redox regulation of light-harvesting complex ii and cab ... · plant physiol. (1 995) 109: 787-795...

Plant Physiol. (1 995) 109: 787-795

Redox Regulation of Light-Harvesting Complex II and cab mRNA Abundance in Dunaliella salina'

Denis P. Maxwell', David E. Laudenbach, and Norman P. A. Huner*

Department of Plant Sciences, The University of Western Ontario, London, Ontario, Canada N6A 5B7

We demonstrate that photosynthetic adjustment at the leve1 of the light-harvesting complex associated with photosystem I1 (LCHII) in Dunaliella salina i s a response to changes in the redox state of intersystem electron transport as estimated by photosystem II (PSII) excitation pressure. To elucidate the molecular basis of this phe- nomenon, LHCll apoprotein accumulation and cab mRNA abundance were examined. Crowth regimes that induced low, but equiv- alent, excitation pressures (either 13"C/20 pmol m-* s-' or 30°C/ 150 pmol m-' s-l) resulted in increased LHCll apoprotein and cab mRNA accumulation relative to alga1 cultures grown under high excitation pressures (either 13"C/150 pmol m-' s-' or 3O0C/25O0 pmol m-' s-l). Thermodynamic relaxation of high excitation pressures, accomplished by shifting cultures from a 13 to a 30°C growth regime at constant irradiance for 12 h, resulted in a 6- and 8-fold increase in LHCll apoprotein and cab mRNA abundance, respectively. Similarly, photodynamic relaxation of high excitation pressure, accomplished by a shift from a light to a dark growth regime at constant temperature, resulted i n a 2.4- to 4-fold increase in LHCll apoprotein and cab mRNA levels, respectively. We conclude that photosynthetic adjustment to temperature mimics adjustment to high irradiance through a common redox sensinglsignaling mechanism. 60th temperature and light modulate the redox state of the first, stable quinone electron acceptor of PSII, which reflects the redox poise of intersystem electron transport. Changes in redox poise signal the nucleus to regulate cab mRNA abundance, which, in turn, determines the accumulation of light-harvesting apoprotein. This redox mechanism may represent a general acclimation mechanism for photosynthetic adjustment to environmental stimuli.

In green algae and higher plants, the light-harvesting complexes are located in the chloroplast thylakoid membrane and are responsible for the capture and transfer of light energy to the photosynthetic reaction centers. The apoproteins of the major Chl a / b LHCII are encoded in the nucleus by the cab multigene family (Buetow et al., 1988; Jansson, 1994). Thus, regulatory signals must be exchanged between the nucleus and chloroplast to maintain optimal levels of the LHCII pigment-protein complexes (Simpson

Dedicated to the memory of Dave Laudenbach who died on June 16, 1995. This research was supported by a Natural Sciences and Engineering Research Council of Canada research grants to N.P.A.H. and D.E.L. D.P.M. was supported, in part, by an Ontario Graduate Scholarship.

Present address: DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48823.

* Corresponding author; e-mail [email protected]; fax 1-519-661-3935.

787

et al., 1986; Taylor, 1989). The cab polypeptides are trans- lated in the cytoplasm on membrane-free polyribosomes as soluble precursor proteins, which are posttranslationally imported into the chloroplast (Keegstra et al., 1989; Dahlin and Cline, 1991; Leheny and Theg, 1994). Upon entry into the chloroplast, these apoproteins are processed to their mature forms and subsequently inserted and assembled into the thylakoid membrane as pigment-protein complexes (Anderson, 1986; Kuhlemeier et al., 1986; Buetow et al., 1988; Chitnis and Thornber, 1988; Mullet, 1988; Keeg- stra et al., 1989; Dahlin and Cline, 1991; Dreyfuss and Thornber, 1994).

Stimulation of cab gene transcription upon exposure of etiolated seedlings to a dark/ light transition is mediated through the photoreceptor phytochrome (Tobin and Sil- verthorne, 1985; Kuhlemeier et al., 1986; Thompson and White, 1991; Bowler and Chua, 1994). Run-on transcription assays on isolated nuclei of the green alga Dunaliella tertiolecta demonstrated that the light-induced accumulation of cab mRNA is due to an increased rate of gene transcription rather than increased mRNA stability (Buetow et al., 1988; Escoubas et al., 1995). Both positive and negative regulatory elements, located within the 5' noncoding region, have been shown to mediate the light response of cab genes (Gilmartin et al., 1990; Gao and Kaufman, 1994). Since low levels of cab mRNA have been detected in dark-grown plants, light does not appear to be an absolute requirement for the transcription of cab genes. However, light does appear to be required for maximal accumulation of cab transcripts (Buetow et al., 1988).

The photoregulation of cab gene expression is also spe- cies dependent. In contrast to angiosperms, pine cotyle- dons accumulate cab gene transcripts and LHCII polypeptides in the dark (Yamamoto et al., 1991). In chlorophytes, the primary structure of the LHCII apoproteins differs from that of higher plants (LaRoche et al., 1991). cab gene transcription in green algae appears to be positively regulated by a blue-light receptor but negatively regulated by phytochrome (Kindle, 1987; Sukenik et al., 1987; LaRoche et al., 1990; Hermsmeier et al., 1991; Jansson, 1994). Further- more, it has been proposed that intermediates of Chl bio-

Abbreviations: F,, maximal Chl a fluorescence with a11 PSII traps open; F,, variable Chl a fluorescence with a11 traps open; F,/F,, maximal photochemical efficiency of with a11 PSII traps open; LHCII, light-harvesting complex associated with PSII; QA, the first, stable quinone electron acceptor of PSII reaction centers; qP, photochemical quenching parameter.

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788 Maxwell et al. Plant Physiol. Vol. 109, 19'35

synthesis may be involved in the regulation of light- responsive genes in Chlamydomonas reinhardtii (Johanning- meier and Howell, 1984; Johanningmeier, 1988; Jasper et al., 1991).

Irradiance stress occurs whenever the light energy ab- sorbed by the photosynthetic apparatus is in excess of that required for photosynthesis and results in the photoinhibition of photosynthesis (Osmond, 1994). PSII appears to be the primary site of photoinhibitory damage (Aro et al., 1993), and thus, plants and green algae adjust the structure and composition of their photosynthetic apparatus to pro- tect PSII (Horton and Ruban, 1994). In green algae, photosynthetic adjustment to irradiance stress involves an increase in the ratio of Chl a / b and a concomitant decrease in LHCII abundance (Melis et al., 1985; Anderson, 1986; Suke- nik et al., 1987, 1988; Mortain-Bertrand et al., 1988; Smith et al., 1990; Davison, 1991; Falkowski and LaRoche, 1991; Harrison et al., 1992; Vasilikiotis and Melis, 1994; Webb and Melis, 1995). It has been suggested that irradiance regulates LHCII apoprotein content either transcriptionally through the inhibition of cab mRNA accumulation and/or posttranslationally through the control of Chl synthesis (Falkowski and LaRoche, 1991; LaRoche et al., 1991; Escou- bas et al., 1995). However, recently, we reported that the growth of Cklorella vulgaris at low temperature and moderate irradiance (5"C/150 pmol m - ' ~ - ~ ) mimics growth of this green alga under high-irradiance stress (27"C/ 2200 pmol mp2 s?). These conclusions were based on the analyses of pigment content and composition, LHCII apoprotein abundance, O, evolution, Chl a fluorescence, and sus- ceptibility to photoinhibition (Maxwell et al., 1995). These results could not be explained independently as either a growth temperature or a growth irradiance response per se. However, these results were correlated with changes in the redox state of Q,. We suggested that this correlation could be explained in terms of temperature- and light- dependent modulation of the redox state QA, as defined by 1 - qp, which equals (QA)reducedl(QA)=educed + (QA)oxidized. The redox state of QA is calculated from the measured qP (van Kooten and Snel, 1990; Bradbury and Baker, 1981). Since light absorption and utilization in photosynthesis integrates temperature-insensitive photochemical reactions with temperature-sensitive biochemical reactions, the redox state of Q, can be modulated either by changing the temperature under conditions of constant irradiance or by changing irradiance under constant temperature conditions (Maxwell et al., 1995).

Allen (1993,1995) proposed that chloroplast gene expression is regulated by the redox poise of intersystem electron transport, which, in turn, results in the capacity to adjust photosystem stoichiometry. This is supported by the recent report of Danon and Mayfield (1994), who showed that the light-dependent translation of psbA mRNA in C. reinkardtii is under redox control. Pearson et al. (1993) suggested that the redox control of chloroplast transcription is mediated by the Cytflb, complex. LaRoche et al. (1991) and Escou- bas et al. (1995) were the first to suggest that irradiance stress mediates LHCII apoprotein and cab mRNA abundance through the redox state of the thylakoid plastoqui-

none pool in D. tertiolecta. In this report, we present data that support the hypothesis that chloroplastic redox poise can regulate nuclear gene expression (Melis et al., 1985; LaRoche et al., 1991). Furthermore, we demonstrate for what to our knowledge is the first time that the modulakion of LHCII apoprotein and cab gene transcript abundance, previously assumed to be strictly an irradiance response in green algae and plants, can be mimicked by an apparent temperature response in Dunaliella salina. We suggest that the mechanisms of photosynthetic adjustment to changes in growth temperature and growth light regimes are similar, because both of these environmental cues share a common redox sensingl signaling pathway for the regulation of cab gene transcription. This sensing/ signaling mechanism is mediated by the redox poise of intersystem electron transport as reflected by changes in excitation pressure. Furthermore, we suggest that this model may represent a general mechanism for photosynthetic adjustment to environmental stress.

MATERIALS A N D METHODS

Crowth Conditions

The halotolerant, unicellular green alga Dunaliella salina was grown axenically according to the method of Naus and Melis (1991) in 1-L Corning tissue culture flasks (Corning, Inc., Corning, NY) containing a hypersaline medium supplemented with 25 mM NaHCO,. Cultures were grown on shakers in controlled environmental growth chambers (Conviron, Winnipeg, Manitoba, Canada) with light sup- plied by a combination of cool-white fluorescent bulbs supplemented with a 500-W halogen lamp (model DEQ, General Electric). The cultures were maintained at a constant growth temperature of either 30 or 13°C. The former were exposed to an irradiance of either 150 or 2500 bLmol mp2 -1 s , whereas the latter were exposed to an irradiance of either 20 or 150 pmol m-' s-' as measured from the center of the culture flasks. A11 cultures were maintained at low Chl concentrations (1-3 pg mL-l) to minimize self- shading. Periodic dilution with fresh growth medium en- sured that cells were harvested in their exponential growth phase.

For the temperature-shift experiment (13°C / 150 bLmol

cultures were transferred from the Corning culture flasks to glass growth tubes and placed in a water-filled aquarium as described by Maxwell et al. (1995). Light was sup- plied by a bank of cool-white fluorescent bulbs, and irradiance, measured in the center of the growth tube, was adjusted by neutra1 density filters. The change in temperature from 13 to 30°C was monitored as a function of time. Equilibration to the new temperature was attained within 5 min of transfer. For the dark experiments, the cultures were wrapped in aluminum foil, the aquarium containing the cultures was draped with a black cloth, and the culture room was maintained in complete darkness to ensure the absence of light.

m-z s -1 to 3O0C/15O pmol m-' s -'), the log-phase cell



Redox Regulation and Light-Harvesting Complex II Abundance 789

Chl Analysis

Cells were harvested by centrifugation and resuspendedin 90% (v/v) acetone. Chl was estimated spectrophoto-metrically using the equations of Jeffrey and Humphrey(1975).

Thylakoid Membrane Isolation and SDS-PAGE

Thylakoids were isolated using the procedure of Smith etal. (1990). All buffers contained 1 mM PMSF, 1 mM aminocaproic acid, and 1 mM benzamidine to inhibit proteolyticactivity. SDS-PAGE was carried out using a Bio-Rad Mini-protean II apparatus according to the method of Maxwell etal. (1995) using a 15% (w/v) separating gel. Lanes wereloaded with equal protein content (20 /u.g) as determined bythe bicinchoninic acid method. Electrophoresis was per-formed at 100 V at room temperature for approximately2.5 h.

Western Blots

Membrane polypeptides separated by SDS-PAGE wereelectrophoretically transferred to nitrocellulose and probedwith monospecific polyclonal antibodies raised againstspinach LHCIIbS (Krol et al., 1995). Blots were developedusing an enhanced chemiluminescence technique follow-ing the manufacturer's protocol (Amersham) and usinggoat anti-rabbit IgG linked to horseradish peroxidase as thesecondary antibody.

RNA Isolation and Blot Hybridization

Total RNA was isolated and electrophoresed accordingto the method of Laudenbach et al. (1988) and transferredto reinforced nitrocellulose in 20 X SSC according to themethod of Thomas (1983). All lanes were loaded with anequal amount of RNA as indicated by staining withethidium bromide. RNA blots were probed with a 1.3-kbEcoRI fragment containing a cDNA cab gene (ZLcab43)cloned from D. salinci (Long et al., 1989). Commerciallyprepared RNA size markers (BRL) were used to estimatethe size of the hybridization signals, cab transcript abun-dance was quantified by densitometric analysis (LKB Ul-troscan XL) of RNA slot blots. Data were normalized byprobing a second, identical set of slot blots with a 3.6-kbDNA probe of the 28S rRNA gene from Chlamydomonasreinhardtii (Duke Chlamydomonas Center stock No. P-92).

Measurement of PSII Excitation Pressure andPhotochemical Efficiency

PSII excitation pressure measurements were made usinga pulse amplitude-modulated fluorescence system (HeinzWalz, Effletrich, Germany). Cell samples were centrifugedrapidly and resuspended in the growth medium to a finalChl concentration of 12 to 20 ^g mL ' and immediatelyexposed to their respective growth temperature andgrowth irradiance in a water-jacketed suspension cuvette(KS 101, Heinz-Walz) equipped with a magnetic stirrer(MKS 101, Heinz-Walz). After the attainment of steady-

state fluorescence under the respective temperature andirradiance regimes, PSII excitation pressure was calculatedas 1 - qP (Bradbury and Baker, 1981; van Kooten and Snel,1990). qP was measured using a flash lamp (KL 1500,Heinz-Walz) that provided saturating flashes (7000m~2 s"1) of 1 s duration. Maximal photochemical efficiencywas determined as the ratio of FV/FM after dark adaptationfor 15 min at room temperature (van Kooten and Snel,1990).

RESULTS

Effect of Growth Regime on Chl Content, Chl a/b Ratio,and PSII Excitation Pressure

PSII excitation pressure is synonymous with the redoxstate of QA, which is equal to (QA)redUced/(QA)reduced +(QA)oxidized- It is estimated in vivo by steady-state Chl afluorescence as 1 — qP (Demmig-Adams et al., 1990). Wedemonstrated previously that PSII excitation pressure canbe increased to a similar extent either by lowering thetemperature at constant irradiance or by increasing theirradiance at constant temperature (Maxwell et al., 1995).The PSII excitation pressure experienced during growthresults in a concomitant adjustment of the Chl a/b ratio andLHCII polypeptide content in C. vulgaris (Maxwell et al.,1995). To determine irradiance and temperature effects onPSII excitation pressure in D. salina, cells were grown un-der conditions such that the Chl/cell and the ratio of Chla/b of cells grown at low temperature (13°C) matched thatof cells grown at high temperature (30°C) under either high(2500 /xmol m~2 s'1) or moderate irradiance (150 ;u,molm~2 s"1) (Fig. 1). To match the Chl content (0.29 pg/cell)and the Chl a/b ratio (approximately 13) for cells grown at30°C/2500 jxmol m"2 s"1 (30/2500), 13°C cells were grown

Figure 1. The effects of growth regime on the appearance of D.salina cultures. Cells were grown at either 30°C/150 yxmol m~2 s"1

(A), 13°C/20 jiimol rrT30°C/2500 ju,mol

(B), 13°C/150 jLtmol m~2 s~' (C), or www.plantphysiol.orgon May 18, 2020 - Published by Downloaded from

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790 Maxwell et al. Plant Physiol. Vol. 109, 1995

at a 17-fold lower irradiance (150 /xmol m~2 s-1) (13/150)(Fig. 1; Table I). Furthermore, the Chl content and the Chla/b ratio of D. salina grown at 13°C and 20 /xmol m~2 s"1

(13/20) were matched by growing cells at 30°C but at an8-fold higher irradiance (150 /xmol m~2 s"1) (30/150) (Fig.1; Table I). Cells with a high ratio of Chl a/b also exhibiteda 2-fold higher ratio of protein/Chl than those cells grownunder conditions that elicited a low Chl a/b ratio (Table I).These results for D. salina are consistent with those for C.vulgaris cultured under similar growth conditions (Max-well et al., 1994, 1995).

D. salina grown at either 13/20 or 30/150 exhibited com-parable PSII excitation pressures, as indicated by the sim-ilar values for 1 - c\P. In addition, both cultures exhibitedsimilar PSII photochemical efficiencies (FV/FM) (Table I). Incontrast, PSII excitation pressures for cells grown at either13/150 or 30/2500 were 3- to 7-fold higher, respectively,than those grown at either 13/20 or 30/150 (Table I). How-ever, the values of 1 — qP for cells grown at 13/150 wereapproximately 50% lower, whereas the FV/FM was 16%higher than for cells grown at 30/2500 (Table I). Similardifferences in excitation pressures and photochemical effi-ciencies also have been observed for C. vulgaris grown at5/150 versus 27/2200, which, in part, can be explained bythe differences in the feedback limitations imposed onphotosynthetic carbon metabolism by growth at low tem-perature and moderate irradiance (13/150) versus growthat high temperatures and saturating irradiance (30/2500)(L.V. Savitch, D.P. Maxwell, and N.P.A. Huner, unpub-lished results).

Effect of PSII Excitation Pressure on LHCII Apoprotein andcab mRNA Abundance

Figure 2A illustrates a Coomassie blue-stained SDS-poly-acrylamide gel of membrane polypeptides isolated from D.salina grown under either high or low excitation pressures.The membrane polypeptide complements of cells grown ateither 30/150 or 13/20 (Fig. 2A, lanes 1 and 2) appeared tobe comparable with respect to levels of LHCII. Further-more, the membrane polypeptide complements of D. salinagrown at either 13/150 or 30/2500 (Fig. 2A, lanes 3 and 4)also were comparable but contained lower levels of LHCIIthan that present in cells grown under low values of 1 — qP(Fig. 2A, lanes 1 and 2). These results were confirmed byimmunoblotting using a monospecific antibody for LHCII(Fig. 2B).

31.0 —

21.5 —

} LHCII

B 1 2 3 4

C 1 2 3 4

3.2 —

1.3 —

Figure 2. The effects of the growth regime on LHCII apoprotein andcab mRNA abundance in D. salina cells. A, Coomassie blue-stainedSDS-PACE gel. Molecular mass markers (kD) are indicated on theleft. The LHCII region is indicated. B, Western blot showing theLHCII region only. C, RNA gel-blot hybridization analysis. Total RNAwas denatured, electrophoresed, transferred to reinforced nitrocellu-lose, and hybridized to the cDNA cab gene probe ZLcab43. The sizeestimate of the transcripts (kb) was derived from RNA size markers.Lanes 1, Cells grown at 30°C/150 /irnol m~2 s~'; lanes 2, cells grownat 13°C/20 jumol m ~2 s ~\- lanes 3, cells grown at 13°C/150m~2 s~'; and lanes 4, cells grown at 30°C/2500 /imol m~2 s~

Table I. The effect of growth regime on the physiological characteristics of D. salina cellsAll data represent the average ± so of four experiments.

Parameter

Chl/cella

Chl a/bProtein/Chlb

1 - qP

a Calculated as

30/1 50

1.96 ±4.45 ±6.06 ±0.10 ±0.72 ±

pg Chl/cell.

0.080.180.250.010.01

b

2.4.6.0.0.

Growth Regime (°i

13/20

.11 ±0.12

.52 ± 0.13

.54 ± 0.28

.09 ± 0.01

.73 ± 0.01

C/fim'ol m 2s

1 3/1 50

')

30/2500

0.24 ± 0.0213.65 ± 0.2213.54 ± 0.770.30 ± 0.020.63 ± 0.03

0131200

.29

.11

.25

.69

.51

± 0.02±0.31± 0.95± 0.02± 0.04

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RNA gel blot hybridization experiments, using a hybrid-ization probe derived from a D. salina LHCII cab gene(Long et al., 1989), were undertaken to examine the relativeabundance of cab mRNA in cells grown under the differenttemperature and light regimes (Fig. 2C). The level of cabmRNA was highest for cells grown at either 30/150 or13/20 (Fig. 2C, lanes 1 and 2) and lowest in cells grown ateither 13/150 or 30/2500 (Fig. 2C, lanes 3 and 4). Further-more, the cab mRNA levels for each growth condition areconsistent with the data presented for LHCII apoproteinabundance. Thus, we hypothesized that the changes in theChl/cell, Chl a/b ratio and the protein/Chl ratio, and LHCIIapoprotein and cab mRNA abundance in D. salina are notinduced by either growth temperature or growth irradi-ance per se but, rather, are a response to changes in theredox state of QA (Table I).

Effects of Thermodynamic Relaxation of PSII ExcitationPressure on LHCII Apoprotein and cab mRNA Abundance

If the redox state of QA regulates LHCII apoprotein andcab mRNA abundance, we hypothesized that cells grown at13/150 and subsequently released from high PSII excita-tion pressure by an increase in temperature at constantirradiance should exhibit a decrease in the Chl a/b ratio, adecrease in 1 - qP, an increase in cab mRNA levels, and aconcomitant increase in LHCII apoprotein abundance. Asillustrated in Figure 3, inset, a shift from 13/150 to 30/150caused PSII excitation pressure to decrease at a rate of 0.075unit h"1. After 12 h (Fig. 3), 1 - qP was similar to valuesexhibited by cells grown under constant conditions of 30 /150 (Table I). Additionally, there was a positive, linearcorrelation between Chl a/b and PSII excitation pressure(Fig. 3). Furthermore, both LHCII abundance and cabmRNA levels increased with time as 1 — qP decreasedbecause of continued exposure to 30/150 (Fig. 4, A and B).

i 2

15

12

_cQ.Oo 6

_cO

0.0 0.1 0.2 0.3

Figure 3. Correlation between the Chl a/b ratio and PSII excitationpressure (1 - qPt in D. salina. At time 0, cells grown at 13°C/150/xmol m~2 s~' were shifted to 30°C/150 /nmol m"2 s ~\ and Chl a/band 1 - qPwere measured as a function of time for 12 h. The insetillustrates the kinetics for the decrease in 1 - qP.

3.2 —

1.3 — ••••HI

Figure 4. The effect of a thermodynamic relaxation of PSII excitationpressure on LHCII apoprotein levels (A), cab mRNA abundance (B),and the Chl a/b ratio (C). At time 0, D. salina cells grown at 13°C/150/xmol m ~2 s ~' were shifted to 30°C/150 /xmol rrT2 s"', and sampleswere collected during a period of 12 h. Lanes 1, Time 0; lanes 2, 15min; lanes 3, 30 min; lanes 4, 1 h; lanes 5, 2 h; lanes 6, 4 h; lanes 7,8 h; lanes 8, 12 h; and lanes 9, 12 h in the dark. A, Western blotanalysis of the LHCII region only. LHCII apoprotein levels werequantified as described in "Materials and Methods." B, RNA gel-blothybridization analysis as described above for Figure 1C. Cab mRNAtranscript levels were quantified by RNA slot-blot hybridization anal-ysis as described in "Materials and Methods." C, Kinetics for thedecrease in Chl a/b and the concomitant increase in LHCII apopro-tein and cab mRNA levels. D, cab mRNA accumulation in the light;•, cab mRNA accumulation in the dark; O, LHCII apoprotein accu-mulation in the light; •, LHCII apoprotein accumulation in the dark;V, Chl a/b in the light; T, Chl a/b in the dark.

The kinetic response for cab mRNA accumulation wasquantified by RNA slot-blot analyses. Using a 3.6-kb DNAprobe of the 28S rRNA gene from C. reinhardtii, we showedthat the level of mRNA for the 28S rRNA in D. salina didnot change as a function of excitation pressure (data notshown). Thus, we used this 3.6-kb DNA probe of the 28SrRNA as an internal standard to normalize the levels of cabmRNA transcripts. As illustrated in Figure 4C, cab mRNA(D) and LHCII polypeptide levels (O) increased at a rate ofabout 0.6-fold rT1 and 0.4-fold h"1, respectively. This re-sulted in a 6- and 8-fold increase in LHCII apoprotein andcab mRNA abundance after 12 h. These rates of increase forcab mRNA and LHCII apoprotein accumulation are signif- www.plantphysiol.orgon May 18, 2020 - Published by Downloaded from




icantly faster than that reported by LaRoche et al. (1991) forD. tertiolecta. As expected, there was a concomitant de-crease in the ratio of Chl a/b (Fig. 4C, V).

Effects of Photodynamic Relaxation of PSII ExcitationPressure on LHCII Apoprotein and cab mRNA Abundance

Our results indicate that cab gene expression is inverselyrelated to 1 - qP, and thus, high levels of cab mRNA shouldbe observed when PSII excitation pressure is minimal. Inthe dark, 1 - qP is zero and hence cab gene expressionshould increase when cells exhibiting high PSII excitationpressure (13/150) are shifted to the dark at either 30 or13°C. As predicted, LHCII apoprotein abundance (Fig. 4C,•) and cab mRNA levels (Fig. 4C, •) exhibited a 4-foldincrease when cells were shifted from 13/150 to the dark at30°C. Furthermore, the Chl a/b ratio decreased from about13 to 4.5 in cells grown at 30°C for 12 h in the dark (Fig. 4C,T). This 12-h dark treatment at 30°C also resulted in a 30%increase in Chl / cell (data not shown). Similarly, cab mRNAincreased 2.4-fold when cells grown at 13/150 were shiftedto 13°C in the dark (Fig. 5) with a concomitant decrease inthe ratio of Chl a/b from about 14 to 8.6 in 24 h. However,as expected, the extent of the increase in cab mRNA andLHCII apoprotein abundance in the dark was lower at 13than at 30°C, because of thermodynamic constraints. Weconclude that neither temperature nor absolute irradiancecan be considered the primary signal for the regulation ofeither cab mRNA abundance, LHCII apoprotein abun-dance, or Chl accumulation in D. salina,

DISCUSSION

Currently, cab mRNA accumulation and LHCII abun-dance are thought to be stimulated by light and regulatedby phytochrome and the blue-light photoreceptor (Tobinand Silverthorne, 1985; Ellis, 1986; Kuhlemeier et al., 1986;Gilmartin et al., 1990; Thompson and White 1991; Harrisonet al., 1992; Bowler and Chua, 1994). However, we suggestthat the data presented in this report, which demonstrate

3.2 —

1.3 — •uFigure 5. The effect of a photodynamic relaxation of PSII excitationpressure on cab mRNA accumulation. At time 0, D. salina cellsgrown at 1 3/150 were shifted to 1 3°C in the dark and samples werecollected during a period of 24 h. RNA gel-blot hybridization anal-ysis was performed as described in Figure 1C. Lane 1, Time 0; lane2, 6 h; lane 3, 12 h; and lane 4, 24 h. The size of the cab transcriptswas estimated using RNA size markers.

modulation of LHCII apoprotein abundance and cabmRNA levels, cannot be rationalized simply as either alight response or a phytochrome response for the followingreasons. First, changes in the Chl a/b ratio were correlateddirectly with changes in PSII excitation pressure (Fig. 2)regardless of whether \ - qP was modulated by changes intemperature at constant irradiance or by changes in irradi-ance at constant temperature. Second, D. salina grown at13°C and an irradiance of 150 jamol m~2 s"1 (Fig. 2C, lane3) exhibited low levels of cab mRNA similar to those of cellsgrown at 30°C with a 17-fold increase in irradiance (2500/xmol m~2 s"1) (Fig. 2C, lane 4). Similarly, cells grown at13°C and an irradiance of 20 /xmol irT2 s"1 (Fig. 2C, lane 2)exhibited levels of cab mRNA comparable to those of cellsgrown at 30°C with an 8-fold increase in irradiance (150jamol m~2 s"1) (Fig. 2C, lane 1). Third, D. salina was cul-tured under a continuous irradiance, which would mini-mize any possible phytochrome effect. Fourth, cells ini-tially grown at 13/150 and then immediately shifted to30/150 exhibited an 8-fold increase in cab mRNA after 12 h(Fig. 4C). Irradiance alone cannot account for this increasein LHCII apoprotein and cab mRNA accumulation. Finally,cells initially grown at 13/150 and then immediatelyshifted to complete darkness at either 30°C (Fig. 4C) or13°C (Fig. 5) exhibited a 4- or 2.4-fold increase in cab mRNAlevels, respectively. Clearly, significant cab mRNA accumu-lation can occur even in the total absence of light in cellspre-exposed to growth conditions that result in high PSIIexcitation pressure.

Comparison of D. salina grown at 30/150 (Fig. 2, lanes 1)with cells grown at 13/150 (Fig. 2, lanes 3) may lead one toconclude that the decreased levels of LHCII polypeptidesand cab mRNA reflect a low growth temperature response.However, if this is due solely to growth temperature, thena reduction of growth irradiance at 13°C should not affecteither LHCII or cab mRNA abundance. On the contrary, areduction in growth irradiance from 150 (Fig. 2, lanes 3) to20 jamol m~2 s-1 (Fig. 2, lanes 2) at 13°C resulted in asignificant increase in both LHCII polypeptides and cabmRNA to levels comparable to those of cells grown at30/150 (Fig. 2, lanes 1). These results may be explained bylow temperature enhancement of mRNA stability. How-ever, the differential accumulation of cab mRNA exhibitedin Figure 2C cannot be due to enhanced stability of mRNAat low temperature, since D. salina grown at 13/150 (Fig. 2,lanes 3) exhibited low levels of cab mRNA compared tocells grown at 13/20 (Fig. 2, lanes 2). Thus, growth tem-perature alone cannot account for the changes in LHCIIapoprotein and cab mRNA abundance.

RNA gel-blot hybridization experiments using a D. salinacab gene as a radiolabeled hybridization probe detected anabundant 1.3-kb mRNA transcript. This is consistent withprevious studies (Long et al., 1989). Lower exposures of theautoradiogram indicate that this 1.3-kb mRNA signal mayconstitute several transcripts of similar size. We also de-tected a less abundant 3.2-kb transcript. Although this hasnot been reported previously in D. salina (Long et al., 1989),a 3.1-kb, low-abundance cab mRNA transcript has beenreported in D. tertiolecta (LaRoche et al., 1990). Since mul- www.plantphysiol.orgon May 18, 2020 - Published by Downloaded from




tiple cab genes (Long et al., 1989) and LHCII apoproteins (Fig. 2B) have been detected in D. salina, the 3.2-kb cab mRNA transcript may encode a different cab gene than the 1.3-kb transcript. Alternatively, the 3.2-kb transcript may represent the unprocessed form of the 1.3-kb transcript. Although the 1.3-kb transcript was 11 times more abundant than the 3.2-kb transcript in cells grown at 13/150, the ratio of the 1.3-kb/3.2-kb transcript decreased to 1.4 when cells were shifted from 13/150 to 13°C in the dark (Fig. 5). A similar change in this ratio did not occur when cells were shifted from 13/150 to 30°C in the dark (Fig. 4, lane 9). Although the nature of this larger transcript remains un- known, it appears to be much more abundant in cells exposed to low temperature in the absence of light.

It has been established that the chloroplast import of the LHCII apoproteins and the subsequent assembly of the LHCII pigment-protein complex are light-driven, energy- dependent processes (Keegstra et al., 1989; Dreyfuss and Thornber, 1994; Leheny and Theg, 1994). Nigericin-induced inhibition of light-dependent protein import is overcome by the addition of ATP (Cline et al., 1985). The results presented in Figures 4 and 5 illustrate that LHCII apoprotein accumulates in thylakoid membranes of D. salina in the dark at either 30 or 13°C in cells that have been pre- exposed to high excitation pressure. Clearly, light per se is not essential either for the import of LHCII apoproteins or for the subsequent assembly of the LHCII pigment-protein complex. Alternative energy sources or the accumulation of excess ATP during exposure to a high redox poise may account for the ability of cells to maintain the capacity for protein import and LHCII assembly in the dark. We suggest that this may represent a nove1 approach to assaying protein import into chloroplasts and LHCII assembly in vivo.

The redox state of QA as estimated by PSII excitation pressure (1 - qP) reflects the balance between the rate of reduction of PSII reaction centers through light absorption and the rate of oxidation of PSII reaction centers through intersystem electron transport. Thus, the redox state of QA reflects the redox poise of intersystem electron transport. We conclude that the changes in the Chl content per cell, the ratio of Chl alb, and LHCII and cab mRNA abundance in response to different temperature/ irradiance growth regimes are regulated by the redox poise of intersystem electron transport as reflected by changes in PSII excitation pressure. This occurs as a consequence of the differential rates of energy absorption through photochemistry versus the thermal sensitive rates of energy utilization through intersystem electron transport and carbon metabolism. The modulation of PSII excitation pressure reflects limitations imposed on photosynthesis as a consequence of changes in temperature or irradiance, which, through feedback mechanisms, alter the redox poise of photosynthetic intersystem electron transport. This may signal alterations in chloroplast (Allen, 1993, 1995) as well as nuclear gene expression, which ultimately affect the structure and function of the photosynthetic apparatus to balance energy absorption and energy utilization. The cyanobacterium Plectonema borya- n u m (S. Falk, D.P. Maxwell, N.P.A. Huner, and D.E. Lau-

denbach, unpublished results), as well as monocotyledon- ous and dicotyledonous plants, also responds photo- synthetically and developmentally to PSII excitation pressure (G.R. Gray and N.P.A. Huner, unpublished results). Since PSII excitation pressure and, thus, the redox poise of intersystem electron transport can be modulated by changing temperature and / or irradiance, the concept of gene regulation by the redox poise of intersystem electron transport may represent a mechanism by which photosynthetic organisms sense changes in ambient temperature as well as irradiance. Furthermore, any environmental stress includ- ing nutrient and water availability potentially can affect the balance between energy absorption and energy utilization. Thus, the redox poise of photosynthetic intersystem electron transport may represent a general redox sensing I signaling mechanism in photosynthetic organisms. This is consistent with recent models describing cab gene regulation upon exposure to high-irradiance stress (Melis et al., 1985; Fujita et al., 1989; Laroche et al., 1990; Escoubas et al., 1995). The data presented in this report indicate that the chloroplast must signal the nucleus to regulate the family of cab genes present in D. salina. Although this has been suggested previously (Melis et al., 1985; Taylor, 1989; La- Roche et al., 1991), the signal transduction pathway has yet to be elucidated. However, a recent report by Escoubas et al. (1995) suggests that the redox state of the plastoquinone pool may be an important intermediate of this signal transduction pathway, whereas Pearson et al. (1993) suggested that the Cyt f/ b, complex may represent the site of redox control. Clearly, further work is required to elucidate the redox signal transduction pathway that links the chloroplast and nucleus.

1

ACKNOWLEDCMENTS

The authors are grateful to N. Nelson for providing the cDNA clone ZLcab43, to A. Grossman for providing the C. reinhardtii rRNA gene probe, to A. Melis for providing a culture of D. salina, and to P. Falkowski for providing access to a manuscript prior to publication.

Received May 4, 1995; accepted July 27, 1995. Copyright Clearance Center: 0032-0889/ 95/ 109/0787/09.

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