Photosynthesis Gene Expression Research Articles

Expression of chloroplast photosynthesis genes during leaf senescence

Transcripts for thylakoid proteins encoded in the chloroplast genome that show decreased synthesis in senescing primary leaves of bean (Phaseolus vulgaris) have been examined by Northern and dot blot analyses. In young, 7‐day‐old leaves pulse‐labelled with 35S‐methionine, all of the major chloroplast DNA‐encoded thylakoid proteins become radiolabelled, including the 68 kDa protein of PSI encoded by psaAl and psbA2, the D‐1, D‐2, CP47 and CP43 proteins of PSII encoded by psbA, psbD, psbB, and psbC, respectively, and cytochrome f and cytochrome b6 encoded by petA and petB, respectively. By contrast, in pulse‐labelled, senescent 28‐day‐old leaves, the only chloroplast‐encoded thylakoid protein showing active synthesis is the D‐1 protein of PSII. Total RNA levels decline by ≅ 9 fold between 7 and 28 days on a fresh weight basis. Dot blot analyses using gene‐specific hybridization probes indicate that steady state transcript levels for all of the chloroplast DNA‐encoded proteins examined maintain the same proportion of total RNA over this period. This means that these transcripts also decline by =9 fold between 7 and 28 days. Thus, reduced synthesis of the 68 kDa protein of PSI, the CP47 and CP43 proteins of PSII, cytochrome f and cytochrome b6 can presumably be attributed, at least in part, to a decline in steady state levels of corresponding message. It is apparent from Northern blot hybridizations that there are also qualitative changes in transcript pattern for petA, psbC and psbD between 7 and 28 days that perhaps reflect alterations in post‐transcriptional processing. These alterations may also contribute to the decreased synthesis of the CP43 protein and cytochrome f. The continued active synthesis of the D‐1 protein of PSII in the senescent 28‐day‐old leaves, despite the =9 fold decrease in the steady state level of its transcript, suggests that the psbA transcript is preferentially translated relative to transcripts for other chloroplastencoded thylakoid proteins.

Localized transposon Tn5 mutagenesis of the photosynthetic gene cluster of Rhodobacter sphaeroides

Four genes essential for bacteriochlorophyll biosynthesis were known to be encoded within a 45 kb region of the Rhodobacter sphaeroides genome, the boundaries of which are defined by puh and puf genes for reaction-centre and light-harvesting LH1 complexes. The cluster is represented by eight overlapping inserts cloned in the mobilizable vector pSUP202. We have used localized transposon Tn5 mutagenesis to characterize this cluster further; a total of 87 independent insertions were generated which identify nine genes for bacteriochlorophyll biosynthesis, six for carotenoid biosynthesis, and puhA encoding the reaction-centre H subunit. This work provides an essential framework for a detailed study of the structure and expression of genes for photosynthesis in this bacterium.

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.

DNA methylation and the differential expression of C4 photosynthesis genes in mesophyll and bundle sheath cells of greening maize leaves

The methylation of nuclear and chloroplast DNAs has been examined in relation to the known differential expression of C4 photosynthesis genes in the bundle sheath and mesophyll cells of etiolated, greening, and fully green maize leaves. We have focused our research on phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBUp2Case) which are coded by nuclear genes, and on the large subunit of RBUp2Case which is coded by a plastid gene. Reversed-phase high performance liquid chromatography revealed several kinds of methylated bases in DNAs of both photosynthetic cell types, with the largest amounts in fully green leaves. The occurrence of selective DNA methylation was investigated by employing an isoschizomeric pair of methyl-sensitive and -insensitive endonucleases followed by Southern hybridizations with specific DNA probes. Notably, there was an inverse correlation between the relative abundance of specific transcripts in a given cell type during greening and the methylation status of the corresponding nuclear or chloroplast gene. Furthermore, a heterologous in vitro transcription system using Escherichia coli RNA polymerase revealed that the plastid gene encoding the RBUp2Case large subunit in both cell types was active as a template in the unmethylated state, whereas it was inactive when methylated. Thus, the selective methylation of both chloroplast and nuclear DNA is likely one component of a multilevel control mechanism for the differential regulation of cell-specific C4 photosynthesis gene expression in greening maize leaves.

An intercistronic stem-loop structure functions as an mRNA decay terminator necessary but insufficient for puf mRNA stability

Segmental differences in stability within the polycistronic transcripts of the puf operon contribute to differential expression of photosynthesis genes in R. capculatus. The comparatively stable 5′ segment of these transcripts ends in a large intercistronic stem-loop structure. We show here that deletion of this RNA hairpin destabilizes the 5′ puf mRNA segment but that its insertion at the 3′ end of the puf operon transcripts fails to stabilize the labile 3′ puf mRNA segment. Evidence is presented that decay of the 3′ segment begins with endonucleolytic cleavage in which the intercistronic stem-loop structure does not participate. We conclude that this RNA hairpin is necessary but insufficient for the stability of mRNA upstream of it, and that it functions in message degradation solely as an mRNA decay terminator that protects upstream mRNA segments from degradation by 3′ exoribonucleases.

Differential expression of photosynthesis genes in R. capsulata results from segmental differences in stability within the polycistronic rxcA transcript

We report that the light-harvesting and reaction center genes in the rxcA locus of R. capsulata are contained within a single operon and that their differential expression results predominantly from marked segmental differences in stability within the polycistronic rxcA transcript. The 3′ portion of this transcript is rapidly degraded to give rise to either of two slowly decaying mRNA remnants, both of which encode only the light-harvesting polypeptides. The greater stability of these remnants accounts for nearly all of the difference between the concentrations of the light-harvesting and reaction center proteins. The unstable 3′ portion of the transcript is delimited by two alternative stem-and-loop structures, which apparently act as barriers to 3′ exoribonucleases and thereby protect the upstream RNA segment. When a DNA fragment containing the rxcA locus was fused to a plasmid promoter and transcribed in E. coli, the long precursor transcript was processed to two short messages of greater stability, as in R. capsulata.