Subunit Of Ribulose Bisphosphate Carboxylase-oxygenase Research Articles

English

A glasshouse experiment was conducted to compare the performance of a stay-green QTL introgression line (RSG 03123) and its parental lines (B35, a stay-green and R16, a high yielding cultivar with rapid senescence) after flowering, when water-limited (WL) or well-watered (WW). Flowering time and plant height were similar in RSG 03123 and the recurrent parent, R16. Under WW conditions, retention of green leaf area (GLA) and chlorophyll was similar in RSG 03123 and R16, whereas under WL conditions RSG 03123 retained significantly (P<0.05) more GLA. The rate of senescence was also lower in RSG 03123 as compared to R16 under WL conditions. B35 displayed a delayed onset of senescence under both WW and WL conditions while both R16 and RSG 01323 displayed an earlier onset of senescence under WL conditions. Photochemical efficiency of photosystem II (ΦPSII), gas exchange characteristics, and total soluble carbohydrates contents, in both leaf and stem, were higher in RSG 03123 than in R16 under WL conditions. These parameters were enhanced in RSG 03123 due to higher retention of chloroplast proteins, such as light-harvesting chlorophyll binding protein complex of PSII (LHCPII), oxygen-evolving complex 33 kDa (OEC33), phosphoenolpyruvate carboxylase (PEPC) and the large subunit of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The stay-green characteristics of RSG 03123 were intermediate between R16 and B35; nonetheless, the results show that stay-green QTL are functional during senescence and improve tolerance to water limitation after flowering. Key words: Stay-green QTL, introgression, drought, photosynthesis.

Transcriptome analyses of Populus x euramericana clone I-214 leaves exposed to excess zinc

Zinc (Zn) is an essential element for plant growth and development, but at high levels this metal can become toxic. Hyperaccumulator species are often not suitable for phytoremediation technologies because they need to be fast growing and have high biomass production, such as those of the Populus genus. Comparative genomics studies of poplars subjected to stress conditions such as heavy metal contamination have generated resources useful for improving the annotation of genes and have provided novel insights in the defense/tolerance mechanisms governing adaptation in non-hyperaccumulator plants. Using a microarray-based comparative analysis, we identified functional gene sets that are differentially regulated in the leaves of Populus × euramericana clone I-214 subjected to an excess but sub-lethal dose of Zn (1 mM). Eco-physiological and chemical analyses confirmed the results obtained in previous similar experiments. A total of 3861 expressed sequence tags (ESTs) were differentially expressed and grouped into two distinct libraries of up-regulated (40%) and down-regulated (60%) putative genes. The annotation of genes and gene products according to the Gene Ontology vocabularies was performed using Blast2GO software. The two transcriptome data sets were used to query all known Kyoto Encyclopedia of Genes and Genomes (KEGG) biosynthetic pathways of the genes identified in this study. The most represented molecular functions and biological processes were nucleotide binding and transcription, transport and response to stress and abiotic and biotic stimuli. The chloroplast, mitochondrion and their membrane systems were the cellular components most affected by excess Zn, as well as the photosynthetic, defense, sulfur and glutathione (GSH) metabolic pathways. The most up-regulated genes encoded electron carriers associated with ferrodoxin, the small subunit of ribulose-bisphosphate carboxylase oxygenase, and enzymes involved in GSH metabolism. This study is the most in-depth transcriptome and gene-annotation analysis of a hybrid poplar to date. The results are presented and critically discussed in terms of poplar response/tolerance to Zn stress for the characterization of non-hyperaccumulator phenotypes and the identification of candidate genes in perennial plants. These genetic findings provide useful information on tree species' adaptation to metal stress and provide powerful tools for the selection and/or genetic manipulation of stress-tolerant poplar clones.

RESPONSE OF THE PHOTOSYNTHETIC APPARATUS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) TO NITRATE, PHOSPHATE, OR IRON STARVATION1

ABSTRACTThe effects of nitrate, phosphate, and iron starvation and resupply on photosynthetic pigments, selected photosynthetic proteins, and photosystem II (PSII) photochemistry were examined in the diatom Phaeodactylum tricornutum Bohlin (CCMP 1327). Although cell chlorophyll a (chl a) content decreased in nutrient‐starved cells, the ratios of light‐harvesting accessory pigments (chl c and fucoxanthin) to chl a were unaffected by nutrient starvation. The chl a‐specific light absorpition coefficient (a*) and the functional absorption cross‐section of PSII (σ) increased during nutrient starvation, consistent with reduction of intracellular self‐shading (i.e. a reduction of the “package effect”) as cells became chlorotic. The light‐harvesting complex proteins remained a constant proportion of total cell protein during nutrient starvation, indicating that chlorosis mirrored a general reduction in cell protein content. The ratio of the xanthophylls cycle pigments diatoxanthin and diadinoxanthin to chl a increased during nutrient starvation. These pigments are thought to play a photo‐protective role by increasing dissipation of excitation energy in the pigment bed upstream from the reaction centers. Despite the increase in diatoxanthin and diadinoxanthin, the efficiency of PSII photochemistry, as measured by the ration of variable to maximum fluorescence (Fv/Fm) of dark‐adapted cells, declined markedly under nitrate and iron starvation and moderately under phosphate starvation. Parallel to changes in Fv/Fm were decreases in abundance of the reaction center protein D1 consistent with damage of PSII reaction centers in nutrient‐starved cells. The relative abundance of the carboxylating enzyme, ribulose bisphosphate carboxylase/oxygenase (RUBISCO), decreased in response to nitrate and iron starvation but not phosphate starvation. Most marked was the decline in the abundance of the small subunit of RUBISCO in nitrate‐starved cells. The changes in pigment content and fluorescence characteristics were typically reversed within 24 h of resupply of the limiting nutrient.

Localization of ribulose-bisphosphate carboxylase-oxygenase and its putative binding protein in the cell envelope of Chromatium vinosum

Antibodies to the large and small subunits of ribulose-bisphosphate carboxylase-oxygenase (RuBisCO; EC 4.1-1.39) and a putative binding protein (PBP) for RuBisCO from Chromatium vinosum have been used to localize these proteins in thin sections. Immunogold techniques employing single and double antibodies establish that RuBisCO and the RuBisCO PBP are concentrated in the cell envelope of C. vinosum.

Preparation of Chlamydomonas Chloroplasts for the in Vitro Import of Polypeptide Precursors

To study the import of polypeptide precursors we have adapted and compared two procedures for the isolation of competent chloroplasts from the green unicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation and elutriation. The chloroplasts actively import the precursor of the small subunit of ribulose bisphosphate carboxylase-oxygenase in vitro.

Expression of two nuclear genes encoding chloroplast proteins during early development of cucumber seedlings

Cloned complementary DNA probes have been used to measure steady-state transcript levels for the small subunit of ribulose bisphosphate carboxylase-oxygenase (SSU) and the chlorophyll a/b-binding protein (LHCP) in cotyledons during early development of cucumber seedlings. Initial accumulation of trancripts to SSU occurs 2d after germination and is independent of light and developmentally programmed. Although transcripts accumulate in dark-grown tissues, their levels increase rapidly in light-grown cotyledons from day 4, coinciding with emergence above the soil, so that by day 6 levels are 2.4 times higher in light-grown compared with dark-grown cotyledons. In contrast, accumulation of transcripts to LHCP occurs only in light-grown cotyledons. Southern blot analysis of genomic DNA indicates that in cucumber there are one and two genes encoding SSU and LHCP, respectively, considerably fewer than in those other plant species that have been examined. Both LHCP genes are expressed in light-grown cotyledons.

Sequence of therbcL gene for the large subunit of ribulose bisphosphate carboxylase-oxygenase from alfalfa

Sequence of the<i>rbc</i>L gene for the large subunit of ribulose bisphosphate carboxylase-oxygenase from alfalfa

Sequence of therbcL gene for the large subunit of ribulose bisphosphate carboxylase-oxygenase from petunia

Sequence of the<i>rbc</i>L gene for the large subunit of ribulose bisphosphate carboxylase-oxygenase from petunia

Sequence of the rbcL gene for the large subunit of ribulose bisphosphate carboxylase-oxygenase from petunia.

Sequence of the rbcL gene for the large subunit of ribulose bisphosphate carboxylase-oxygenase from petunia.

Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase.

The small subunits of spinach ribulosebisphosphate carboxylase-oxygenase were isolated by mild acid precipitation of the hexadecameric holoenzyme. About one-third of the small subunits remained in the supernatant while the remainder, and all of the large subunits, were precipitated and irreversibly denatured. The spinach small subunits were able to reassemble with the large subunit octamer of ribulosebisphosphate carboxylase-oxygenase from the cyanobacterium, Synechococcus ACMM 323, prepared as described previously (Andrews, T. J., and Ballment, B. (1983) J. Biol. Chem. 258, 7514-7518) to produce a catalytically active, hybrid enzyme. The heterologous small subunits bound an order of magnitude less tightly than homologous small subunits and the specific activity of the hybrid, when fully saturated with foreign small subunits, was about half that of the homologously reassembled or native Synechococcus enzyme. In addition, the Km(CO2) of the hybrid was about twice as high. However, the degree of partitioning between carboxylation and oxygenation was identical for the hybrid, the homologously reassembled, and the native Synechococcus enzymes and clearly less in favor of carboxylation than partitioning by the spinach enzyme. Therefore, this important facet of catalysis by ribulosebisphosphate carboxylase-oxygenase appears to be specified exclusively by the large subunit.

Sequence of a genomic DNA clone for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco.

We have cloned and sequenced a gene for the small subunit (SS) of ribulose bis-phosphate carboxylase-oxygenase from Nicotiana tabacum. The tobacco gene is most closely related to the SS genes from the dicots soybean and pea, and less so to the monocots wheat and Lemna; the deduced amino acid sequence of the mature protein is in all cases more closely conserved than is its chloroplast transit sequence. Unlike the genomic sequences of the two monocots, which have one intron, and the two other dicots, which have two introns, the tobacco gene has three introns. The third tobacco intron lies within a highly conserved region of the protein. Its position coincides with the boundary of a 12 amino acid insertion in the SS genes of higher plants, relative to those of blue green algae. The 5' flanking end of the gene carries 67 bp inverted repeats, which flank a series of eight direct repeats; the direct repeats themselves each carry inverted repeats. The 3' untranslated end of this gene differs by only 2 bp from that of an N. sylvestris SS gene.

Two mRNA species differing by 258 nucleotides at the 5′ end are formed from the barley chloroplast rbcL gene

The region of the barley chloroplast genome encoding the 5′ third of the rbcL gene has been subcloned in M13 vectors and sequenced by the dideoxy chain termination method. Some of the recombinant M13 clones have been used as a template for the synthesis of highly radioactive single stranded DNA probes. These are specifically hybridizing to short regions at the 5′ end of the two transcripts, which were previously found to be encoded by the rbcL region in barley cpDNA. The two transcripts differ in size by more than 200 nucleotides, are encoded by the same strand and contain the full coding capacity for the large subunit of ribulose bisphosphate carboxylase-oxygenase. The short transcript is predominant in dark grown seedlings. Of two probes employed in this study one hybridized only to the long and the other to both rbcL mRNA species. Thereby it was possible to quantitate the rbcL mRNAs by hybridization with the probes and subsequent S1-nuclease digestion of the single stranded molecules. I found this mRNA to be aproximately 1.38% of total plastid RNA (excluding the small rRNAs and the tRNAs) in dark grown seedlings and 2.20% in greened seedlings. The two probes were also used in primer extension experiments using the two transcripts as template. After hybridizing the probe to the mRNA reverse transcription was performed in the presence of cold deoxynucleotides. The reverse transcribed strands were separated on sequencing gels. The probe which acted as a specific primer for the short transcript was extended by 29 nucleotides, and the probe specific for the longer transcript was extended by 91 nucleotides. The end point of the latter transcript was only found with template RNA from plastids of greened barley seedlings. Thus it was shown that the 5′ ends of the two transcripts begin at position-58 and-316 from the ATG triplet of the rbcL reading frame. A comparison of the sequences upstream from the ATG codon of the rbcL gene of barley with those of maize, spinach and tobacco revealed completely conserved prokaryote type transcription promotors in front of the 5′ end of the long transcript, while no such promotors are evident close to the 5′ end point of the short transcript. The two monocot species differ from the dicot species by an insertion of about 140 nucleotides in the mRNA leader sequence.

The function of the small subunits of ribulose bisphosphate carboxylase-oxygenase.

When ribulose bisphosphate carboxylase-oxygenase from Synechococcus (strain RRIMP N1) was precipitated under mildly acidic conditions, most of its small subunits remained in solution. The precipitated enzyme readily redissolved at neutral pH and remained as an octamer of large subunits with some small subunits still attached. Optimum pH for this separation was 5.3 and disulfide-reducing reagents were not necessary. The fraction of small subunits removed by a single precipitation increased with increasing NaCl concentration. Catalytic activity of small subunit-depleted enzyme was linearly proportional to the fraction of small subunits remaining, while the carboxylase:oxygenase activity ratio and the affinity for CO2 remained constant. When isolated small subunits were added back to depleted enzyme preparations at neutral pH, reassociation occurred with return of catalytic activity. Under the usual assay conditions at pH 7.7, the binding constant of the small subunits was estimated to be about 10(-9) M. The small subunits also bound avidly to surfaces. However, loss of small subunits from the enzyme during the course of purification was minimal. The results are consistent with a simple model in which only those large subunits which have a small subunit bound to them are catalytically competent. Thus, an essential, even if indirect, role for the small subunits in catalysis is indicated.

Effects of Reducing Agent on the Conformation of the Isolated Subunits of Ribulose Bisphosphate Carboxylase-Oxygenase from Cold-Hardened and Unhardened Rye

Summary The large and small subunits of ribulose-1,5-bisphosphate carboxylase-oxygenase from cold-hardened and unhardened winter rye were isolated by gel filtration through Sephadex G200. The amino acid composition of the large subunits of the enzyme from cold-hardened and unhardened rye were not significantly different nor was the amino acid composition of the small subunits. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the isolated large subunits of the enzyme from cold-hardened and unhardened plants in the presence and absence of β-mercaptoethanol resulted in an apparent differential, reversible structural change associated with this polypeptide. This differential conformational change in the large subunit was further supported by circular dichroism spectroscopy. In contrast to the sodium dodecyl sulfate polyacrylamide gel electrophoresis data, the circular dichroic data indicated a significant, reversible conformational change in the isolated small subunits in the presence and absence of β-mercaptoethanol. However, this effect was similar in the small subunits of the enzyme of either cold-hardened or unhardened Puma rye.