Regulation Of psbA Gene Expression Research Articles

Organization of chloroplast psbA-trnH intergenic spacer in dicotyledonous angiosperms of the family umbelliferae

Chloroplast intergenic psbA-trnH spacer has recently become a popular tool in plant molecular phylogenetic studies at low taxonomic level and as suitable for DNA barcoding studies. In present work, we studied the organization of psbA-trnH in the large family Umbelliferae and its potential as a DNA barcode and phylogenetic marker in this family. Organization of the spacer in Umbelliferae is consistent with a general pattern evident for angiosperms. The 5'-region of the spacer situated directly after the psbA gene is more conserved in length compared to the 3'-region, which has greater sequence variation. This pattern can be attributed to the maintenance of the secondary structural elements in the 5'-region of the spacer needed for posttranscriptional regulation of psbA gene expression. In Umbelliferae only, the conserved region contains a duplication of the fragment corresponding to the loop of the stem-loop structure and an independent appearance of identical sequence complementarities (traits) necessary to stabilize the stem-loop structure in different lineages. The 3'-region of the spacer nearest to trnH ranges greatly in size, mainly due to deletions, and the decrease in spacer length is a general trend in the evolution psbA-trnH in Umbelliferae. The features revealed in spacer organization allow us to use it as phylogenetic marker, and indels seem to be more informative for analyses than nucleotide substitutions. However, high conservation among closely related taxa and occurrence of homoplastic inversions in the stem-loop structure limit its application as DNA barcode.

Expression of psbA Genes is Regulated at Multiple Levels in the Cyanobacterium Synechococcus sp. PCC 7942

In cyanobacterium Synechococcus sp. PCC 7942 the photosystem II reaction-center protein D1 is encoded by three psbA genes. The psbAI gene encodes D1:1 protein, the form used for acclimated growth, and psbAII and psbAIII genes encode the stress-induced form, D1:2 protein. Strong light and low temperature have been shown to induce the expression of psbAII/III genes and down-regulate the expression of psbAI gene. Recently, we reported the involvement of reduced thiols in the up-regulation of psbAII/III genes. In this study, we have analyzed the regulation of psbA gene expression in Synechococcus further, at both the transcriptional and post-transcriptional levels. We show that the inhibitors of the photosynthetic electron-transfer chain, which have different effects on the redox state of the plastoquinone (PQ) pool, have similar effect on the transcription of psbA genes. The inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) do not cause any changes in psbA gene expression when added under low-light conditions, but dramatically reduce the high-light induction of psbAII/III genes when added upon a high-light shift. Moreover, when the thiol reductant, dithiothreitol, is added to Synechococcus cells together with DCMU concomitant with the high-light shift, no inhibition of psbAII/III gene up-regulation takes place, indicating that the thiol redox state rather than the redox state of the PQ pool regulates psbA gene transcription. We also provide evidence for post-transcriptional regulation of psbA gene expression, particularly, inhibition of translation of psbAI transcripts at high light, and demonstrate that the D1 protein synthesis and degradation processes are coregulated in Synechococcus.

RNA binding-proteins interact specifically with the Arabidopsis chloroplast psbA mRNA 5' untranslated region in a redox-dependent manner.

The 5' untranslated region (5'UTR) of the psbA mRNA (psbA encodes the PSII reaction center protein, D1) is a key site for RNA-protein interactions in the post-transcriptional regulation of gene expression. In this study, we mapped the major psbA mRNA 5'-terminus at -77 nt, and two minor termini clusters centered at -48 and -64 nt, upstream from the psbA translational start codon of Arabidopsis thaliana. RNA mobility shift, RNase protection and UV-crosslinking assays were used to characterize the interaction of chloroplast proteins with the RNA 5'UTR. RNA-protein interactions depended upon a thermolabile secondary structure and specific sequences in a 35 nt region of the 5'UTR, which were 80% conserved with the psbA 5'UTRs from five other plants. Major and minor proteins of 43- and 30-kDa, respectively, were detected by UV-crosslinking to RNA. Oxidizing conditions abolished the association of the proteins with the 5'UTR, while RNA-binding activity was recovered upon incubation with a reductant. Based on these findings, we hypothesize that post-transcriptional regulation of psbA gene expression in chloroplasts of vascular plants involves redox-dependent interactions between specific sequences in the 5'UTR and 43- and 30-kDa RNA-binding proteins.

Expression of psbA genes is regulated at multiple levels in the cyanobacterium Synechococcus sp. PCC 7942.

In cyanobacterium Synechococcus sp. PCC 7942 the photosystem II reaction-center protein D1 is encoded by three psbA genes. The psbAI gene encodes D1:1 protein, the form used for acclimated growth, and psbAII and psbAIII genes encode the stress-induced form, D1:2 protein. Strong light and low temperature have been shown to induce the expression of psbAII/III genes and down-regulate the expression of psbAI gene. Recently, we reported the involvement of reduced thiols in the up-regulation of psbAII/III genes. In this study, we have analyzed the regulation of psbA gene expression in Synechococcus further, at both the transcriptional and post-transcriptional levels. We show that the inhibitors of the photosynthetic electron-transfer chain, which have different effects on the redox state of the plastoquinone (PQ) pool, have similar effect on the transcription of psbA genes. The inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) do not cause any changes in psbA gene expression when added under low-light conditions, but dramatically reduce the high-light induction of psbAII/III genes when added upon a high-light shift. Moreover, when the thiol reductant, dithiothreitol, is added to Synechococcus cells together with DCMU concomitant with the high-light shift, no inhibition of psbAII/III gene up-regulation takes place, indicating that the thiol redox state rather than the redox state of the PQ pool regulates psbA gene transcription. We also provide evidence for post-transcriptional regulation of psbA gene expression, particularly, inhibition of translation of psbAI transcripts at high light, and demonstrate that the D1 protein synthesis and degradation processes are coregulated in Synechococcus.

A genetically engineered increase in fatty acid unsaturation in Synechococcus sp. PCC 7942 allows exchange of D1 protein forms and sustenance of photosystem II activity at low temperature.

Photosystem II (PSII)-reaction-center protein D1 is encoded by three psbA genes in Synechococcus sp. PCC 7942. The psbAI gene encodes D1 form I (D1:1) and the psbAII and psbAIII genes encode the transiently expressed D1 form II (D1:2). We have studied the role of membrane-lipid unsaturation in the expression of psbA genes at low temperature, using a Synechococcus transformant with an increased unsaturation level of membrane lipids. Transfer of the cells from 32 degrees C to 25 degrees C under growth light resulted in the exchange of D1:1, the prevailing form, for D1:2 in the wild-type bacterium and the transformant, with no loss of PSII activity. Lowering the temperature further to 18 degrees C caused a drastic decrease in PSII activity in the wild-type bacterium, whereas the transformant was much less affected. Similar decreases in psbAI transcripts and loss of D1:1 occurred in both strains at 18 degrees C, with concomitant accumulation of psbAII/III transcripts, the latter event being especially prominent in the wild-type bacterium. However, the wild-type bacterium was incapable of accumulating D1:2 to compensate for the loss of D1:1, which resulted in disassembly of PSII at low temperature. These results imply translational rather than transcriptional regulation of psbA gene expression in Synechococcus 7942 at low temperature, and demonstrate the crucial role of the degree of membrane-lipid unsaturation in promotion of exchange of the D1 protein forms and thus the sustenance of PSII function at low temperature.

CARACTERÍSTICAS DE LAS SPIN-OFF ACADÉMICAS EN COSTA RICA: UN ESTUDIO EMPÍRICO

Efficient splicing in vivo of most self-splicing group I introns is believed to require proteins, raising the possibility that splicing could be regulated; however, examples of such regulation have been lacking. The Chlamydomonas reinhardtii chloroplast psbA gene contains four large group I introns that self-splice efficiently in vitro, but only under nonphysiological conditions. The psbA gene encodes the D1 protein of photosystem II, which is synthesized at very high rates in the light in order to replace photodamaged protein. We show that psbA pre-mRNAs, containing one or more introns, accumulate in wild-type cells in the dark, apparently due to rate-limited splicing. Analysis of the pre-RNAs indicates that splicing of the four introns does not follow a strict order. Exposure of cells to light induced rapid (15-20 min) decreases in precursor levels of approximately 3-5-fold (depending on the intron), which were accompanied by transient increases in free intron levels. Because light also stimulated psbA transcription approximately 2-fold over the same period, the data suggests that light increases the splicing efficiency of psbA introns approximately 6-10-fold. Similar estimates of the extent of light stimulation were obtained by analyzing precursor decay rates in the presence of actinomycin D. The effect of light is specific for psbA introns, because levels of unspliced 23S pre-RNA did not decrease. The light-induced increase in psbA pre-RNA processing was abolished by inhibitors of photosynthetic electron transport, but not by the ATP synthesis inhibitor, carbonylcyanide m-chlorophenylhydrazone, which actually promoted pre-RNA processing in the dark. Finally, nonphotosynthetic mutants, including the tscA-lacking photosystem I mutant, H13, did not show evidence of light-stimulated RNA processing. However, the light response was restored in photosynthetic transformants of H13 that had been complemented with the tscA gene. These data suggest strongly that light coordinately stimulates splicing of all four psbA introns. Moreover, they demonstrate that this response to light is mediated by photosynthetic electron transport. The implications of these results for the regulation of psbA gene expression are discussed.