Photosynthetic Gene Expression Research Articles

Regulatory factors controlling photosynthetic reaction center and light-harvesting gene expression in Rhodobacter capsulatus

Most species of photosynthetic bacteria synthesize their photosynthetic apparatus only under conditions of reduced oxygen tension. To a large extent, this phenomenon is dependent upon anaerobic induction of photosynthesis gene expression. Here we report an example of a regulatory gene, regA, that is involved in transactivating anaerobic expression of the photosynthetic apparatus. We show that RegA is itself responsible for differential induction of light-harvesting and reaction center gene expression relative to operons for photopigment biosynthesis. Surprisingly, strains disrupted for regA were found to retain normal photosynthetic growth capabilities under high light intensities. We further show that photosynthetic growth in the absence of transactivating structural gene expression is a consequence of the superoperonal organization of the photosynthetic gene cluster.

Maize rbcS promoter activity depends on sequence elements not found in dicot rbcS promoters.

Although the molecular mechanisms of dicot photosynthetic gene regulation have been pursued actively, comparable studies of monocot regulation have been slow to come forth. We show here that monocot (maize and wheat) but not dicot (pea, tobacco, and Arabidopsis) ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene promoters are active in maize mesophyll protoplasts. The evolutionarily conserved GT and G boxes of dicot rbcS promoters are not essential for light-responsive expression in monocot leaf cells. Instead, at least six constitutive and light-sensitive regulatory elements are likely important for maize rbcS expression. Synergism between upstream and downstream promoter elements is required. Whereas in dicots, light triggers coupled leaf development and photosynthetic gene expression, in monocots, light regulation of rbcS is uncoupled from leaf development. Light regulation of maize rbcS may be divided into direct and indirect contributions mediated by different regulatory elements. Because wheat and maize rbcS promoters show sequence homologies and similar expression patterns in monocot and dicot leaf cells, it appears likely that monocots share conserved regulatory elements irrespective of whether they utilize the C3 or C4 pathway for carbon fixation.

Expression of photosynthetic genes from the C4 plant, maize, in tobacco

Previous results from this laboratory have demonstrated the presence of genes for phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase in C3 plants. The structure and light-enhanced expression of these genes is very similar to that of the genes found in the C4 plant, maize. In order to investigate whether or not the regulation of these genes is similar in C3 and C4 plants, we have constructed chimeric genes using beta-glucuronidase as a reporter gene under the control of the maize promoters of the genes for phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose bisphosphate carboxylase (RuBisCO). The chimeric genes were introduced into tobacco, a C3 plant. These genes were expressed primarily in leaf and stem tissue and the expression was enhanced by light. Thus, as in C4 plants, the genes are expressed in a tissue-specific and light-inducible manner in the C3 plant. Since the expression of these genes is restricted to specific cells in leaf tissue of C4 plants, we also investigated the spatial pattern of expression of the chimeric genes using histochemical analysis of beta-glucuronidase activity. High level expression of all of these genes was found in mesophyll cells. This included the small subunit of RuBisCO, which is not expressed in mesophyll cells but in bundle sheath cells in C4 plants. This report describes similarities between C3 and C4 plants in regulating the expression of these genes.

Expression of photosynthetic genes is distinctly different between chloroplasts and amyloplasts in the liquid-cultured cells of sycamore (Acer pseudoplatanus L.).

A nonphotosynthetic, white-wild cell line of sycamore (Acer pseudoplatanus L.) contains amyloplasts as the only kind of plastid, whereas a photosynthetically competent green variant cell line contains only chloroplasts. Transcripts of both nuclear and plastid genes for photosynthetic components in the white cells were not detectable in contrast to those in the green cells. To investigate the limiting step (s) behind these diminished levels of transcripts, we have performed in vivo pulse-chase labeling of RNA in both cell types. These studies indicated that the rates of incorporation of [3H]uridine and nucleotide pool sizes were indistinguishable between the two cell lines. Transcripts of certain nuclear (rbcS, cab, psbO) and plastid (rbcL) genes in the white cell were not detectable. We infer from these data that transcriptional regulation entails an important role in controlling photosynthetic RNA levels. Related analyses exploiting plastid run-on transcription have provided supporting evidence that the transcription of the amyloplast genome in the white cell is greatly suppressed in contrast to that of the chloroplast genome in the green cell. The results support a model of selective suppression of photosynthesis genes in nonphotosynthetic higher plant cells, and indicate that gene expression in such a system is primarily controlled at the transcriptional level.

DNA methylation is a determinative element of photosynthesis gene expression in amyloplasts from liquid-cultured cells of sycamore (Acer pseudoplatanus L.).

Transcriptional regulation has been shown to operate as a selective control mechanism of expression of photosynthetic genes in the nonphotosynthetic plastids, amyloplasts, of a white-wild cell line of sycamore (Acer pseudoplatanus L.). To elaborate the mechanisms governing the transcriptional regulation at the molecular level, we have examined the template activity of the amyloplast DNA compared to the chloroplast DNA by using the in vitro run-off transcription assay system with extracts of the two plastid types. The results of these assays clearly indicate that most of the amyloplast DNA regions do not serve as a template for the in vitro transcription regardless of the plastid extracts; this is in contrast to the chloroplast DNA which serves as an active template. It is highly likely that the template activity of amyloplast DNA per se is the modulating element of transcriptional regulation. Parallel experiments determining the DNA base content by HPLC analysis have shown that a variety of methylated bases, especially 5-methylcytosine, are localized in the DNA regions containing suppressed genes of the amyloplast genome. In sharp contrast, methylated bases were undetectable in the expressed gene regions of amyloplast and whole chloroplast genomes. The overall findings strongly support the notion that DNA methylation is involved in the selective suppression of photosynthetic genes in the nonphotosynthetic plastids of cultured sycamore cells.

Nitrogen-dependent regulation of photosynthetic gene expression

Nitrogen-limited Chlamydomonas reinhardtii is chlorotic and very deficient in chlorophyll a/b light-harvesting complexes (LHC). Rates of synthesis of photosynthetic proteins, but especially the LHC apoproteins, are reduced 10- to 40-fold. Moderately high levels of chloroplast transcripts accumulate in nitrogen-limited cells, and there is a correlation between chloroplast DNA levels and chloroplast mRNA abundance. In contrast, nuclear transcripts encoding LHCII and ribulose 1,5-bisphosphate carboxylase small subunits are markedly reduced. Thus, nitrogen availability affects chloroplast protein synthesis by inhibition of translation and, to a lesser extent, chloroplast DNA amplification. Regulation of nuclear-encoded photosynthetic proteins by nitrogen is achieved through mechanisms affecting transcription and/or mRNA stability.

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed.

Cell position and light influence C4 versus C3 patterns of photosynthetic gene expression in maize.

C4 plants such as maize partition photosynthetic activities in two morphologically distinct cell types, bundle sheath (BS) and mesophyll (M), which lie as concentric layers around veins. We show that both light and cell position relative to veins influence C4 photosynthetic gene expression. A pattern of gene expression characteristic of C3 plants [ribulose bisphosphate carboxylase (RuBPCase) and light-harvesting chlorophyll a/b binding protein in all photosynthetic cells] is observed in leaf-like organs such as husk leaves, which are sparsely vascularized. This pattern of gene expression reflects direct fixation of CO2 in the C3 photosynthetic pathway, as determined by O2 inhibition assays. Light induces a switch from C3-type to C4-type gene expression patterns in all leaves, primarily in cells that are close to a vein. We propose that light causes repression of RuBPCase expression in M cells, by a mechanism associated with the vascular system, and that this is an essential step in the induction of C4 photosynthesis.

Transcription of oxygen-regulated photosynthetic genes requires DNA gyrase in Rhodobacter capsulatus.

The regulation of the photosynthetic genes by DNA supercoiling in Rhodobacter capsulatus has been studied by using gyrase inhibitors in vivo and by measurement of mRNA levels of more than a dozen genes. The results demonstrate that the levels of mRNA for light-harvesting (I, II) and reaction center (L, M, H) proteins, bacteriochlorophyll biosynthetic enzymes, ribulose-bisphosphate carboxylase (EC 4.1.1.39), and the mRNAs from the open reading frames Q and R decreased immediately and dramatically upon addition of novobiocin and coumermycin. In contrast, the mRNAs for carotenoid biosynthetic enzymes, the cytochrome bc1 complex, and constitutively expressed mRNA under aerobic conditions for light-harvesting I and for reaction center (L, M) proteins are less sensitive to the inhibitors. In accordance with these results, the biosynthesis of bacteriochlorophyll is markedly repressed by gyrase inhibitors novobiocin, coumermycin, nalidixic acid, and oxolinic acid either under anaerobic conditions or during a shift from aerobic to anaerobic conditions. The synthesis of light-harvesting (I, II) bacteriochlorophyll complexes is also inhibited by novobiocin and coumermycin. The kinetics of specific mRNA changes and the differential sensitivity of anaerobic and aerobic genes to the gyrase inhibitors strongly suggest that DNA supercoiling is involved in the differential expression of photosynthetic genes in response to the level of oxygen in R. capsulatus.

Cellular pattern of photosynthetic gene expression in developing maize leaves.

Leaf development in C4 plants such as maize involves the differentiation of two photosynthetic cell types [bundle sheath (BS) and mesophyll (M)] to form Kranz-type leaf anatomy. This cellular dimorphism partitions photosynthetic activities so that each enzyme of the C4 pathway accumulates only in the appropriate cell type. We have exploited this property to study BS and M cell interactions in developing maize leaves. Our previous studies showed that C4 proteins appear concurrently with the appearance of Kranz anatomy. To look at earlier events in BS and M cell development we have used three of the corresponding C4 mRNAs as cell-specific markers. We have followed, in situ, the accumulation of malic enzyme (ME), phosphoenolpyruvate carboxylase (PEPCase), and ribulose bisphosphate carboxylase (RuBPCase) mRNAs in developing leaves of both normal and mutant argentia (ar) maize. We have isolated a partial cDNA clone for maize ME to examine ME mRNA expression. We show that throughout the development of light-grown seedlings, all three mRNAs accumulate in a cell-specific fashion in both normal and ar leaves. The pattern of C4 mRNA accumulation longitudinally along the veins, laterally across the leaf, and locally around individual veins reveals the spatial and temporal sequence of BS and M cell development. BS cell-specific mRNAs accumulate around developing veins before Kranz anatomy is evident morphologically. Our analysis of the ar mutant, in which C4 mRNA appearance is delayed relative to the appearance of Kranz anatomy, demonstrates first that BS and M cells develop in clusters across the leaf blade and second that BS cells surrounding any individual vein are activated asynchronously. We discuss our results in relation to models and mechanisms of BS and M cell development.

Oxygen-regulated mRNAs for light-harvesting and reaction center complexes and for bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus during the shift from anaerobic to aerobic growth

The stability and regulation by oxygen of mRNAs for the photosynthetic apparatus in Rhodobacter capsulatus have been studied by using proflavin to inhibit transcription and by shifting cells from anaerobic to aerobic conditions. The results from the inhibition experiments show that the mRNA for the light-harvesting LH-II polypeptides (beta, alpha) is more stable than that for the light-harvesting LH-I polypeptides (beta, alpha) during anaerobic growth, whereas the mRNAs for the reaction center polypeptides L (RC-L), M (RC-M), and H (RC-H) are less stable than both the LH-I and LH-II mRNAs. When photosynthetic cells are shifted from anaerobic to aerobic conditions, an immediate decrease in the levels of mRNA for the LH-I, LH-II, RC-L, RC-M, and RC-H proteins was observed. The level of mRNA for the LH-II proteins, however, is more sensitive to oxygen and is reduced faster than the level of mRNA for the LH-I proteins. These results suggest that oxygen represses the expression of genes coding for the light-harvesting antenna and reaction center complexes and may selectively accelerate the degradation of mRNA for the LH-II proteins. The mRNAs for several enzymes in the bacteriochlorophyll biosynthetic pathway are regulated by oxygen in a similar manner. The mRNAs for carotenoid biosynthetic enzymes, however, are regulated by oxygen in a different way. We have found that the amounts of mRNAs for carotenoid biosynthetic enzyme, relative to the amounts of mRNAs for LH and RC, increased during the shift from anaerobic to aerobic conditions. We have particularly shown that although the expression of most photosynthetic genes in R. capsulatus is repressed by oxygen, the crtA gene, located in the BamHI H fragment of the R' plasmid pRPS404 and responsible for the oxidation of spheroidene to spheroidenone, responds to oxygen in an opposite fashion. This exzymatic oxidation may protect the photosynthetic apparatus from photooxidative damage.

Cell-Specific Photosynthetic Gene Expression in Maize Determined Using Cell Separation Techniques and Hybridization in Situ

Bundle sheath strands and mesophyll cell extracts have been isolated from maize (Zea mays L.) leaves using a mechanical disruption-filtration technique. Northern blot analysis showed that phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) mRNA accumulates only in mesophyll cells. The mechanisms regulating the cell-specific expression of this gene must, therefore, be at either the level of RNA transcription or that of mRNA turnover. The first successful application of hybridization to mRNA molecules in photosynthetic plant tissue sections is described. Results obtained from this in situ study corroborate our finding that PEPCase mRNA accumulates only in mesophyll cells as well as the previously reported (Link, G, DM Coen, L Bogorad 1978 Cell 15: 725-731) finding that the accumulation of ribulose 1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) large subunit mRNA is restricted to bundle sheath cells. Demonstrating the differential accumulation of PEPCase mRNA and RuBPCase mRNA by utilizing the in situ hybridization technique paves the way for its use as a powerful tool in relating cellular differentiation to gene expression during plant development.

Photosynthetic Gene Expression and Cellular Differentiation in Developing Maize Leaves

We have exploited the positional gradient of cellular differentiation in Zea mays leaves to study the accumulation of mRNAs encoding subunits of the two CO(2)-fixing enzymes and the major chlorophyll-binding protein. These three proteins are differentially compartmentalized in the two photosynthetically active cell types of the leaf. Previous studies have shown that accumulation of the two carboxylases commences 2 to 4 cm from the base of the leaf (Mayfield SP, WC Taylor Planta 161: 481-486) at a position where bundle sheath and mesophyll cells show morphological evidence of maturation. The light-harvesting chlorophyll a/b protein accumulates progressively from the leaf base, as does its mRNA, in spite of its localization in mesophyll cells after cellular differentiation occurs. While small quantities of phosphoenolpyruvate carboxylase mRNA are detectable in the basal region of the leaf, significant mRNA accumulation is coincident with that of the polypeptide at 4 to 6 cm from the leaf base, the region where bundle sheath and mesophyll cells exhibit fully differentiated morphologies. mRNAs encoding the small and large subunits of ribulose 1,5-bisphosphate carboxylase accumulate to significant levels before bundle sheath cells are fully differentiated and before their polypeptides are detectable. Cytological examination indicates that this is the position at which the maturation of intermediate vascular bundles is first evident. Cytosolically localized small subunit mRNA and chloroplast-localized large subunit mRNA are complexed with polyribosomes at all positions of the leaf.