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First published online October 29, 2004; 10.1104/pp.104.038919 Plant Physiology 136:3737-3750 (2004) © 2004 American Society of Plant Biologists Plastid Regulation of Lhcb1 Transcription in the Chlorophyte Alga Dunaliella tertiolecta1Environmental Biophysics and Molecular Ecology Program, Institute of Marine and Coastal Sciences, New Brunswick, New Jersey 08901–8521(Y-B.C., M.K., P.G.F.); and Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada E3B 6E1 (D.G.D.)
We identify four novel DNA-binding complexes in the nuclear-encoded Lhcb1 promoter of the chlorophyte alga Dunaliella tertiolecta that are regulated by photosynthetic pathways in the plastid. The binding activities of three of the complexes were positively correlated with time-dependent changes in Lhcb1 transcript abundance, implicating their roles as transcriptional enhancers in a retrograde signal transduction pathway. Using a combination of inhibitors, uncouplers, and antimycin A, and by following the kinetic pattern of gene regulation, we infer two different sensors in the signal transduction pathway. On short time scales of 0.5 to about 4 h, the transthylakoid membrane potential appears to be a critical determinant of gene expression, whereas on time scales of 8 h or longer, the redox state of the plastoquinone pool becomes increasingly more important. The differentiation of these two types of signals was observed in parallel effects on gene transcription and on the patterns of DNA-binding activities in the Lhcb1 promoter. These signals appear to be transduced at the nuclear level via a coordinated ensemble of DNA-binding complexes located between –367 and –188 bp from the start codon of the gene. The regulation of these elements allows the cell to up- or down-regulate the expression on Lhcb1 in response to changes in irradiance.
All photosynthetic organisms can adjust the rate of photon absorption to optimize photosynthetic electron transport (PET) in relation to changes in spectral irradiance. Long and persistent shifts in photon flux density (PFD) often involve changes in gene expression that modify the activity, concentration, and structure of the photosynthetic machinery (Falkowski and LaRoche, 1991  ; Anderson et al., 1995; Durnford and Falkowski, 1997). In comparison with higher plants, photoacclimation is far more pronounced in unicellular algae, where not only the magnitude and rates of the responses are extraordinary, but also the response can easily be distinguished from developmental processes (Falkowski and Chen, 2003). In particular, species in the marine unicellular chlorophyte genus Dunaliella can undergo an almost 10-fold change in the abundance of antenna chlorophyll (Chl)-binding proteins on a per cell basis within a single generation (Sukenik et al., 1988). In eukaryotic algae, these changes involve transcription and translation of both nuclear and chloroplast genes. Consequently, tight interorganellar regulatory networks are required to coordinate gene expression in these two compartments. In this paper, we examine how changes in the abundance of the nuclear-encoded Lhcb gene in response to changes in PFD are sensed by the plastid and how the signals are correlated with binding factors in the promoter region.
At least three signaling pathways involved in plastid regulation of nuclear gene expression have been described for plant cells. The first is mediated by tetrapyrrole biosynthesis, the second requires plastid protein synthesis, and the third involves electron carrier redox poise and potentially other photosynthetic signals (for review, see Rodermel, 2001
Among the thylakoid electron carriers, the plastoquinone (PQ) pool is considered to be an ideal candidate for signaling excess or insufficient PSII activity relative to the capacity for carbon fixation (Falkowski et al., 1986
Using a variety of PET and other inhibitors, Escoubas et al. (1995)
The mechanism(s) by which a chloroplast redox signal is relayed to the nucleus remains unknown. Plastid redox signals may be transmitted via one or more plastid factors, the existence of which was postulated decades ago (Bradbeer et al., 1979 In this study, we measured the kinetics of Lhcb1 transcript abundance and DNA-binding activity associated with a 180-bp Lhcb1 promoter region in D. tertiolecta following light transitions, or following the addition of a variety of inhibitors, or combinations of the two types of treatments. Our results reveal, for the first time to our knowledge, that the binding activity of multiple DNA-binding complexes, associated with the Lhcb1 promoter region, are affected by changes in light intensity, redox poise of electron transfer components between PSII and PSI, and the transthylakoid membrane potential. The relationship between the Lhcb1 transcript abundance and the binding activities of these complexes implicate the roles of the latter as transcriptional enhancers in a retrograde signal transduction pathway. The results further suggest that two types of chloroplastic signals, the redox state of PQ pool as potentially sensed by the cytochrome b6f complex and the transthylakoid membrane potential, cooperatively regulate Lhcb1 expression.
DNA-Binding Patterns in the Lhcb1 Promoter Region To dissect the binding properties of the Lhcb1 promoter, we generated a set of six 30-bp long oligo DNA probes that sequentially represent the 180-bp promoter region (from –367 to –188 bp; Fig. 1). Based on electrophoretic mobility shift assay (EMSA) of whole-cell protein extracts, we identified four different binding complexes that we call HLF, LF1, LF2, and LF3 (Fig. 2). The largest complex, HLF, bound to all six probes, while the second largest complex, LF1, bound to all except Oligo 3. One of the two smaller complexes, LF2, bound strongly to Oligo 3 (–307 to –278 bp) and Oligo 5 (–247 to –218 bp), whereas LF3 bound primarily to Oligo 5. These results were confirmed using more than one dozen PCR-generated probes in the same promoter region (data not shown). Binding of all complexes is specific; they can be outcompeted by their respective unlabeled oligo DNA, but not by nonspecific competitors such as sonicated salmon sperm DNA (Fig. 2).
There are significant sequence similarities between two sections in the targeted promoter region: from –367 to –288 bp and from –270 to –227 bp (Fig. 1). We identified three tentative binding sites in these two nonoverlapping promoter regions. The first, a TCTAA box, has a consensus sequence TCTAAHGT and is located between –365 to –357 bp (within the Oligo 1 probe) and –266 to –258 bp (within the Oligo 4 probe). In both cases, this site is 12 to 15 bp upstream of two other putative binding sites. The second contains a CACT box with a consensus sequence CARRCACTSGRA and overlaps the third, a GGAA box, which has a core sequence ASMMYYGGAA. Among the three putative binding motifs, only the TCTAA box is also found in the Lhcb1 promoter in C. reinhardtii and in Lhcb2/Lhcb3 promoters in Arabidopsis.
The effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the time course change of Lhcb1 transcript abundance in cells grown under different light conditions is shown in Figure 3. In both high-light (HL)- and low-light (LL)-acclimated cultures, the addition of 200 nM DCMU (which leads to an approximately 50% reduction in PET through PSII) resulted in a rapid decrease of Lhcb1 transcript abundance in the first 2 to 4 h. In HL-acclimated cells, the Lhcb1 transcript levels recovered and increased nearly 2-fold above the previous steady-state level. The pattern of change in Lhcb1 transcript abundance is consistent with that of Chl a/cell (data not shown). Though the addition of DCMU to HL cultures resulted in increased Lhcb1 transcript abundance, the pattern differed both in the magnitude and kinetics compared to the control HL-to-LL shift. In the latter, the relative Lhcb1 transcript levels increased immediately following the light transition, and the level was tripled within the first 4 h, about two times higher than the peak level reached in DCMU-treated cells after 24 h (Fig. 3).
We further tested the ability of DCMU to mimic LL conditions by adding the inhibitor prior to a shift from LL to HL. If the reduction of the PQ pool were the sole signal responsible for the up-regulation of Lhcb1 transcription, the HL-induced decline in Lhcb1 transcript abundance should be attenuated. However, DCMU failed to prevent decreases in Lhcb1 transcript abundance (Fig. 4). These results strongly suggest that the redox state of the PQ pool per se is not the only photosynthetic signal that regulates Lhcb1 expression.
Effects of Uncouplers on Lhcb1 Transcript Abundance The initial decrease in the relative abundance of Lhcb1 following the addition of DCMU raises the possibility that signals other than the PQ pool redox status are involved in the regulation of Lhcb1 transcription. In both HL- and LL-acclimated cultures, Lhcb1 transcript abundance decreased within 1 h following the addition of 400 nM nigericin or 2 µM valinomycin and reached a nadir within 2 h (Fig. 5). Over the next 4 to 6 h, transcript levels rose to approximately 150% of the previous steady-state level. The kinetic patterns of change in Lhcb1 transcript abundance were similar for the two uncouplers.
Effects of Antimycin A on Lhcb1 Transcript Abundance
The results from the uncoupler treatments suggest the transthylakoid membrane potential, i.e. the sum of proton gradient and the electric potential, may be involved in the short-term regulation of Lhcb1 transcription. We further examined this hypothesis by analyzing the effects of antimycin A on Lhcb1 transcript abundance. Antimycin A inhibits cyclic electron transport around PSI (for review, see Bendall and Manasse, 1995
Effects of DBMIB on Lhcb1 Transcript Abundance In both HL- and LL-acclimated cultures, the addition of 1.5 µM 2,5-dibromo-6-methyl-3-isopropyl-1,4-benzochinon (DBMIB) led to declines in Lhcb1 transcript abundance (Fig. 7). The transcripts were virtually undetectable 4 h following the addition of the inhibitor and remained so for the rest of incubation. The pattern of the changes in Lhcb1 transcript abundance was consistent with that of Chl a/cell (data not shown).
While the short-term changes of Lhcb1 transcript abundance are similar between those induced by DBMIB and a shift from LL to HL, the longer-term changes differ. During the acclimation from LL to HL, Lhcb1 transcript abundance partially recovered after 6 h, reaching a new and lower steady-state level. We further examined the Lhcb1 changes in HL cells transferred to LL with DBMIB added just before the light shift. Kinetic fluorescence measurements with a fast repetition rate (FRR) fluorometer (Kolber et al., 1998 ) indicated that DBMIB induced an immediate and dramatic reduction of the PQ pool, followed by a moderate recovery within 2 h. DBMIB effectively prevented Lhcb1 transcript abundance from increasing due to the reduced irradiance (Fig. 8). These results suggest that the signal from the reduced PQ pool repressed the up-regulation of Lhcb1 transcription that would have otherwise occurred had the shift been done in the absence of the inhibitor.
Effects of Various Treatments on the Redox Status of PQ Pools Transient Chl fluorescence kinetics was measured to assess the oxidation rate of QA– and the redox status of the PQ pool. The changes in the percentages of oxidized PQ to the PQ pool following the additions of DCMU and DBMIB resembled those following light transitions from HL to LL and from LL to HL respectively (Fig. 9). Addition of DCMU increased oxidized PQ by approximately 50% more than that following the HL-to-LL transition. Addition of DBMIB reduced oxidized PQ by approximately 50% less than that by an LL-to-HL transition. Additions of nigericin, valinomycin, and antimycin A increased oxidation of the PQ pool; however, their effects appeared insignificant. Overall, the differences of effects on the redox status of PQ pools by different treatments between the short and the longer term were very small.
Photoacclimation Involves Changes of DNA-Binding Patterns in the Lhcb1 Promoter Within 2 h after an LL-acclimated culture was transferred to the HL condition, Lhcb1 transcript levels decreased more than 10-fold (Fig. 4). The change in transcript abundance was concurrent with substantial decreases of LF1, LF2, and LF3 binding (Fig. 10A). Lhcb1 transcript levels reached the lowest level within 6 h following the shift and then quickly recovered to 40% of the initial level. The rapid recovery of Lhcb1 transcript abundance is concurrent with the dramatic increase of DNA-binding activities of LF1, LF2, and LF3 complexes (Figs. 4 and 10A). Lhcb1 transcript levels reached a new, 40% lower, steady-state level 12 h following the shift. EMSA revealed that after 24 h, the binding activities of all four complexes were much lower than those in LL cells. The strong positive correlation between Lhcb1 levels and the binding activities of LF1, LF2, and LF3 in the Lhcb1 promoter region are also closely coupled in time, suggesting that these binding complexes enhance Lhcb1 transcription. On the other hand, binding activities of LF factors (Fig. 10A) did not increase significantly while Lhcb1 transcript abundance nearly tripled following an HL-to-LL shift (Fig. 3). Instead, the increased Lhcb1 transcript abundance is coupled with a decreasing trend of the HLF-binding activity (Fig. 10A), suggesting a complicated role of HLF as a transcriptional repressor in regulating Lhcb1 transcription.
Effects of Photosynthetic Inhibitors and Uncouplers on DNA-Binding Activities in the Lhcb1 Promoter Region We further investigated the relationship between Lhcb1 transcript abundance and the DNA-binding patterns in the presence of PET inhibitors and uncouplers. The DNA-binding activities of LF1, LF2, and LF3 declined substantially 3 h after the DCMU addition to HL-acclimated cells, followed by dramatic increases 9 h later (Fig. 10B). The Lhcb1 transcript abundance and the binding activities of LF1 and LF2 in the Lhcb1 promoter region are not only positively related but also closely coupled in time, once again suggesting that LF factors may enhance Lhcb1 transcription. The changes of binding activities of LF2 and LF3 following the addition of nigericin or valinomycin to HL cultures were similar but much smaller than those induced by DCMU (data not shown). While there were few changes in both the overall binding pattern and activities of individual complexes in cells treated by antimycin A alone, the binding activities of HLF, LF1, LF2, and LF3 were substantially enhanced in cells treated by both antimycin A and DCMU (Fig. 10C). The addition of 1.5 µM DBMIB to the LL culture led to an overall steady decrease of the binding activities of all complexes throughout the incubation, with HLF and LF1 activities virtually eliminated in the end, except that the binding of LF1 and LF2 increased at the 9-h time point (Fig. 10B). The overall patterns of changes in DNA-binding activities of all complexes are similar between those induced by the LL-to-HL shift and those by the DBMIB, yet their corresponding longer-term changes in Lhcb1 transcript abundance differ (Fig. 7).
Phosphatases, including calf intestine alkaline phosphatase, The effects of the thiol-oxidizing agent, azodicarboxylic acid bis-dimethylamide (diamide), and the thiol-modifying agent, N-ethyl maleimide (NEM), on the activities of the four DNA-binding complexes are shown in Figure 11. In vitro binding activities of HLF, LF1, LF2, and LF3 were all eliminated when whole-cell protein extracts were incubated with NEM before the addition of the oligo probes (Fig. 11). This was accompanied by the appearance of a new DNA-binding complex that migrated between LF1 and LF2. When NEM was added after the binding reaction was completed, however, the binding activity of HLF was enhanced while that of LF1 was reduced. Adding NEM after the completion of the binding reaction also resulted in less inhibition on the activities of other binding complexes than adding it prior to the binding reaction. The differences in sequence in which NEM was added affected DNA-binding activities, suggesting that the thiol group(s) in the complexes are not only directly involved in the binding in vitro, but can also enhance and reduce binding affinity of the HLF complex. While NEM dramatically inhibits in vitro binding activities of LF1, LF2, and LF3, diamide exhibits much less of an effect, especially if it is added after the binding reactions were completed (Fig. 11). However, diamide and NEM had a similar effect on HLF-binding activity. Taken together, the results of thiol group-modifying reagents treatment suggest that the assembly and binding of transcription factors to Lhcb1 promoter is thiol group dependent.
Chloroplastic Signals Affecting DNA-Binding Activities in the Lhcb1 Promoter Region
Four DNA-binding complexes were detected when the Lhcb1 promoter fragments were incubated with whole-cell protein extracts isolated from D. tertiolecta. The binding activity of the HLF complex is generally stronger in HL cells than in LL cells based on the experimental results conducted on exactly the same culture as the one used by Escoubas et al. (1995)
Our EMSA results indicate that larger binding complexes, such as HLF and LF1, were formed when the same protein sample was incubated with the nonoverlapping promoter fragments in the 180-bp region (Fig. 2). Escoubas et al. (1995)
Light-mediated transcription of Lhc genes in higher plants is regulated by the binding of regulatory proteins to upstream promoter regions (Argüello-Astorga and Herrera-Estrella, 1998
The nuclear-localized Lhcb genes are tightly regulated by light intensity in both algae and higher plants. Escoubas et al. (1995) On longer time scales however, the redox state of the PQ pool clearly regulates the transcription of Lhcb1 gene. DBMIB always results in both short-term and longer-term decreases in Lhcb1 transcript levels in both HL and LL culture (Fig. 7) and prevented increases in Lhcb1 transcription during HL-to-LL transitions (Fig. 8). Transient fluorescence measurements indicated that in all DBMIB treatments, the PQ pool was rapidly reduced within the first 1 to 2 h before some recovery (Fig. 9), suggesting that the reduced PQ pool and/or cytochrome b6f are among the chloroplastic signals responsible for down-regulation of Lhcb1 transcription. DCMU, which always led to an oxidized PQ pool, induced increases in both Lhcb1 transcript levels and Chl synthesis 6 to 8 h following the addition of the inhibitor (Fig. 3). Although the short-term decline in Lhcb1 transcript abundance immediately following DCMU addition could be mediated by a different chloroplastic signal and/or changes in transcript stability, the longer-term DCMU effects on Lhcb1 transcription and Chl synthesis phenotypically resemble those by an HL-to-LL shift. These results suggest that a signal initiated from the oxidized PQ pool regulates longer-term photoacclimative responses.
There is, however, strong evidence that the redox state of the PQ pool is not the only photosynthetic signal that regulates Lhcb1 transcription, especially in the short term. Despite the fact that DCMU additions always resulted in a rapidly oxidized PQ pool (Fig. 9), both Lhcb1 transcript levels (Fig. 3) and Chl synthesis decreased substantially within the first 4 to 6 h (data not shown). Furthermore, when added to an LL culture prior to a shift to HL, DCMU failed to prevent decreases in both Lhcb1 transcript level (Fig. 4) and Chl synthesis, although the inhibitor prevented the PQ pool from being reduced by the elevated light intensity. DCMU's inability to prevent Lhcb genes from being down-regulated by HL was also reported in C. reinhardtii (Teramoto et al., 2002
Like DCMU, addition of nigericin and valinomycin to LL and HL cultures resulted in sizable decreases in Lhcb1 transcript levels within the first 2 h, which then recovered and climbed to a new steady-state level at about 50% higher than the previous level (Fig. 5). Since nigericin uncouples photophosphorylation by dissipating
The role of ATP and/or
While the combined effects of DCMU and antimycin A resemble those by DBMIB, there is one major difference between the two scenarios: the PQ pool and cytochrome b6f in DBMIB-treated cells are much more reduced than those in cells treated by both DCMU and antimycin A (Fig. 9). Their opposite effects on Lhcb1 transcription clearly point out the importance of the redox status of the PQ pool as a chloroplastic signal, as well as the notion that different signals regulate nuclear Lhcb1 transcription in a cooperative fashion. However, the effect of DCMU plus antimycin A largely resembles the acclimation to LL in terms of the simultaneous reduction of
Our EMSA results revealed that the binding affinity of all four complexes clearly involves thiol group modification in vitro (Fig. 11), a phenomenon that has been reported for several transcription factors in higher plants and animal cells (for review, see Sun and Oberley, 1996
Although the lack of a transformation system limits our ability to modify the promoter region of Lhcb1 in D. tertiolecta, our results reveal, for the first time to our knowledge, three complexes that bind to the Lhcb1 promoter are closely coupled to the chloroplastic signals and are correlated with enhanced transcription of the gene in vivo. Sequence analysis of the targeted Lhcb1 promoter region revealed multiple and repetitive sequences as possible binding sites for these DNA-binding complexes. Results from our time course study of Lhcb1 transcription affected by light transitions and various photosynthesis inhibitors indicate the nuclear Lhcb1 gene expression may be cooperatively regulated by multiple chloroplastic signals. Specifically, our results suggest that the short-term expression of Lhcb1 is primarily regulated by transthylakoid membrane potential signals triggered by  pH/ATP pool, while the longer-term expression is primarily regulated by the redox signals initiated from the PQ pool.
Materials
Restriction, modifying, and amplifying enzymes were purchased mainly from Promega (Madison, WI) and used according to vendor's instructions. Chemicals for making medium and various buffer solutions and reagents were purchased from Fisher Scientific (Hampton, NH) and Sigma (St. Louis). [
Dunaliella tertiolecta (clone DUN, Provasoli-Guillard Culture Collection, West Boothbay Harbor, ME) was grown in 20-L-size batch culture using filtered off-shore North Atlantic seawater amended with f/2 nutrients (Guillard and Ryther, 1962
At each sampling point, 90 to 150 mL culture (3–10 x 107 cells) was harvested by centrifugation. The cells were immediately lysed in an RNA extraction buffer (1.2% [w/v] SDS, 30 mM EDTA, 50 mM Tris-HCl, pH 8.0, 220 mM NaCl, and 50 mM
Northern-blot analysis was performed to determine the time course abundance of Lhcb1 (previously known as cab1; LaRoche et al., 1991
Culture harvest and protein extraction were conducted essentially as previously described (Escoubas et al., 1995
Our entire study of DNA-binding activities in D. tertiolecta Lhcb1 promoter was centered on a 180 bp region (–367 to –188 bp). Six 30-bp long double-stranded oligo DNA (Oligo 1–6) were constructed by annealing six pairs of commercially synthesized single-stranded oligos (Integrated DNA Technologies), which sequentially represent both strands of the 180-bp upstream region of Lhcb1. These oligo DNA were isolated via agarose gel electrophoresis and subsequently purified using Centrex MF-0.4 micro-centrifuge filters (Schleicher & Schuell) and Microcon-10 devices (Millipore, Bedford, MA). The purified oligo DNA were quantified and used as either labeled probes or unlabeled competitors in EMSA. The locations and sizes of the oligo DNA are presented in Figure 1.
EMSA was used to study DNA-binding activities in the Lhcb1 upstream region of D. tertiolecta cultures grown under different conditions. In all EMSA, the labeled oligo DNA probes were added at over-saturating level so that the DNA protein-binding activities were limited only by the amount of the DNA-binding proteins presented in the binding mixtures. The binding reaction and the EMSA were performed based on previously described protocols with minor modifications (Escoubas et al., 1995
The light transition studies were conducted to examine the changes of DNA-binding activities in the Lhcb1 upstream region in responding to the change of light intensities. Two one-step transition experiments from LL (70–100 µmol quanta m–2 s–1) to HL (1,200–1,400 µmol quanta m–2 s–1) and from HL-to-LL transitions were carried out. For each transition experiment, 20-L batch culture of D. tertiolecta was grown and acclimated to the initial light intensity. At the beginning of the experiment, the culture was equally divided into two 10-L subcultures, with one remaining under the initial light conditions while the other was transferred to a different light condition. The incubation lasted for 8 h following the light shift. Samples for Chl, cell density, and RNA analysis were taken at the mid and end point of the incubation. The biomass from each subculture was harvested for protein extraction at the end of the experiment.
Results from preliminary experiments based on measurements of variable Chl fluorescence, Chl, and cell density were used to choose the proper concentrations for each inhibitor, ensuring their effective inhibition on photosynthesis while still allowing cells to grow. Stock solutions of photosynthetic electron transfer inhibitors DCMU and DBMIB were made in dimethyl sulfoxide. Stock solutions of photophosphorylation uncouplers nigericin and valinomycin were made in ethanol, as was the PSI cyclic electron transfer inhibitor antimycin A. Based on the dose response analyses, we used the following concentration for each inhibitor: DCMU, 200 nM; DBMIB, 1 µM initial with repletion of 250 nM every 4 to 5 h to remedy the labile nature of DBMIB; nigericin, 400 nM; valinomycin, 2 µM; and antimycin A, 1 µM. At the zero time of each inhibitor experiment, batch cultures of D. tertiolecta acclimated to HL or LL conditions were divided into the treatment subculture that receives the inhibitor and the control subculture that receives the solvent (in which the inhibitors stock solutions were made) with the level identical to what was added to the treatment subculture. Variable fluorescence of each subculture was monitored frequently throughout the entire incubation using a pulse amplitude-modulated fluorometer to ensure the effectiveness of the inhibitors on photosynthetic electron transfer. Samples for Chl, cell density, RNA, and protein were taken according to predetermined time intervals. For the combination treatments involving both light transitions and inhibitor addition, the inhibitors were added to the cultures just prior to the light shift.
The photosynthetic performance and redox status of the PQ pool were assessed using the FRR fluorometer (Kolber et al., 1998
We are very grateful to Ms. Mary Ann Tran, Mr. Carlos Gonzales, and Ms. Katerina Zrotalova for participation in the experiments. We thank Drs. Mario Giordano, Yoram Gerchman, and Yi Sun for helpful discussions. We are indebted to Mr. K. Wyman for technical help, to Dr. Dan Tchernov for graphics preparation, and to Ms. Emmeline Romana and Ivy Jones for administrative support. Received January 10, 2004; returned for revision June 28, 2004; accepted August 3, 2004.
1 This work was supported by the U.S. Department of Energy (contract no. DEAC02–76CH00016). 
2 Present address: Institute of Microbiology, Laboratory of Photosynthesis, Opatovicky mlyn, 379 81 Trebon, Czech Republic. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.038919. * Corresponding author; e-mail falko{at}imcs.rutgers.edu; fax 732–932–4083.
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TOP
ABSTRACT
RESULTS
-protein phosphatase, and protein Tyr phosphatase, did not significantly affect in vitro binding activities of any of the complexes (data not shown). 
, and ATP production, triggering signals to rapidly down-regulate Lhcb1 transcription. While such negative signals may overwrite the up-regulation redox signals from the oxidized PQ pool, the balance is reversed in the longer term as the redox state of the latter electron carrier becomes dominant. 
-32P]dATP and [
-32P]ATP (3,000 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston) and AP Biotech (Piscataway, NJ). Primers and oligo DNA were synthesized and purified by Integrated DNA Technologies (Coralville, IA). 
-mercaptoethanol). The subsequent RNA processing was carried out following commonly used protocols with minor modification (Sambrook et al., 1989
-amino-n-caproic acid, 1 mM benzamidine chloride, 100 µM sodium metabisulfite, 1 mg/mL aporotinin, and 1x General Protease Inhibitor Cocktails by Sigma) was added to both extraction and storage buffers. 
470nm (functional cross section of photosynthetic units) were determined as described previously (Kolber et al., 1998
, where Fs is the steady-state fluorescence intensity and 
is the maximum fluorescence in the light. 
