psbA Transcripts Research Articles

Positive Regulation of psbA Gene Expression by cis-Encoded Antisense RNAs in Synechocystis sp. PCC 6803

The D1 protein of photosystem II in the thylakoid membrane of photosynthetic organisms is encoded by psbA genes, which in cyanobacteria occur in the form of a small gene family. Light-dependent up-regulation of psbA gene expression is crucial to ensure the proper replacement of the D1 protein. To gain a high level of gene expression, psbA transcription can be enhanced by several orders of magnitude. Recent transcriptome analyses demonstrated a high number of cis-encoded antisense RNAs (asRNAs) in bacteria, but very little is known about their possible functions. Here, we show the presence of two cis-encoded asRNAs (PsbA2R and PsbA3R) of psbA2 and psbA3 from Synechocystis sp. PCC 6803. These asRNAs are located in the 5' untranslated region of psbA2 and psbA3 genes. Their expression becomes up-regulated by light and down-regulated by darkness, similar to their target mRNAs. In the PsbA2R-suppressing strain [PsbA2R(-)], the amount of psbA2 mRNA was only about 50% compared with the control strain. Likewise, we identified a 15% lowered activity of photosystem II and a reduced amount of the D1 protein in PsbA2R(-) compared with the control strain. The function of PsbA2R in the stabilization of psbA2 mRNA was shown from in vitro RNase E assay when the AU box and the ribosome-binding site in the 5' untranslated region of psbA2 mRNA were both covered by PsbA2R. These results add another layer of complexity to the mechanisms that contribute to psbA gene expression and show PsbA2R as a positively acting factor to achieve a maximum level of D1 synthesis.

Modeling RNA polymerase competition: the effect of σ-subunit knockout and heat shock on gene transcription level

BackgroundModeling of a complex biological process can explain the results of experimental studies and help predict its characteristics. Among such processes is transcription in the presence of competing RNA polymerases. This process involves RNA polymerases collision followed by transcription termination.ResultsA mathematical and computer simulation model is developed to describe the competition of RNA polymerases during genes transcription on complementary DNA strands. E.g., in the barley Hordeum vulgare the polymerase competition occurs in the locus containing plastome genes psbA, rpl23, rpl2 and four bacterial type promoters. In heat shock experiments on isolated chloroplasts, a twofold decrease of psbA transcripts and even larger increase of rpl23-rpl2 transcripts were observed, which is well reproduced in the model. The model predictions are in good agreement with virtually all relevant experimental data (knockout, heat shock, chromatogram data, etc.). The model allows to hypothesize a mechanism of cell response to knockout and heat shock, as well as a mechanism of gene expression regulation in presence of RNA polymerase competition. The model is implemented for multiprocessor platforms with MPI and supported on Linux and MS Windows. The source code written in C++ is available under the GNU General Public License from the laboratory website. A user-friendly GUI version is also provided at http://lab6.iitp.ru/en/rivals.ConclusionsThe developed model is in good agreement with virtually all relevant experimental data. The model can be applied to estimate intensities of binding of the holoenzyme and phage type RNA polymerase to their promoters using data on gene transcription levels, as well as to predict characteristics of RNA polymerases and the transcription process that are difficult to measure directly, e.g., the intensity (frequency) of holoenzyme binding to the promoter in correlation to its nucleotide composition and the type of σ-subunit, the amount of transcription initiation aborts, etc. The model can be used to make functional predictions, e.g., heat shock response in isolated chloroplasts and changes of gene transcription levels under knockout of different σ-subunits or RNA polymerases or due to gene expression regulation.ReviewersThis article was reviewed by Dr. Anthony Almudevar, Dr. Aniko Szabo, Dr. Yuri Wolf (nominated by Dr. Peter Olofsson) and Prof. Marek Kimmel.

Photoperiod and temperature influence cadmium’s effects on photosynthesis-related gene transcription in Chlorella vulgaris

Routine metal compound toxicity tests are performed at one constant photoperiod and temperature. There is little knowledge about the interactions between metal compound toxicity and photoperiod or temperature. The purpose of this study was to analyze the effects of photoperiod and temperature on cadmium (Cd) toxicity in the fresh alga, Chlorella vulgaris, both singly and in combination. Exposure to 2 or 4μM Cd alone significantly decreased the transcription of the photosynthesis-related genes psbA, psaB and rbcL. Three-way ANOVA analysis showed that both temperature and photoperiod interacted with the dosage of Cd to influence the abundance of psbA and psaB, but not rbcL. Specifically, psbA transcription was more sensitive to Cd under long photoperiods or high temperatures than under short photoperiods or low temperatures. Because photoperiod and temperature have certain impacts on the toxicity of metal compounds, these two environmental factors should be given more attention in laboratory research.

Diversity of active marine picoeukaryotes in the Eastern Mediterranean Sea unveiled using photosystem-II psbA transcripts

In vast areas of the oceans, most of the primary production is performed by cells smaller than 2-3 mum in diameter (picophytoplankton). In recent years, several in situ molecular studies showed a broad genetic diversity of small eukaryotes by sequencing 18S rRNA genes. Compared with photosynthetic cyanobacteria that are dominated by two genera, Prochlorococcus and Synechococcus, marine photosynthetic picoeukaryotes (PPEs) are much more diverse, with virtually every algal class being represented. However, the genetic diversity and ecology of PPEs are still poorly described. Here, we show using in situ molecular analyses of psbA transcripts that PPEs in the Eastern Mediterranean Sea are highly diverse, probably very active, and dominated by groups belonging to the red algal lineages, Haptophyta, Heterokontophyta (also called Stramenopiles), and Cryptophyta.

A supervised learning approach for taxonomic classification of core-photosystem-II genes and transcripts in the marine environment

BackgroundCyanobacteria of the genera Synechococcus and Prochlorococcus play a key role in marine photosynthesis, which contributes to the global carbon cycle and to the world oxygen supply. Recently, genes encoding the photosystem II reaction center (psbA and psbD) were found in cyanophage genomes. This phenomenon suggested that the horizontal transfer of these genes may be involved in increasing phage fitness. To date, a very small percentage of marine bacteria and phages has been cultured. Thus, mapping genomic data extracted directly from the environment to its taxonomic origin is necessary for a better understanding of phage-host relationships and dynamics.ResultsTo achieve an accurate and rapid taxonomic classification, we employed a computational approach combining a multi-class Support Vector Machine (SVM) with a codon usage position specific scoring matrix (cuPSSM). Our method has been applied successfully to classify core-photosystem-II gene fragments, including partial sequences coming directly from the ocean, to seven different taxonomic classes. Applying the method on a large set of DNA and RNA psbA clones from the Mediterranean Sea, we studied the distribution of cyanobacterial psbA genes and transcripts in their natural environment. Using our approach, we were able to simultaneously examine taxonomic and ecological distributions in the marine environment.ConclusionThe ability to accurately classify the origin of individual genes and transcripts coming directly from the environment is of great importance in studying marine ecology. The classification method presented in this paper could be applied further to classify other genes amplified from the environment, for which training data is available.

The Role of Phosphorylation in Redox Regulation of Photosynthesis Genes psaA and psbA during Photosynthetic Acclimation of Mustard

The long-term response (LTR) to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB (encoding the P700 apoproteins of photosystem I) and psbA (encoding the D1 protein of photosystem II) and requires the action of plastid-localized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase (PEP) is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.

Differential regulation of psbA and psbD gene expression, and the role of the different D1 protein copies in the cyanobacterium Thermosynechococcus elongatus BP-1

In Thermosynechococcus elongatus BP-1 , which is the preferred organism in recent structural studies of PSII, three psbA and two psbD genes code for three D1 and one D2 protein isoforms, respectively. The regulation and function of these genes and protein products is largely unknown. Therefore, we used quantitative RT-PCR to follow changes in the mRNA level of the respective genes, in combination with biophysical measurements to detect changes in the electron transport activity of Photosystem II under exposure to different visible and UV light, and temperature conditions. In cells which are acclimated to 40 μmol m − 2 s − 1 growth light conditions at 40 °C the main populations of the psbA and psbD transcripts arise from the psbA1 and psbD1 genes, respectively. When the temperature is raised to 60 °C psbA1 becomes the single dominating psbA mRNA species. Upon exposure of the cells to 500 μmol m − 2 s − 1 intensity visible light psbA3 replaces psbA1 as the dominating psbA mRNA species, and psbD2 increases at the expense of psbD1. UV-B radiation also increases the abundance of psbA3, and psbD2 at the expense of psbA1 and psbD1, respectively. From the different extent of total D1 protein loss in the absence and presence of lincomycin it was estimated that the PsbA3 protein isoform replaces PsbA1 in about 65% of PSII centers after 2 h of high light acclimation. Under the conditions of different psbA transcript distributions chlorophyll fluorescence and thermoluminescence measurements were applied to monitor charge recombination characteristics of the S 2Q A − and S 2Q B − states. We obtained faster decay of flash-induced chlorophyll fluorescence in the presence of DCMU, as well as lower peak temperature of the Q and B thermoluminescence bands when PsbA3 replaced PsbA1 as the main D1 protein isoform. The relevance of dynamic changes in the abundance of psbA and psbD transcript levels, as well as D1 protein isoforms in the acclimation of T. elongatus to changing environmental conditions is discussed.

The psbA gene family responds differentially to light and UVB stress in Gloeobacter violaceus PCC 7421, a deeply divergent cyanobacterium

Gloeobacter violaceus PCC 7421 is a slow-growing cyanobacterium which lacks thylakoid membranes, but whose five-membered psbA gene family encodes three isoform variants of the PsbA (D1) reaction center protein of Photosystem II. Under standard culture conditions Gloeobacter exhibits photosystem II electron transport, but several clear modifications in the redox potential of key cofactors bound by the PsbA protein are manifested in the flash-fluorescence characteristics. In other cyanobacteria dynamic expression of multiple psbA genes and turnover of PsbA isoforms is critical to counter excitation stress. We found that each of Gloeobacter's five psbA genes is expressed, with transcript abundances spanning 4.5 orders of magnitude. psbAI ( glr2322) and psbAII ( glr0779), encoding identical PsbA:2 form proteins, are constitutively expressed and dominate the psbA transcript pool under control conditions. psbAIII ( gll3144) was strongly induced under photoinhibitory high irradiance stress, thereby contributing to a large increase in the psbA transcript pool that allowed cells to maintain their PsbA protein pools and then recover from irradiance stress, within one cellular generation. In contrast, under comparable photoinhibition provoked by UVB the cells were unable to maintain their psbA transcript and PsbA protein pools, and showed limited subsequent recovery. psbAIV ( glr1706) and psbAV ( glr2656), encoding two divergent PsbA isoforms, showed consistent trace expression but were never quantitatively significant contributors to the psbA transcript pool.

A Bacterial Transgene for Catalase Protects Translation of D1 Protein during Exposure of Salt-Stressed Tobacco Leaves to Strong Light

During photoinhibition of photosystem II (PSII) in cyanobacteria, salt stress inhibits the repair of photodamaged PSII and, in particular, the synthesis of the D1 protein (D1). We investigated the effects of salt stress on the repair of PSII and the synthesis of D1 in wild-type tobacco (Nicotiana tabacum 'Xanthi') and in transformed plants that harbored the katE gene for catalase from Escherichia coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both wild-type and katE-transformed plants, but the effect of salt stress was less significant in the transformed plants than in wild-type plants. In the presence of lincomycin, which inhibits protein synthesis in chloroplasts, the activity of PSII decreased rapidly and at similar rates in both types of leaf disc during photoinhibition, and the observation suggests that repair of PSII was protected by the transgene-coded enzyme. Incorporation of [(35)S]methionine into D1 during photoinhibition was inhibited by salt stress, and the transformation mitigated this inhibitory effect. Northern blotting revealed that the level of psbA transcripts was not significantly affected by salt stress or by the transformation. Our results suggest that salt stress enhanced photoinhibition by inhibiting repair of PSII and that the katE transgene increased the resistance of the chloroplast's translational machinery to salt stress by scavenging hydrogen peroxide.

Engineered ectopic expression of the psbA gene encoding the photosystem II D1 protein in Synechocystis sp. PCC6803

A genetic vector-recipient system was developed to engineer expression of the wild-type psbA2 gene encoding the photosystem II (PSII) D1 protein only from a non-native location (ectopic) in the Synechocystis sp. PCC6803 and the result was a new strain, designated MK1. While MK1 accumulates near normal levels of PSII under low light conditions, it is very sensitive to photoinhibition. This phenotype can be traced to impaired PSII repair capacity. Based upon the hypothesis that the non-native transcriptional activity of the re-introduced psbA gene was insufficient to sustain the translation rate necessary for normal PSII repair rates, we conducted a quantitative analysis of the relationship between psbA transcript abundance on the rate of recovery from photoinhibition. Analysis of MK1 and two other engineered strains, with intermediate levels of psbA mRNA, indicated that transcript levels are indeed limiting the engineered strains. Furthermore, transcript levels may become limiting even in the wild-type, but only under very high light conditions when the demands for D1 replacement synthesis are maximal. The work extends the original studies of Komenda and colleagues (Komenda et al. (2000) Plant Mol Biol 42(4):635-645) and sets the stage for alternative approaches to engineering non-native expression of the D1 protein.

Cool temperatures interfere with D1 synthesis in tomato by causing ribosomal pausing

Photodamage occurs when leaves are exposed to light in excess of what can be used for photosynthesis and in excess of the capacity of ancillary photoprotective as well as repair mechanisms. An important site of photodamage is the chloroplast encoded D1 protein, a component of the photosystem II (PSII) reaction center. Even under optimal growth irradiance, D1 is photodamaged necessitating rapid turnover to prevent the accumulation of photodamaged PSII reaction centers and consequent inhibition of photosynthesis. However, this on-going process of D1 turnover and replacement was impeded in the chilling-sensitive tomato (Solanum lycopersicum) plants when exposed to high-growth light at cool temperature. The decrease in D1 turnover and replacement was found not to be due to changes in the steady-state level of the psbA message. While the recruitment of ribosomes to psbA transcript, initiation of D1 translation, and the association of polysomes with the thylakoid membrane occurred normally, chilling temperatures caused ribosomal pausing during D1 peptide elongation in tomato. The pause locations were non-randomly located on the D1 transcript. The interference with translation caused by ribosomal pausing allowed photodamaged PSII centers to accumulate leading to the consequent inhibition of photosynthesis.

Cooperation of group 2 σ factors, SigD and SigE for light-induced transcription in the cyanobacterium Synechocystis sp. PCC 6803

A light-inducible σ factor of RNA polymerase, SigD, can contributes to the light-induced transcription of psbA in the cyanobacterium Synechocystis sp. PCC 6803. Here, another light-induced σ factor, SigE, was characterized together with SigD. Results indicated that SigE also contributes to light-induced transcription on the cpcBACD, psbA, petBD and psaAB promoters whose potential sequences are of the Escherichia coli σ70-type. SigD and SigE interfere with each other’s expression. A rhythmic expression, in which the periodic peak of SigE exhibits a 24-h interval according to the upcoming night, was observed at the protein level. The cooperation of group 2 σ factors, SigD and SigE, for light-induced transcription was discussed.

Glycinebetaine Counteracts the Inhibitory Effects of Salt Stress on the Degradation and Synthesis of D1 Protein during Photoinhibition in Synechococcus sp. PCC 7942

Glycinebetaine (hereafter referred to as betaine) is a compatible solute that accumulates in certain plants and microorganisms in response to various types of stress. We demonstrated previously that when the cyanobacterium Synechococcus sp. PCC 7942 (hereafter Synechococcus) is transformed with the codA gene for choline oxidase, it can synthesize betaine from exogenously supplied choline, exhibiting enhanced tolerance to salt and cold stress. In this study, we examined the effects of salt stress and betaine synthesis on the photoinhibition of photosystem II (PSII). Salt stress due to 220 mm NaCl enhanced photoinhibition of PSII and betaine protected PSII against photoinhibition under these conditions. However, neither salt stress nor betaine synthesis affected photodamage to PSII. By contrast, salt stress inhibited repair of photodamaged PSII and betaine reversed this inhibitory effect of salt stress. Pulse-chase-labeling experiments revealed that salt stress inhibited degradation of D1 protein in photodamaged PSII and de novo synthesis of D1. By contrast, betaine protected the machinery required for degradation and synthesis of D1 under salt stress. Neither salt stress nor betaine affected levels of psbA transcripts. These observations suggest that betaine counteracts the inhibitory effects of salt stress, with resultant accelerated repair of photodamaged PSII.

Transcription of a ‘photosynthetic’ T4‐type phage during infection of a marine cyanobacterium

The transcription of S-PM2 phage following infection of Synechococcus sp. WH7803, a marine cyanobacterium, was analysed by quantitative real-time PCR. Unlike the distantly related coliphage T4, there were only two (early and late) instead of three (early, middle and late) classes of transcripts during the developmental cycle of the phage. This difference is consistent with the absence from the S-PM2 genome of T4-like middle mode promoter sequences and the transcription factors associated with their recognition. Phage S-PM2 carries the 'photosynthetic' genes psbA and psbD that encode homologues of the host photosystem II proteins D1 and D2. Transcripts of the phage psbA gene appeared soon after infection and remained at high levels until lysis. Throughout the course of infection, the photosynthetic capacity of the cells remained constant. A considerable transient increase in the abundance of the host psbA transcripts occurred shortly after infection, suggesting that the host responds to the trauma of phage infection in a similar way as it does to a variety of other environmental stresses. The very substantial transcription of the phage psbA gene during the latter phase of phage infection suggests that S-PM2 has acquired this cellular gene to ensure that D1 levels and thus photosynthesis are fully maintained until the infected cell finally lyses. Unexpectedly, transcripts of a phage-encoded S-layer protein gene were among the earliest and most abundant detected, suggesting that this partial homologue of a host protein plays an important role in the S-PM2 infection process.

Cyanobacterial psbA families in Anabaena and Synechocystis encode trace, constitutive and UVB-induced D1 isoforms

Cyanobacteria cope with UVB induced photoinhibition of Photosystem II by regulating multiple psbA genes to boost the expression of D1 protein (in Synechocystis sp. PCC6803), or to exchange the constitutive D1:1 protein to an alternate D1:2 isoform (in Synechococcus sp. PCC7942). To define more general patterns of cyanobacterial psbA expression, we applied moderately photoinhibitory UVB to Anabaena sp. PCC7120 and tracked the expression of its five psbA genes. psbAI, encoding a D1:1 protein isoform characterized by a Gln130, represented the majority of the psbA transcript pool under control conditions. psbAI transcripts decreased upon UVB treatment but the total psbA transcript pool increased 3.5 fold within 90 min as a result of sharply increased psbAII, psbAIV and psbAIII transcripts encoding an alternate D1:2 protein isoform characterized by Glu130, similar to that of Synechococcus. Upon UVB treatment the relaxation of flash induced chlorophyll fluorescence showed a characteristic acceleration of a decay phase likely associated with the exchange from the D1:1 protein isoform encoded by psbAI to the alternate D1:2 isoform encoded by psbAIV, psbAII and psbAIII. Throughout the UVB treatment the divergent psbA0 made only a trace contribution to the total psbA transcript pool. This suggests a similarity to the divergent psbAI gene from Synechocystis, whose natural expression we demonstrate for the first time at a trace level similar to psbA0 in Anabaena. These trace-expressed psbA genes in two different cyanobacteria raise questions concerning the functions of these divergent genes.

Effects of long-term ionic and osmotic stress conditions on photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803.

Salinity is considered to be one of the most severe problems in worldwide agricultural production, but the published investigations give contradictory results of the effect of ionic and osmotic stresses on photosynthesis. In the present study, long-term effects of both ionic and osmotic stresses, especially on photosynthesis, were investigated using the moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803. Our results show that the PSII activity and the photosynthetic capacity tolerated NaCl but a high concentration of sorbitol completely inhibited both activities. In line with these results, we show that the amount of the D1 protein of PSII was decreased under severe osmotic stress, whereas the levels of PsaA / B and NdhF3 proteins remained unchanged. However, high concentrations of sorbitol stress led to a drastic decrease of both psbA (encoding D1) and psaA (encoding PsaA) transcripts, suggesting that severe osmotic stress may abolish the tight coordination of transcription and translation normally present in bacteria, at least in the case of the psaA gene. Taken together, our results indicate that the osmotic stress component is more detrimental to photosynthesis than the ionic one and, furthermore, under osmotic stress, the D1 protein appears to be the target of this stress treatment.

Irradiation-Regulated Differential Accumulation of Chloroplast-Encoded Transcripts in Mature Leaves of Populus deltoides

Steady state transcript levels of psbA, rbcL, atpB, and psbN genes accumulated differentially in mature leaves of a field-grown tree Populus deltoides during natural diurnal cycle and in dark adaptation followed by irradiation. The rbcL transcript accumulated independent of irradiation under diurnal conditions, which demonstrated a correlation with the phenomenon of midday depression in photosynthesis. The psbA and atpB transcripts accumulated more in dark whereas psbN accumulated more in light. Diurnal rhythm of gene expression in mature chloroplasts was independent of development related changes.

Blue light-induced transcription of plastid-encoded psbD gene is mediated by a nuclear-encoded transcription initiation factor, AtSig5.

Light is one of the most important environmental factors regulating expression of photosynthesis genes. The plastid psbD gene encoding the photosystem II reaction center protein D2 is under the control of a unique blue light responsive promoter (BLRP) that is transcribed by a bacterial-type plastid RNA polymerase (PEP). Promoter recognition of PEP is mediated by one of the six nuclear-encoded sigma factors in Arabidopsis. The replacement of the plastid sigma factor associated with PEP may be the major mechanism for switching of plastid transcription pattern in response to environmental and developmental signals. This study demonstrates that AtSig5 is a unique sigma factor that is essential for psbD BLRP activity. A T-DNA insertional mutant with reduced AtSIG5 expression resulted in loss of primary transcripts from the psbD BLRP. Furthermore, transient overexpression of AtSig5 in dark-adapted protoplasts specifically elevated psbD and psbA transcription activities. On the other hand, overproduction of AtSig2 enhanced the transcription of psbA gene and trnE operon, but not psbD transcription. The AtSIG5 gene is phylogenetically distinct from other plastid sigma factors, and its expression is induced exclusively by blue light. We propose that AtSig5 acts as a mediator of blue light signaling that specifically activates the psbD BLRP in response to blue light in Arabidopsis.

The antioxidant status of photosynthesizing leaves under nutrient deficiency: redox regulation, gene expression and antioxidant activity in Arabidopsis thaliana.

Redox signals provide important information on plant metabolism during development and in dependence on environmental parameters and trigger compensatory responses and antioxidant defence. The aim of the study was to characterize the redox and antioxidant status of photosynthesizing leaves under N, P and S deficiency on a comparative basis. Therefore, redox signals, indicators of the cellular redox environment and parameters of antioxidant defence were determined and related to general growth parameters, namely (1) transcript levels of all chloroplast encoded genes; (2) ascorbate and glutathione; (3) activities of catalase (CAT) and ascorbate peroxidase (APX); and (4) transcript amounts of eight peroxiredoxins, three catalases and three ascorbate peroxidases. The results reveal distinct patterns of redox responses dependent on the type of nutrient deficiency. (1) Nitrogen deprivation caused up-regulation of psbA, psbC, petA, petG and clpP transcripts, down-regulation of psbG, psbK and ndhA, a five-fold increase in ascorbic acid, a severe drop in CAT and APX activities, although cat1 mRNA levels were increased in young and old leaves. (2) With the exception of psbA and psaJ transcripts, P-starvation induced a general trend to decreased mRNA abundance of plastome genes; ascorbate and glutathione levels were increased, as was the activity of APX and CAT. In accordance with that result, transcripts of all cat genes and stromal apx, as well as prxIIC, prxIID, were elevated under P deprivation. (3) Sulphur depletion increased transcripts of petA, petB, petD, petG, ndhJ and rpo-genes. mRNAs of psbG, psbK, atpA, atpB, atpE and atpF were decreased. Glutathione levels dropped to less than 25% of control, in parallel activities of APX were stimulated in young leaves. Transcripts of many antioxidant enzymes were unaltered or decreased, only cat2 mRNA was increased. It is concluded that N-, P- and S-nutrient deprivation trigger distinct redox changes and induce oxidative stress with a rather defined pattern in the context of nutrient-specific alterations in metabolism.

CSP41a, a multifunctional RNA-binding protein, initiates mRNA turnover in tobacco chloroplasts.

Expression of chloroplast stem-loop binding protein (CSP)41a, a highly conserved chloroplast endoribonuclease, was reduced >90% by the expression of antisense RNA in Nicotiana tabacum. The most striking effects of this silencing were two- to sevenfold decreases in the degradation rates of rbcL, psbA, and petD transcripts in lysed chloroplast extracts. These results are consistent with the hypothesis that CSP41a participates in initiating mRNA turnover through endonucleolytic cleavages. Surprisingly, rbcL and psbA mRNAs accumulated to similar levels in wild-type and antisense lines. This suggested that decreased degradation was compensated by reduced transcription, which was confirmed using run-on transcription assays. The collective accumulation of petD-containing mRNAs in antisense plants decreased by 25% compared to wild-type controls. However, the relative levels of petD processing intermediates in wild-type and antisense plants did not differ, and there were no changes in petD 3'-end maturation, suggesting that CSP41a is not required for petD RNA processing. CSP41a is a Mg2+-dependent enzyme; therefore, extracts from antisense plants were tested at different Mg2+ concentrations. These experiments showed that the half-life of rbcL decreased as the Mg2+ concentration was reduced, and at <1 mm free Mg2+, conditions where CSP41a is nearly inactive in vitro, the rbcL degradation rate was similar in wild-type and antisense extracts, suggesting that CSP41a is normally bypassed under these conditions. Mg2+ has been shown to mediate RNA stability during chloroplast biogenesis, and our data suggest that regulation of CSP41a activity by Mg2+ is a component of this process.