I. Redox regulation of photosynthesis via analysis of Synechocystis mutants

Our future research on global transcriptional regulation will be divided into 3 categories: i. alteration of environmental conditions; ii. analysis of regulatory mutants; and iii. analysis of membrane transport mutants. The environmental conditions include iron-deficiency, nitrogen deficiency and differences in redox states within the cell. To this end, we have begun producing mutants in genes that code for proteins involved in the production of cyanophycin, an unusual nitrogen-storage (a non-ribosomally synthesized polypeptide, poly arg-asp) that represents a key form of stored nitrogen in certain cyanobacteria.

We are also interested in identifying genes that are involved with redox control of photosynthesis and pigment-related genes. We have identified 2 genes, rppA (slr0797) and rppB (slr0798), which represent a two-component regulatory system that control the synthesis and stoichiometry of PSII and PSI genes, in addition to photopigment-related genes. rppA (regulator of photosynthesis and photopigment-related gene expression) and rppB exhibit strong sequence similarity to prokaryotic response regulators and histidine kinases, respectively. In wild type, the steady-state mRNA levels of PSII reaction center genes increased when the plastoquinone (PQ) pool was oxidized and decreased when the PQ pool was reduced, whereas transcription of the PSI reaction center genes were affected in an opposite fashion. Such results suggested that the redox poise of the PQ pool is critical for regulation of the photosystem reaction center genes. In ΔrppA, an insertion mutation of rppA, the PSII gene transcripts were highly upregulated relative to the wild type under all redox conditions, whereas transcription of phycobilisome-related genes and PSI genes was decreased. The higher transcription of the psbA gene in ΔrppA was manifest by higher translation of the D1 protein and a concomitant increase in O2 evolution. The results demonstrated that RppA helps regulate the stoichiometry of PSI and PSII and is repsonsive to changes in redox poise. We would plan to repeat this type of experiment with the microarray and compare transcription in the wild-type vs. ΔrppA. We will also perform such comparative studies in strains lacking the ability to produce cyanophycin or that fail to degrade this compound.

1. Redox state of the PQ pool controls photosynthesis and PBS-related gene expressionChanges in the redox state of components in the electron transport chain have been implicated in controlling the transcriptional activators of photosynthetic gene expression in cyanobacteria. To test if RppA is a regulator that responds to redox poise, we have treated Synechocystis sp. strain PCC6803 wild-type and ΔrppA cultures under different light conditions and with two specific inhibitors of electron transport, DCMU and DBMIB. DCMU blocks electron transfer from the PSII primary acceptor QA to the PQ pool, and DBMIB prevents the electron transfer from PQ pool to cytb6/f. Our results indicated that, in wild-type cells, the steady-state mRNA levels of PSII genes increased when the PQ pool was oxidized by DCMU, decreased when the PQ pool was reduced by DBMIB, and were extremely depressed when glucose was present (Fig. 3A). In contrast, transcription of the PSI reaction center operon was depressed when the PQ pool was oxidized, and net accumulation of the psaA-psaB transcripts increased when the PQ pool was reduced The inverse effects of DCMU and DBMIB strongly suggest that the balance between the reduced and the oxidized forms of the PQ pool is involved in the signal transduction of PS genes expression. Compared to the wild type, expression of photosynthesis genes in ΔrppA was less sensitive to PQ redox variation, especially the PSII reaction center genes. We conclude that the rppA gene is normally involved in the establishment of the appropriate stoichiometry between the photosystems and can sense changes in the PQ redox poise.

Our results relate to a number of important areas of photosynthesis research, including redox control of gene expression and the effect of photoinhibition on PSII gene expression. Similar results in regard to redox control of chloroplast gene expression were obtained recently by Pfannschmidt et al. This paper supported and expanded upon the model of Allen (2) that relates the transcriptional activity of PSI and PSII genes to the redox poise of the PQ pool. Overall, their results indicated that when either photosystem becomes rate limiting for photosynthesis, the transcription of genes for its specific reaction-center proteins become induced. Their results in chloroplasts from mustard seedlings, and ours in cyanobacteria, are virtually identical in this regard. Another detailed study of redox control of psbA expression in Synechocystis (1) also concluded that such transcription is under redox control. These authors specifically emphasized that accumulation of QA- activates psbA transcription. We have explicitly not tried to differentiate between QA- and PQ pool redox state or the thiol redox state at this early stage of our studies, since it is difficult to determine which would be the actual signal. It is possible that QA- could be involved directly with the mechanism to indicate that PSII centers need to be replaced. However, it is less certain if this is the direct signal for the transcriptional regulation of PSI genes or those involved with phycobilisomes. For simplicity, we have just referred to the redox poise of the PQ pool as we begin the process of sorting out the control mechanism in the ΔrppA, as well as in other mutants.

Another very fruitful line of investigation has been initiated by Grossman and colleagues (14, 17, 48). This group isolated mutants of Synechococcus sp. strain PCC 7942 that were defective in the degradation of phycobilisomes during sulfur- or nitrogen-limited growth. They identified NblA, a small polypeptide that is critical for phycobilisome degradation during this nutrient deprivation (14). In Synechococcus sp. strain PCC 7942, nblA transcripts were very low in nutrient-replete cells, and their abundance increased about 50-fold during sulfur or nitrogen deprivation (14). The present study showed that, in Synechocystis sp. strain PCC 6803, RppA strongly depressed nblA transcription. The steady-state mRNA level of nblA was dramatically increased when RppA was absent. This may be one reason why ΔrppA contained less PC than the wild type, since over-expression of nblA could trigger the PBS-degradative process. Interestingly, nblA transcription is also upregulated in the RppB mutant, but to a lesser degree than in ΔrppA (data not shown). These results suggest that phosphorylation is essential for RppA activation, and RppA could be phosphorylated by other kinases in addition to RppB. In ΔrppA, the accumulation of nblA transcripts increased greatly under both PQ oxidizing and reducing conditions (Fig. 5A). The regulation of RppA on nblA transcription is eliminated when glucose is present and when cells were transferred to dark or to high light. These data implied that nblA transcription is under different controls under diverse environmental stress conditions. NblR was identified to be an essential inducer of nblA expression in Synechococcus sp. strain PCC 7942 under nitrogen and sulfur deprivation conditions (48). NblR had all of the characteristics of a response regulator that is controlled by the intracellular redox state. Based on this data, we currently conclude that RppA and NblR work differently towards controlling nblA expression, although they may have overlapping functions. We will soon be able to study their related functions by constructing double mutants in Synechocystis sp. strain PCC 6803 that are deficient in both RppA and NblR.

In cyanobacteria, the light-harvesting antenna consists of the phycobilisomes and chlorophyll. The majority of Chl molecules are associated with PSI, whereas the phycobilisomes are generally the major light-harvesting antenna for PSII. The state transitions are associated with the movement of the phycobilisomes from PSII to PSI when PSII has been provided with too much excitation energy (45). Our results are very similar to that of Alfonso et al. (1) in that PSI and PBS-related genes were not under the same level of redox control as the PSII reaction-center genes. Our results indicated that, in the wild type, transcription of PSI and PBS-related genes decreased in the presence of DCMU and increased in the presence of DBMIB and glucose. It is of interest that regulation of the PBS-related genes is closer to that of PSI than to those of PSII. This suggests that the main function of the redox-regulated signal is to allow for the degradation and resynthesis of PSII. Under these circumstances, new transcription of PBS-related genes and PBS synthesis could complicate the repair mechanism and would be more appropriate at a later time. In ΔrppA, transcription of the PBS structural genes (apcABC and cpcBA) was significantly reduced under both oxidizing and reducing conditions and, especially, in the presence of glucose. Once again, the high rate of PSII synthesis may require that the transcription of the light-harvesting proteins be reduced significantly. 2. Light conditions affect transcription and translation of the PSII reaction center components.Exposure of oxygenic photosynthetic organisms to high light causes photoinhibition of photosynthesis. Photoinhibition is associated with an inactivation of PSII electron transport and subsequent degradation of the PSII reaction-center proteins (4, 29). In Synechococcus sp. strain PCC 7942, there are two forms of D1 protein: D1:1 and D1:2. It has been demonstrated that PSII reaction centers containing D1:2 have a higher intrinsic resistance to photoinhibition and are more photochemically efficient than PSII centers with D1:1 (9, 12, 13, 30). In Synechocystis sp. strain PCC 6803, only one form of D1 has been identified. The rapid degradation of D1, and possibly D2, is balanced by an induction of gene expression at the high-light intensity that compensates for the loss of these proteins and maintains a functional PSII. In Synechocystis sp. strain PCC 6803, the accumulation of psbA and psbDII transcripts was enhanced by a shift to high-light intensities (Fig. 3B). Like in Synechococcus, the primary function of the monocistronic psbDII locus in Synechocystis may be to produce extra D2 protein to maintain a functional PSII at high-light intensity without increasing synthesis of the more stable psbC gene product (8). The oxygen evolution of both Synechocystis sp. strain PCC 6803 wild type and ΔrppA increased 35-45% in photoautotrophic and photomixotrophic growth conditions, respectively, under high light for 2 h (Fig. 6). The protein synthesis inhibitor chloramphenicol caused the O2-evolution activity to be lost completely within 2 h under high-light irradiation, indicating that rapid de novo protein synthesis is required in order to maintain PSII activity. Fast D1 degradation and synthesis were confirmed by the pulse-chase and immunoblot experiments. Both experiments indicated that the rate of D1 synthesis was faster and the steady-state level was higher in ΔrppA than in the wild type. This phenomenon was more obvious when cells were grown in the presence of glucose. It is important to note that, although D1 and D2 synthesis was enhanced, the synthesis of CP43 and CP47 was not, especially in ΔrppA. This suggests that there can be more PSII centers, but with less antennae Chl on average. Thus, the O2-evolution per mg Chl in ΔrppA would appear higher than the wild type as is seen in Table 1 and Fig. 6.

REFERENCES

Alland, D., I. Kramnik, T. R. Weisbrod, L. Otsubo, R. Cerny, L. P. Miller, W. R. J. Jacobs, and B. R. Bloom. 1998. Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): The effect of isoniazid on gene expression in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 95:13227-13232.

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