigments and processes downstream of PSII, respectively.

Nitrogen Limitation

N-limitation induces dramatic changes in the photosynthetic biomass and

physiology of S. costatum. Reductions in maximal quantum efficiency (φC,max)

are largely driven by reductions in photosystem II conversion efficiency (Fv/Fm(0))

because N-limitation leads to direct insults to reaction center proteins.

Reductions in Fv/Fm(0) also lead to increases in PSII cross sectional area (σPSII(0))

because of the partial interconnectedness of photosynthetic units. Both Fv/Fm-E

and φC-E efficiency curves, which are shifted to lower irradiances under N-

limitation, are consistent with altered σPSII-E curves. These results suggest that

photosynthetic efficiency is dominated by PSII-specific processes. Relative P-E

curves (and associated α*) are also consistent with σPSII-E. However, there are

also reductions in Pbmax that are partially driven by reductions in 1/τPSU.

Reductions in 1/τPSU are likely due to previously described changes in RuBisCO

content and function under N-limitation and are not robustly quantified by PSII

measurements. Combined, these results support a general regulatory role of

166

PSII in photosynthetic efficiency under N-limitation. In the context of non-steady

state N-limitation, PSII specific processes describe well φC-E and relative P-E

curves, but do not describe well absolute P-E curves because processes not

linked to PSII such as RuBisCO concentration and turnover appear to be

regulating Pbmax (Chapter V).

CONCLUSIONS

These results demonstrate that under a variety of environmental situations

characteristic of natural oceanic variability, photosystem II describes well the

relative structure of photosynthesis-irradiance and quantum efficiency-irradiance

curves except at very low irradiance (Table VI-1). However, other processes not

associated with photosystem II (or quantified by fluorescence) and unique to

each environmental situation can significantly impact the magnitude of maximal

photosynthetic rates and efficiency. In addition, other environmental conditions

not examined here that do significantly affect photophysiology such as

phosphorus-limitation may not severely influence PSII, thus leading to poor PSII-

photophysiology relationships (Geider et al., 1993). Combined these results

demonstrate the utility of photosystem II properties as a function of background

irradiance in describing the irradiance structure of photosynthetic rates and

efficiency, but also point to some of the limitations of fluorescence techniques.

167

Table VI-1: Relative success of PSII in describing photophysiological variability (+ is well described, - is poorly described)

Source of Variability Species efficiency

magnitude efficiency structure

rates magnitude

rates structure

low light S. costatum n/a - n/a -

D. tertiolecta n/a + n/a +

non-photochemical quenching

C. reinhardtii - + - +

photoacclimation S. costatum - + - +

nitrogen-limitation S. costatum + + - +

168

Appendix 1: Definition of Symbols and Abbreviations

Symbol Typical Units Property

*ia m2 mg Chl a-1

mean chlorophyll a-specific absorption coefficient normalized to incident

irradiance spectra (see equation II-3)

1/τPSU sec-1 photosynthetic unit turnover time

A unitless fraction of open reaction centers (ex.

Akp)

E&A mol Chl a mol O2-1 Emerson and Arnold number

Ef µmol quanta m-2 flash intensity

Ek µmol quanta m-2

sec-1 Talling Constant, or photosynthesis

saturation intensity (Pbmax/α*)

Fm, φFm a.u. saturated or maximal fluorescence yield

Fo, φFo a.u. initial fluorescence yield

Fv, φFv a.u. Fm-Fo, variable fluorescence

Fv/Fm, φFv/Fm unitless PSII photosynthetic conversion

efficiency

kd sec-1 rate constant of non-radiative decay

kf sec-1 rate constant of fluorescence

kp sec-1 rate constant of photochemistry

kt sec-1 rate constant of excitation transfer to

PSI

MT sec-1 multiple turnover

n mol O2 mol Chl a-1 photosynthetic unit concentration

NPP nonphotosynthetic protective pigments

NPQ nonphotochemical quenching

npq1 nonphotochemical quenching mutant 1

169

npq2 nonphotochemical quenching mutant 2

PAR or PAREo

µmol quanta m-2

sec-1 photosynthetically active radiation (400-

700nm)

Pbmax mg C mg Chl a-1 hr-1 or mol O2 g Chl a-1

hr-1

light saturated chlorophyll a normalized photosynthetic rate

Pbs

mg C mg Chl a-1 hr-1 or mol O2 g Chl a-1

hr-1

light saturated chlorophyll a normalized photosynthetic rate if no photoinhibition

(see equation II-1)

P-E photosynthesis-irradiance

PMT photomultiplier tube

PSI photosystem I

PSII photosystem II

PSU photosynthetic unit

PUR or PUREo

µmol quanta m-2

sec-1 photosynthetically usable radiation

(400-700nm) (see equation II-4)

ST single turnover

WT wild type

Y/Ymax unitless relative O2 flash yield

α*

(mg C mg Chl a-1 hr-

1) (µmol quanta m-2 sec-1)-1 or (mol O2 g Chl a-1 hr-1) (µmol quanta m-2 sec-1)-1

photosynthetic light utilization coefficient

β

(mg C mg Chl a-1 hr-

1) (µmol quanta m-2 sec-1)-1 or (mol O2 g Chl a-1 hr-1) (µmol quanta m-2 sec-1)-1

photoinhibition parameter (see equation II-1)

φ mol C or O2 mol

quanta-1 quantum yield

170

φf unitless fluorescence yield

φp unitless photochemical yield of PSII (see

Fv/Fm)

φX,max mol X mol quanta-1 where X=O2 or C

maximum quantum yield

σcell µm2 cell-1 cellular absorption cross section

σChla m2 mg Chl a-1 chlorophyll a absorption cross section

σPSII(X) a.u. relative PSII absorption cross section in

the presence of background light X

σPSU m2 µmol quanta-1 photosynthetic unit cross section

(note 167 Å2 quanta-1= m2 µmol quanta-

1)

171

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BIOGRAPHY Zackary Ian Johnson Born 20 April 1972, Iowa City, Iowa, USA Education: Massachusetts Institute of Technology, Cambridge, MA (1990-1994), B.S. Duke University, Durham, NC (1994-2000), Ph.D. (Botany) Professional positions:

Laboratory Assistant, WHOI, Woods Hole, MA (1993) Laboratory Assistant, MIT, Cambridge, MA (1991-94)

Visiting Scientist, Brookhaven National Laboratories, Upton, NY (1997) Teaching Assistant / Laboratory Instructor / Seminar Leader (Biological

Oceanography, Chemical Ecology, Marine Ecology, Marine Ecosystems, Invertebrate Zoology, Oceans and Climate Change), Duke University Marine Laboratory, Beaufort, NC (1995-2000)

Selected Major Research Vessel Experience:

Phytoplankton Population / Optical Properties, R/V Endeavor (1992) Mesoscale Iron Addition / Limitation (IRONEX2), R/V Melville (1995) JGOFS Arabian Sea #6/7, R/V Thomas G. Thompson (1995) Zonal Biogeochemical Fluxes of Western Pacific, R/V Thompson (1996)

Selected Publications: Barber, R. T., Marra, J., Bidigare, R. R., Codispoti, L. A., Halpern, D., Johnson,

Z., Latasa, M., Goericke, R., Smith, S., (in press). Primary productivity and its regulation in the Arabian Sea during 1995. Deep-Sea Research II .

Johnson Z and P Howd (2000) Marine Photosynthetic Performance Forcing and

Periodicity for the Bermuda Atlantic Time Series, 1989-1995, Deep Sea Research I 47(8), 1485-1512

Johnson Z, ML Landry, RR Bidigare, SL Brown, L Campbell, J Gunderson, J

Marra, C Trees (1999) Energetics and growth kinetics of a deep Prochlorococcus spp. population in the Arabian Sea, Deep-Sea Research II 46, 1719-1943

Barber, R. T., Borden, L., Johnson, Z., Marra, J., Knudson, C., Trees, C., (1997)

Ground truthing modeled kpar and on deck primary productivity incubations with in situ incubations. Ocean Optics XIII SPIE 2963, 834-389.

Johnson Z (1997) Modeled inherent scattering properties of small light-limited

phytoplankton: implications for deep phytoplankton size class distributions. Ocean Optics XIII SPIE 2963, 862-7.