This article was downloaded by:[Loreto, Francesco]On: 7 November 2007Access Details: [subscription number 783886388]Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Plant InteractionsPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t716100758

Photosynthetic properties of an orchid community incentral ItalyDomenico Serafini a; Federico Brilli a; Paola Pinelli b; Sebastiano Delfine b;Francesco Loreto aa CNR - Istituto di Biologia Agroambientale e Forestale, Monterotondo Scalo(Roma), Campobasso, Italyb Universita' del Molise - Dipartimento SAVA, Campobasso, Italy

Online Publication Date: 01 December 2007To cite this Article: Serafini, Domenico, Brilli, Federico, Pinelli, Paola, Delfine,Sebastiano and Loreto, Francesco (2007) 'Photosynthetic properties of an orchidcommunity in central Italy', Journal of Plant Interactions, 2:4, 253 - 261To link to this article: DOI: 10.1080/17429140701668544

URL: http://dx.doi.org/10.1080/17429140701668544

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expresslyforbidden.

The publisher does not give any warranty express or implied or make any representation that the contents will becomplete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should beindependently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with orarising out of the use of this material.

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007 ORIGINAL ARTICLE

Photosynthetic properties of an orchid community in central Italy

DOMENICO SERAFINI1, FEDERICO BRILLI1, PAOLA PINELLI2,

SEBASTIANO DELFINE2, & FRANCESCO LORETO1

1CNR � Istituto di Biologia Agroambientale e Forestale, Monterotondo Scalo (Roma), and 2Universita’ del

Molise � Dipartimento SAVA, Campobasso, Italy

(Received 22 August 2007; accepted 6 September 2007)

AbstractChlorophyllous Mediterranean orchids share a habitat endangered by climate change and land use change. These orchidsare characterized by two mechanisms of carbon assimilation, being autotrophic carbon fixation through photosynthesissupplemented by heterotrophic carbon fixation from mycorrhizal fungi. We investigated whether photosynthesis may sustainautotrophy of several species of orchids co-occurring in the same habitat (the understory of a chestnut forest in theApennines range) along a vegetative season, and how photosynthesis responds to environmental parameters in the differentspecies. Combined analysis of gas-exchange, chlorophyll fluorescence, optical properties, chlorophylls concentration, andRibulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco) activity were carried out to characterize the photosyntheticapparatus of the orchid species. Both in vivo and in vitro measurements indicated that in all orchids, in natural conditionsand over the entire vegetative season (May to July), a detectable amount of carbon, typical of autotrophic shade leaves, isfixed. It is therefore suggested that these orchids are predominantly autotrophic. As an exception, however, Limodorumabortivum, a co-occurring orchid in the examined habitat, is unable to photosynthesize at rates compatible with autotrophy.At the low light intensity experienced in the understory habitat all orchids exhibited a similar quantum yield, butphotosynthesis of Dactylorhiza saccifera and Cephalanthera longifolia was stimulated by light intensities higher than ambient,indicating that these species may better use sunflecks reaching the understory vegetation. Photosynthesis of all orchids,including Limodorum, positively responded to increasing CO2 concentration and temperature. Whether this will lead to alarger photosynthetic carbon fixation because of present and future climate change needs to be assessed with long-termexperiments also including the impacts of climate on mychorrizal activity and host plants.

Keywords: Conservation biology, fluorescence, global change, mixotrophy, orchid species, photosynthesis

Introduction

Orchids are native to every continent and they form

the world’s largest family of flowering plants (Dress-

ler 1981). Orchids are often epiphytes and auto-

trophic (belonging to different photosynthetic types,

C3, and facultative CAM, depending on climatic

conditions) in the tropical areas. In the Mediterra-

nean area, however, orchids are terrestrial and

evolved different mechanisms to resist limiting con-

ditions (especially aridity and elevated tempera-

tures). Among theses mechanisms, the evolution of

a parasitism characterized by the maintenance of

heterotrophy also in the adult and even in the

reproductive stage, may be common and has been

poorly investigated. In fact, myco-heterotrophy

(Leake 1994), a particular trophism between orchids

and their mycorrhizal fungi, has been often retrieved

in a-chlorophyllous plants and in those with photo-

synthetic rates insufficient to sustain growth (Taylor

& Bruns 1997). In orchids synthesizing chlorophyll,

myco-heterotrophy may be replaced by autotrophy

once plants become competent to photosynthesize,

or may cooperate with photosynthesis in supplying

organic matter for plant growth. The simultaneous

presence of these two metabolisms (therefore named

mixotrophy) has been suggested by recent studies in

chlorophyllous orchids (Gebauer & Meyer 2003,

Selosse et al. 2004, Julou et al. 2005). In agreement

with this suggestion, measurements of photosynth-

esis in chlorophyllous orchids have evidenced photo-

synthetic rates very low both in the partially myco-

heterotrophic genus Cephalanthera (Julou et al.

2005) and in Limodorum (Girlanda et al. 2006),

indicating that photosynthesis may not supply en-

ough substrate for growth and development of

chlorophyllous Mediterranean orchids.

Mixotrophy can make Mediterranean orchids

particularly vulnerable to climatic changes and to

Correspondence: Francesco Loreto, CNR � Istituto di Biologia Agroambientale e Forestale Via Salaria Km. 29,300 � 00016 Monterotondo

Scalo (Roma), Italy. E-mail: francesco.loreto@ibaf.cnr.it

Journal of Plant Interactions, December 2007; 2(4): 253�261

ISSN 1742-9145 print/ISSN 1742-9153 online # 2007 Taylor & Francis

DOI: 10.1080/17429140701668544

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

the consequent habitat shifts, as they depend on two

different sources of carbon. The inhibition of photo-

synthesis has been shown to negatively affect vege-

tative and reproductive functions in Dactyloriza

maculata (Vallius 2001). The Apennines range is

believed to be particularly fragile to global change.

The expected increase of temperature and decrease

of rainfall (projected to be in 100 years�3.58Cand�15%, respectively, [Piervitali & Colacino

1999]) is predicted to dramatically shift in elevation

or even extinguish the habitats of mountain vegeta-

tion in the Apennines (Stanisci et al. 2005). Orchid

presence constitutes a good indicator of stability in

the Mediterranean ecosystem, while their disappear-

ance is a bioindication of the occurrence of ecologi-

cal change, and should be prevented with the

adoption of adequate conservation programs, and

global change mitigation practices (Cozzolino et al.

2006). Characterization of the physiology of orchid

photosynthesis, especially in relation to climate and

plant phenology, is of particular importance for the

implementation of conservation programs.

In this work we explored the photosynthetic

properties of a community of orchids sharing the

same habitat, and specifically colonizing the unders-

tory of chestnut forests in central Italy. In particular,

we aimed at understanding whether: (a) Different

orchid species within the same habitat are character-

ized by a similar photosynthetic behavior; (b) auto-

trophic carbon fixation is extended over the

vegetative season of the orchids or is characteristic

of a limited part of plant life; and (c) orchid

photosynthesis depends on environmental factors,

and especially responds to current temperature and

CO2 rise (Intergovernamental Panel on Climate

Change [IPCC] 2001).

Materials and methods

Plant material, sites description, and experimental

planning

Orchids growing in chestnut (Castanea sativa P.

[Mill.]) forests in the Apennines range near Teano

(Napoli, latitude: 41 308 N, altitude: 400�500 m)

were used in this experiment. These orchids are

terrestrial, putatively mixotrophic, and share a

habitat characterized by volcanic soil, low light

diffusing in the forest understory, and humid and

mild winter and dry and hot summer. The mean

annual precipitation is around 1500 mm (of which

only around 200 mm in summer) and the average

temperature is 20.88C in summer, according to the

data-base of the Ufficio Idrografico e Mareografico

di Napoli (Vacca et al. 2003). The community was

constituted by the following chlorophyllous orchid

species: Cephalanthera longifolia, Dactylorhiza sacci-

fera, Limodorum abortivum, Platanthera chlorantha

and Serapias vomeracea.

Experiments were carried out (a) in situ on a field

campaign in the last week of May; and (b) on plants

transplanted together with an intact monolith of 50 l

of original soil under a chestnut grove, and grown in

the field at CNR-IBAF experimental station (Mon-

terotondo, Roma, latitude 42 058, altitude 200 m), in

light, temperature and humidity conditions similar

to those occurring in Teano. Measurements in Roma

were carried out three times to follow the seasonality

of photosynthesis: before May, during June, and

after July flowering.

Both in vivo and in vitro measurements were

repeated at least three times on three different leaves

of different plants. Measurements were repeated on

two consecutive years and, unless otherwise noted,

means9SE. of the measurements collected on the

same plants on two consecutive years are shown.

Differences between means were analyzed with

ANOVA and Tukey’s test using Graph Pad Prism 4

software (Graph Pad Software, San Diego, Califor-

nia). Different letters indicate differences between

species significant at pB0.01 (single letter), or 0.05

(double letters). The best fits of the light and CO2

responses were executed with Sigmaplot 2002 soft-

ware (Systat). A x2 non parametric test was used to

show significant differences between linear best-fits

as indicated by the different lines of Figure 4.

Gas-exchange and fluorescence measurements

In vivo measurements were carried out on both sites

with a portable gas-exchange system (Li-Cor 6400,

Li-Cor, Lincoln, Neb, USA) which allows measure-

ments of the exchange of water and CO2 between leaf

and air and simultaneous measurements of chloro-

phyll fluorescence. Single leaves were enclosed in the

6 cm2 gas-exchange cuvette (for the scale-like leaves

of Limodorum a round shaped 200 cm3 conifer

cuvette, Li-Cor 6400-05) and sampled under grow-

ing conditions of temperature, light, humidity and

CO2 concentration in air. Both in Teano and in

Roma these values ranged from 23�288C, 100�400

mmol m�2 s�1 of incident light intensity (except

when in presence of sunflecks, see Figure 3),

40�60% relative humidity, and 370�400 ppm of

CO2 in the air. In additional experiments in Roma,

environmental parameters were controlled by the Li-

Cor 6400 system to generate light (0�1300 mmol

m�2 s�1 PPFD) CO2 (0�1300 ppm CO2) and

temperature (25�318C) responses. Leaves were al-

lowed to adapt to the changing condition for 10 min

(light response) or 30 min (CO2 and temperature

responses) Photosynthesis, transpiration, stomatal

conductance, and intercellular CO2 concentration

(Ci) were calculated from gas-exchange measure-

ments following the formulations outlined in von

Caemmerer and Farquhar (1981). Fluorescence

measurements were carried out to estimate the

maximal quantum yield in dark-adapted leaves, as

indicated by the ratio between variable and maximal

254 D. Serafini et al.

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

fluorescence (Fv/Fm), and the quantum yield in

illuminated leaves, as indicated by the parameter

DF/Fm’ (the ratio between maximal � steady state

fluorescence and maximal fluorescence). The para-

meter DF/Fm’ was then used to calculate the rate of

linear electron transport driving photosynthesis and

photorespiration (Jf) as shown by Loreto et al.

(1994). Details on nomenclature and connotation

of fluorescence parameter are given in van Kooten

and Snel (1990). All gas-exchange measurements

were carried out between 10:00 and 14:00 h.

Chlorophyll measurements

In July, after gas-exchange and fluorescence mea-

surements, some leaves were cut to carry out

biochemical measurements. Chlorophylls were ex-

tracted from leaf discs (2 cm2) ground to a fine and

homogeneous powder with 3 ml of methanol (100%)

for 4 h at 48C. Particulates were removed by

centrifugation at 12,000 g and 58C for 10 min,

and the supernatant was removed and used for

pigment determinations. For chlorophyll determina-

tion the absorbance was measured at 470, 646.8 and

663.2 nm with a spectrophotometer (Perkin Elmer,

Norwalk, CT, USA) and the extinction coefficients

and the equations reported by Lichtenthaler (1987)

were used to calculate the concentration of chlor-

ophyll a and b.

Rubisco measurements and estimation of kinetic

properties of the enzyme.

Rubisco (Ribulose-1,5-bisphosphate carboxylase/

oxygenase) activity was determined with the radio-

metric method described by Di Marco and Tricoli

(1983). This method enables the measurement of

the maximal activity of the enzyme. Values for the

photosynthetic parameters Vcmax (maximum velo-

city of carboxylation at RuBP-saturated rate of

Rubisco), and Jmax (maximum rate of electron

transport) were obtained by fitting the mechanistic

model of CO2 assimilation proposed by Farquhar

et al. (1980) to individual responses of photosynth-

esis to Ci, using the method developed by de Pury

and Farquhar (1997).

Results

In the measurements performed in the chestnut

forest of Teano, measurable and comparable photo-

synthesis and stomatal conductance rates were found

in all species with the exception of Limodorum

(Figure 1a, 1b). In the measurements carried out in

Roma these indications were confirmed, although the

rates of photosynthesis were in general slightly

reduced (Figure 1c, 1d). This might reflect an

incomplete adaptation to the new environment

(e.g., a response to the absence of symbiosis with

the new host trees), although no major difference in

plant development and growth was observed between

the plants growing at the two sites. Photosynthesis

and stomatal conductance rates during and after

flowering remained as high as before flowering

(Figure 1c, 1d, the statistical treatment did not

show significant differences in the means of the single

species during the three measurements) indicating no

seasonal trend of gas-exchange during the vegetative

cycle of all orchids. As in the measurements of Teano,

however, the rates of photosynthesis and stomatal

conductance of Limodorum leaves were significantly

lower and barely measurable all over the season.

The following experiments were carried out in the

Roma set, before flowering in May. A similar

quantum yield was observed in all orchids exposed

to low light intensities, as indicated by the slope of

the linear response of photosynthesis to light inten-

sities between 0 and 150 mmol photons m�2 s�1

(Figure 2), and by the fluorescence measurements in

dark-adapted leaves (Fv/Fm around 0.75 in all

leaves, data not shown). When exposed at light

intensities higher than those experienced during

growth (about 200 mmol photons m�2 s�1), how-

ever, photosynthesis was stimulated in Dactylorhiza

and Cephalanthera (Figure 2). In Platanthera and

Serapias photosynthesis saturated already at growth

light intensities. In Limodorum, photosynthesis was

also light-dependent for light intensities up to growth

conditions while saturated at brighter intensities. In

both field sites we detected a considerable number of

sunflecks (800�1500 mmol m�2 s�1 PPFD) reach-

ing the orchids during our gas-exchange measure-

ments. Measurements of selected leaves of the two

species that showed a light-induced stimulation of

photosynthesis, confirmed that photosynthesis and

electron transport rates of Dactylorhiza and Cepha-

lanthera can actually increase in response to this

rapid (from 1�10 min) exposure to bright light, while

this is not the case, for instance, with Limodorum

leaves (Figure 3).

Photosynthesis of all orchids also responded to

CO2, as expected for autotrophic, shade plants

(Figure 4). In Dactylorhiza, Cephalanthera, Pla-

tanthera and Limodorum, photosynthesis was sup-

pressed at a lower intercellular CO2 (Ci) than in

Serapias. However, photosynthesis of Serapias leaves

was more sensitive to increasing CO2 than in the

other orchids, as shown by the steep slope of the

linear response of photosynthesis at low Ci in this

species (indicated by the dashed line in Figure 4),

and by the observation that photosynthesis of

Serapias leaves did not saturate even at very high

intercellular CO2 concentration. Photosynthesis was

also slightly stimulated at elevated CO2 in Limo-

dorum, the orchid characterized by very low photo-

synthetic rates.

Photosynthesis was temperature-dependent in all

orchid species, and the highest rates were recorded at

the highest leaf temperature (Figure 5). When

Orchid photosynthesis 255

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

Pho

tosy

nthe

sis,

µm

ol m

–2 s

–1

0

2

4

6

8

0

1

2

3

4

5

6

Cephalanthera Serapias Dactylorhiza Platanthera Limodorum Cephalanthera Serapias Dactylorhiza Platanthera Limodorum

A (Teano)

C (Roma)

Sto

mat

al c

ondu

ctan

ce, m

ol m

–2 s

–1

0,00

0,05

0,10

0,15

0,20

B (Teano)

May June JulyMay June July0,00

0,02

0,04

0,06

0,08

0,10

0,12

D (Roma)

aa

aab

c

a

a

aab

c

Figure 1. Photosynthesis (A, C) and stomatal conductance (B, D) of orchids co-occurring in the habitat of a chestnut forest understory in

central Italy. Field measurements taken at two different sites, Teano (A, B) and Roma (C, D) are shown. In Roma, measurements were

repeated on three different months on the same specimens. The five bars shown each month in the Roma experiments represent the

different orchid species in the same order shown in panels A and B. Measurements are shown as means9SE (n�4). In the upper panels

different letters indicate differences of photosynthesis and stomatal conductance between species significant at pB0.01 (single letter), or

0.05 (double letters) (ANOVA and Tukey’s test). Environmental conditions during the measurements are reported in the text.

Light intensity, µmol m–2 s–1

0 200 400 600 800 1000 1200 1400

Pho

tosy

nthe

sis,

µm

ol m

–2 s

–1

–2

0

2

4

6

8

10Dactylorhiza Cephalanthera PlatantheraSerapiasLimodorum a

a

b

bc

d

Figure 2. Light response of photosynthesis of orchid leaves. Means9SE (n�6) are shown. When SE is not visible, the symbols size is larger

than the error bar. At a light intensity of 1000 mmol photons m�2 s�1 different letters indicate differences between species significant at pB

0.01 (single letter), or 0.05 (double letters) (ANOVA and Tukey’s test). Slopes of the linear response of photosynthesis to light intensity

were not significantly different, as assessed by x2 non-parametric test, in the different species (best fits not shown).

256 D. Serafini et al.

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

exposed to a simultaneous increase of temperature

and CO2 (308C and 800 ppm, respectively), photo-

synthesis was stimulated to a very high extent with

respect to growth conditions (258C and 370 ppm of

CO2). This stimulation was observed in species with

contrasting photosynthetic rates such as Limodorum,

Cephalanthera and Dactylorhiza. In all cases, how-

ever, the stimulation was not significantly higher

than that recorded upon exposure to either elevated

CO2 or elevated temperature, which indicates that

the effects of these two environmental factors are

likely not additive (data not shown).

In vitro measurements showed a similar chloro-

phyll ratio in all orchid species (Table I). Dactylor-

hiza leaves are characterized by red patchy areas

interspersed with the natural pigmentation. The red

patches were characterized by a much higher con-

centration of anthocyanin and by a lower amount of

chlorophyll a and b than the normally pigmented

areas of Dactylorhiza (data not shown).

All orchids were also characterized by detectable

maximal activity of Rubisco but the maximal activity

of this enzyme was very low in Limodorum leaves

(Table I). Serapias leaves, in contrast, showed the

Time of day, hh.min

0

1

2

3

4

5

6

7DactylorhizaCephalantheraLimodorum

11.30 12.00 12.30 13.00 13.30 14.000

200

400

600

800

1000

1200

Pho

tosy

nthe

sis,

µm

ol C

O2

m–2

s–1

Ligh

t int

ensi

ty, µ

mol

pho

tons

m–2

s–1

Figure 3. Response of orchid photosynthesis to sunflecks occurring on May 2006 in the chestnut forest near Roma. Measurements were

taken every 10 min on three different leaves (one for each species). Light intensity is shown by the continuous line and photosynthesis of the

three orchid species is shown by symbols. Light intensity variations of up to950 mmol photons m�2 s�1 are smoothened by the graphic

programme and are not reported.

Intercellular CO2 concentration (Ci), ppm

0 200 400 600 800 1000

Pho

tosy

nthe

sis,

µm

ol m

–2 s

–1

–4

–2

0

2

4

6

8

10

12

14

Dactylorhiza Cephalanthera PlatantheraSerapiasLimodorum

Figure 4. CO2 response of photosynthesis of orchid leaves. The relationship between photosynthesis and the intercellular CO2

concentration, estimated from gas-exchange measurements, is shown. Means9SE (n�6) are reported. A x2 non parametric test was

used to show significant differences between linear best-fits of the initial slope of the relationship. Significantly different slopes are indicated

by the dotted line (Limodorum) the long dashed line (Serapias) and the short dashed line (all other orchids).

Orchid photosynthesis 257

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

highest rates of Rubisco activity, the highest calcu-

lated parameters for the enzyme (Vcmax and Jmax),

and the highest linear electron transport rates (Jf)

(Table I).

Discussion

The chlorophyllous orchids sharing the same habitat

in chestnut forest understories of central Italy are

characterized by photosynthetic rates comparable to

those of shade plants (Boardman 1977). Our results

therefore suggest that these orchid species can be

predominantly autotrophic at least along the vege-

tative cycle including pre-floral, floral and post-floral

stages. Experiments showing a 13C abundance in

Cephalanthera plant organs intermediate between

that expected from full heterotrophy and full auto-

trophy, previously suggested that 33% (Abadie et al.

2006) or even the majority of carbon (Gebauer &

Meyer 2003) is contributed by mychorrizal fungi in

Cephalanthera. A similar conclusion was reached by

Julou et al. (2005), as in their study the photosyn-

thetic rates in natural conditions of chlorophyllous

individuals of Cephalanthera damasonium were below

compensation point (i.e., photosynthesis was nega-

tive). We did not perform measurements of isotope

abundance that could partition carbon fixed hetero-

trophycally from that acquired photosynthetically

(Abadie et al. 2006). However, in our experiment

Cephalantera plants generally photosynthesized very

efficiently, at rates generally higher than 4 mmol m�2

s�1, which suggests a high autotrophy also for some

species of this orchid genus. Perhaps a higher

contribution of the heterotrophic carbon fixation to

the mixotrophy is only limited to a very early stage of

development, which was not covered by our mea-

surements. Alternatively, the environmental condi-

tions of central Italy, characterized by very mild

winter and dry summer, may dramatically change

the physiology of this orchid genus, and in particular

its association with mycorrhizal fungi. Limodorum

appears to be the only chlorophyllous orchid of the

habitat investigated whose photosynthesis is not

sufficient to sustain growth efficiently. Previous

Table I. Pigment concentrations, Rubisco activity, calculated maximal velocity of carboxylation (Vcmax) and maximal electron transport

rate (Jmax), and electron transport rate measured by fluorescence (Jf) in leaves of Dactylorhiza, Cephalanthera, Platanthera, Serapias and

Limodorum, characterized by different photosynthetic rates (see Figure 1, data are from leaves collected in Roma after measurements carried

out in July). Means9SE (n�4) are reported. Different letters indicate differences between species significant at pB0.01 (single letter), or

0.05 (double letters) (ANOVA and Tukey’s test).

Parameter Dactylorhiza Cephalanthera Platanthera Serapias Limodorum

Chlorophyll a (mg cm�2) 30.593.7a 27.791.3a 26.893.0a 28.692.2a 22.993.4ab

Chlorophyll b (mg cm�2) 17.493.5a 14.891.9a 13.992.4a 15.994.0a 11.793.1ab

Chlorophyll a/b 1.7490.21a 1.8890.11a 1.9290.12a 1.8090.15a 1.9590.28a

Rubisco activity (mmol m�2 s�1) 10.392.2a 7.292.9ab 8.291.6ab 12.991.4a 0.591.1c

Vcmax (mmol m�2 s�1) 25.595.1ab 21.494.3b 20.193.3b 33.193.6a 0.790.5c

Jmax (mmol m�2 s�1) 50.794.0ab 44.995.9b 33.595.4c 61.093.4a 4.690.6d

Jf (mmol m�2 s�1) 32.693.8a 34.095.2a 27.792.2b 35.493.0a 4.191.0c

Temperature, °C24 25 26 27 28 29 30 31 32

Pho

tosy

nthe

sis,

µm

ol m

–2 s

–1

0

2

4

6

Dactylorhiza Cephalanthera PlatantheraSerapiasLimodorum

a

aaa

a

b

bbb

a

Figure 5. Temperature response of photosynthesis in orchid leaves. Means9SE (n�6) are shown. Different letters indicate differences

between species significant at pB0.01 at the two contrasting temperatures, 25 and 318C (ANOVA and Tukey’s test).

258 D. Serafini et al.

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

indications about this behavior (Girlanda et al.

2006) are confirmed by this larger data-set. Mea-

surements of mycorrhizal carbon contribution to the

orchid organic compounds may reveal their sub-

stantial role in supporting mixotrophy along the

entire vegetative period.

The investigated orchids were shade adapted,

growing under very dense chestnut canopies, at light

intensities rarely exceeding 400 mmol m�2 s�1, and

generally below 200 mmol m�2 s�1 (Figure 3).

Accordingly, all species presented a very efficient

system of light capture by the photochemistry of

photosynthesis. A high quantum yield was estimated

by the steep linear response of photosynthesis to low

light intensities, and by the maximal fluorescence

yield in dark-adapted leaves. However, photosynth-

esis of Dactylorhiza and Cephalanthera responded to

light intensities more elevated than those experi-

enced during growth, while the other three orchid

species saturated photosynthesis at growth light

intensities (Figure 2). This difference was also

observed in response to rapid sunflecks naturally

occurring in the field (Figure 3). Thus Dactylorhiza

and Cephalanthera can use more efficiently the light

that reaches the understory vegetation. Whether this

depends on a more efficient transfer of the energy in

the photochemical apparatus of these two species, or

on a faster activation of the biochemistry of carbon

fixation, or on a faster stomatal opening (Valladares

et al. 1997) remains to be studied. The possibility to

efficiently use the light provided by sunflecks may

positively affect the carbon fixed by these orchid

species along sunny days, as previously demon-

strated for other understory plant species (Naum-

burg & Ellsworth 2002). It may also give to these

orchids an evolutionary advantage if future condi-

tions will be characterized by less dense canopies

perhaps in response of increasing aridity in Central

Italy (IPCC 2001). However, whether such an

advantage for carbon acquisition will be balanced

by negative feedbacks (e.g., lower level of myco-

heterotrophy or increasing competition with other

plant species invading the forest understory) remains

to be ascertained.

We noted that the quantum yield of red patches,

characterized by a large presence of anthocyanins,

interspersed with normally (green) pigmented leaf

areas of Dactylorhiza was lower than in the green

areas, at all light intensities (data not shown). Thus,

once the lower yield of the anthocyanin-rich areas is

taken into account, the efficiency of light use in

Dactylorhiza leaves is even higher than estimated in

our study.

Photosynthesis of all orchids was CO2-dependent.

Net photosynthesis was suppressed when intercellu-

lar CO2 was well above zero (Figure 4). This high

compensation point indicates the presence of photo-

respiration and, therefore, a mechanism C3 of

photosynthesis in all orchids. The higher compensa-

tion point of Serapias may indicate a higher con-

tribution of photorespiration or mitochondrial

respiration in this species. However, no clear inter-

specific differences of mitochondrial respiration were

observed in dark-adapted leaves (Figure 2), and we

suggest that high photorespiration rates may be

occurring in some species, possibly because of low

diffusion of CO2 at the Rubisco sites (Evans &

Loreto 2000). Also confirmation of the occurrence

of large rates of photorespiration came from the

observations that: (a) Photosynthesis was largely

stimulated under CO2 level higher than ambient in

all orchids, and (b) this stimulation persisted even at

very high levels of Ci. The second observation

supports the indication that further resistances may

largely decrease CO2 concentration after entering

the leaves and before acquisition by chloroplasts.

The CO2 response was particularly evident in

Serapias as for this species no saturation of photo-

synthesis was observed.

The Rubisco activity, assessed in vivo by the slope

of the linear relationship between photosynthesis

and Ci (at low Ci, [Farquhar et al. 1980]), and by

the in vitro assay, was similar in all orchids, with the

exception of Limodorum and Serapias. In the first

species Rubisco activity was found to be very low,

while in Serapias a high activity of the enzyme was

measured. Among the orchid species of the commu-

nity, Serapias appears to have the largest potential to

exploit increasing levels of CO2 in the atmosphere,

as demonstrated by the high Rubisco activity and

calculated velocity of carboxylation and electron

transport rate. In contrast, the low Rubisco activity

may be one of the reasons why Limodorum photo-

synthesis is much lower than photosynthesis of all

other orchids co-occurring in the same habitat.

However, a large stimulation of photosynthesis

upon exposure to elevated CO2 and/or temperature

was also observed in Limodorum, indicating that,

especially in this orchid whose autotrophy has been

often questioned (Girlanda et al. 2006), the potential

photosynthetic rate of carbon fixation may be

dramatically larger than in nature. Whatever is the

limitation of photosynthesis in nature in the different

orchids, our experiments suggest that the rate of

autotrophic carbon fixation of orchids living in the

understory of central Italy may benefit from cur-

rently rising levels of CO2 (IPCC 2001).

It should be mentioned that actual growth at

elevated CO2 and temperature may cause the onset

of several feedback mechanisms, in part or in toto

canceling the stimulation of photosynthesis we have

noticed upon short-term exposure to these condi-

tions. Long-term exposure to elevated CO2 may lead

to adjustments such as the down-regulation of

Rubisco activity (Sage et al. 1989), or may cause

the onset of nutritional limitations, typically nitrogen

limitations ((Long et al. 2004), which may further

negatively feedback on the relationship between

Orchid photosynthesis 259

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

orchids, symbionts, and the host-species. It should

be also mentioned that drought stress, another

important factor in the Mediterranean area, and

probably exacerbated by current and future climate

change (Piervitali & Colacino 1999), was not con-

sidered in the present work and may offset the

possible positive feedback of rising CO2 and tem-

perature on orchid photosynthesis. Our short-term

experiments do not account for these long-term

effects, but may be important to indicate how the

future climate will affect these orchid species who are

currently considered at risk of extinction (Cozzolino

et al. 2006), and may play an important role in the

conservation of genetic diversity of Mediterranean

forests.

Contrary to our expectations, based on the

observation that temperature conditions in the

orchid habitat were relatively cold compared to the

other areas of the Apennines (Vacca et al. 2003),

orchid photosynthesis was also stimulated by in-

creasing temperature. Temperature increase should

further favor photorespiration over photosynthesis

because of the higher solubility of oxygen with

respect to CO2, and this effect may only be

compensated by increasing Rubisco activity and/or

by higher CO2 availability at the chloroplasts (Ber-

nacchi et al. 2001). Transpiration and intercellular

CO2 concentration were not affected by temperature

(data not shown) but since mesophyll conductance

may increase at elevated temperatures (Bernacchi

et al. 2002), a higher CO2 concentration in the

chloroplasts may explain why photosynthesis is

stimulated at increasing temperatures in our orchids.

Conclusion

In summary, our experiments indicate that, under

the current environmental conditions, orchids shar-

ing the same habitat in the central Apennines range

are all characterized by a C3 photosynthetic meta-

bolism and, with the exception of Limodorum, are

largely autotrophic along the vegetative period,

before, during and after flowering. Our data also

indicate that photosynthesis of chlorophyllous orch-

ids of the Mediterranean forest increases at increas-

ing temperature and CO2 concentration, and that

Dactylorhiza and Cephalanthera may also take advan-

tage of light intensities higher than those experienced

in their understory habitat.

Acknowledgements

This work was supported by the project of the Italian

Ministry for Scientific Research (MIUR) ‘Orchidee

micoeterotrofiche dell’area Mediterranea’. We thank

Donata Cafasso and Giuseppe Santarelli for helping

with plant classification and collection and with in

vivo measurements, and Fabrizio Pietrini for helping

with biochemical measurements.

References

Abadie J-C, Puttsepp U, Gebauer G, Faccio A, Bonfante P,

Selosse M-A. 2006. Cephalanthera longifolia (Neottieae, Orch-

idaceae) is mixotrophic: A comparative study between green

and nonphotosynthetic individuals. Can J Bot 84:1562�1477.

Bernacchi CJ, Singsaas EL, Portis AR, Long SP. 2001. Improved

temperature response functions for models of Rubisco-limited

photosynthesis. Plant Cell Environ 24:253�259.

Bernacchi CJ, Portis AR, Nakano H, von Caemmerer S, Long SP.

2002. Implications for the determination of Rubisco enzyme

kinetics and for limitations to photosynthesis in vivo. Plant

Physiol 130:1992�1998.

Boardman NK. 1977. Comparative Photosynthesis of Sun and

Shade Plants. Annu Rev Plant Physiol 28:355�377.

Cozzolino S, Nardella AM, Impagliazzo S, Widmer A, Lexer C.

2006. Hybridization and conservation of Mediterranean orch-

ids: Should we protect the orchid hybrids or the orchid hybrid

zones? Biol Conserv 129:14�23.

de Pury DGG, Farquhar GD. 1997. Simple scaling of photo-

synthesis from leaves to canopies without the errors of big-leaf

models. Plant Cell Environ 20:537�557.

Di Marco G, Tricoli D. 1983. RuBP carboxylase determination by

enzymic estimation of D-3-PGA formed. Photosynth Res

4:145�149.

Dressler RL. 1981. The orchids. London, UK: Harvard Uni-

versity Press.

Evans JR, Loreto F. 2000. Aquisition and diffusion of CO2 in

higher plant leaves. In: Leegood RC, Sharkey TD, von

Caemmerer S, editors. Photosynthesis: Physiology and meta-

bolism. Amsterdam, The Netherlands: Kluwer Academic

Publishers. pp 321�351.

Farquhar GD, von Caemmerer S, Berry JA. 1980. A biochemical

model of photosynthetic CO2 assimilation in leaves of C3

species. Planta 149:78�90.

Gebauer G, Meyer M. 2003. 15N and 13C natural abundance of

autotrophic and myco-heterotrophic orchids provides insight

into nitrogen and carbon gain from fungal association. New

Phytol 160:209�223.

Girlanda M, Selosse MA, Cafasso D, Brilli F, Delfine S, Fabbian

R, Ghignone S, Pinelli P, Segreto R, Loreto F, Cozzolino S,

Perotto S. 2006. Inefficient photosynthesis in the Mediterra-

nean orchid Limodorum abortivum is mirrored by specific

association to ectomycorrhizal Russulaceae. Mol Ecol 15:491�504.

Intergovernamental Panel on Climate Change (IPCC). 2001.

Climate change 2001: IPCC third assessment report. Inter-

governamental Panel on Climate Change. Cambridge Univer-

sity Press, Cambridge. On-line at: www.grida.no/climate/

ipcc_tar/.

Julou T, Burghardt B, Gebauer G, Berveiller D, Damesin C,

Selosse M-A. 2005. Mixotrophy in orchids: insights from a

comparative study of green individuals and nonphotosynthetic

individuals of Cephalanthera damasonium. New Phytol 166:639�653.

Leake JR. 1994. The biology of myco-heterotrophic (‘saprophy-

tic’) plants. New Phytol 127:171�216.

Lichtenthaler HK. 1987. Chlorophylls and carotenoids: pigments

of photosynthetic biomembranes. In: Methods in Enzymology

Vol. 148. London, UK: Academic Press Inc. pp 350�382.

Long SP, Ainsworth EA, Rogers A, Ort DR. 2004. Rising

atmospheric carbon dioxide: Plants FACE the future. Ann

Rev Plant Biol 55:591�628.

Loreto F, Di Marco G, Tricoli D, Sharkey TD. 1994. Measure-

ments of mesophyll conductance, photosynthetic electron

transport and alternative electron sinks of field grown wheat

leaves. Photosynth Res 41:397�403.

Naumburg E, Ellsworth DS. 2002. Short-term light and leaf

photosynthetic dynamics affect estimates of daily understory

photosynthesis in four tree species. Tree Physiol 22:393�401.

Piervitali E, Colacino M. 1999. Temperature and precipitation

trends in Italy during the last century. In: Visconti G, Beniston

260 D. Serafini et al.

Dow

nloa

ded

By:

[Lor

eto,

Fra

nces

co] A

t: 06

:53

7 N

ovem

ber 2

007

M, Iannorelli ED, Barba D, editors. Global change and

protected areas. Advances in global change research, 9.

Dordrecht, The Netherlands: Kluwer Academic Publishers.

pp 216�222.

Sage RF, Sharkey TD, Seemann JR. 1989. Acclimation of

photosynthesis to elevated CO2 in five C3 species. Plant Physiol

89:590�596.

Selosse MA, Faccio A, Scappaticci G, Bonfante P. 2004.

Chlorophyllous and achlorophyllous specimens of Epipactis

microphylla (Neottieae, Orchidaceae) are associated with ecto-

mycorrhizal septomycetes, including truffles. Micr Ecol

47:416�426.

Stanisci A, Pelino G, Blasi C. 2005. Vascular plant diversity and

climate change in the alpine belt of the central Apennines

(Italy). Biodiver Conserv 14:1301�1318.

Taylor DL, Bruns TD. 1997. Independent, specialized invasions

of ectomycorrhizal mutualism by two nonphotosynthetic orch-

ids. Proc Natl Acad Sci USA 94:4510�4515.

Vacca A, Adamo P, Pigna M, Violante P. 2003. Genesis of Tephra-

derived soils from the Roccamonfina volcano, South Central

Italy. Soil Sci Soc Am J 67:198�207.

Valladares F, Allen MT, Pearcy RW. 1997. Photosynthetic

responses to dynamic light under field conditions in six tropical

rainforest shrubs occurring along a light gradient. Oecologia

111:505�514.

Vallius E. 2001. Factors affecting fruit and seed production

in Dactylorhiza maculata (Orchidaceae). Bot J Linn Soc

135:89�95.

Van Kooten O, Snel JFH. 1990. The use of chlorophyll

fluorescence nomenclature in plant stress physiology. Photo-

synth Res 25:147�150.

von Caemmerer S, Farquhar GD. 1981. Some relationships

between the biochemistry of photosynthesis and the gas

exchange of leaves. Planta 153:376�387.

Orchid photosynthesis 261