Small Subunit Of Ribulose Bisphosphate Research Articles

Achieving High Expression of Cry in Green Tissues and Negligible Expression in Endosperm Simultaneously via rbcS Gene Fusion Strategy in Rice.

To allay excessive public concern about the safety of transgenic foods, and to optimize insect-resistant genes expression to delay the evolution of resistance in pests, we developed a promising strategy to fuse the GOI (gene of interest) with OsrbcS (rice small subunit of ribulose bisphosphate carboxylase/oxygenase) in transgenic rice, which acted as a carrier, driven by the OsrbcS native promoter to sequester its expression in green tissues. Using eYFP as a trial, we reported a high-level accumulation of eYFP in green tissue and almost none in the seed and root of the fused construct compared to the non-fused construct. After applying this fusion strategy in insect-resistant rice breeding, recombinant OsrbcS-Cry1Ab/Cry1Ac expressed rice plants conferred high resistance to leaffolders and striped stem borers, among which two single-copy lines possessed normal agronomic performance in the field. Specifically, Cry1Ab/Cry1Ac protein levels in single-copy construct transgenic lines ranged from 1.8 to 11.5 µg g-1 in the leaf, higher than the Actin I promoter-driven control, T51-1, about 1.78 µg g-1 in the leaf, but negligible (only 0.00012-0.00117 µg g-1) in endosperm by ELISA analysis. Our study provided a novel approach to creating Cry1Ab/Cry1Ac-free endosperm rice with a high level of insect-resistant protein in green tissues through the simultaneous usage of the OsrbcS promoter and OsrbcS as a fusion partner.

Characterization of the rice RbcS3 promoter and its transit peptide for use in chloroplast-targeted expression

Here, we report characterization of an RbcS promoter and its transit peptide for use in chloroplast-targeted expression. RbcS3 that encodes a small subunit of ribulose bisphosphate carboxylase/oxygenase is of great interest due to its green tissue-specific expression such as leaf and leaf sheaths. We isolated the RbcS3 promoter and its transit peptide sequence and linked to GFP for characterization in transgenic plants. Our qRT-PCR analysis on leaves and roots of transgenic plants revealed that GFP transcript levels were higher by fivefold in leaves of RbcS3:TP3:GFP transgenic lines than in those of the RbcS3:GFP transgenic lines, suggesting that the transcript levels were increased by chloroplast-targeted expression, as shown in previously characterized RbcS1:TP1 system. The transit peptide sequence TP3 was sufficient to target GFP to chloroplasts as observed in protoplasts of RbcS3:TP3:GFP transgenic lines. GFP fluorescence in RbcS3:GFP plants was detected only in the cytosol. The GFP protein levels in leaves of RbcS3:TP3:GFP plants were 0.12–0.23 % of the total soluble leaf proteins. These levels were higher or comparable to those of PGD1:GFP plants (GFP driven by a constitutive promoter PGD1). Taken together, these results indicate that the fusion of the RbcS3 promoter to its transit peptide, RbcS3:TP3, provides a chloroplast-targeting expression system.

Fidelity of targeting to chloroplasts is not affected by removal of the phosphorylation site from the transit peptide.

Phosphorylation of the transit peptide of several chloroplast-targeted proteins enables the binding of 14-3-3 proteins. The complex that forms, together with Hsp70, has been demonstrated to be an intermediate in the chloroplast protein import pathway in vitro[May, T. & Soll, J. (2000) Plant Cell 12, 53-63]. In this paper we report that mutagenesis (in order to remove the phosphorylation site) of the transit peptide of the small subunit of ribulose bisphosphate carboxylase/oxygenase did not affect its ability to target green fluorescent protein to chloroplasts in vivo. We also found no mistargeting to other organelles such as mitochondria. Similar alterations to the transit peptides of histidyl- or cysteinyl-tRNA synthetase, which are dual-targeted to chloroplasts and mitochondria, had no effect on their ability to target green fluorescent protein in vivo. Thus, phosphorylation of the transit peptide is not responsible for the specificity of chloroplast import.

Mutations in a sequence near the N-terminus of the small subunit alter the CO2/O2specificity factor for ribulose bisphosphate carboxylase/oxygenase

Seven unique substitutions have been introduced by site-directed mutagenesis into the first conserved region of the small subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans 6301. After expression of each large, altered-small subunit gene tandem in Escherichia coli, two substitutions in the small subunit tyr17→asp17 (Y17D) and arg10→gly10 (R10G) result in little or no carboxylase activity. For the latter substitution, no L8S8enzyme complex could be detected suggesting that this mutation prevents assembly. Mutant enzymes containing the following substitutions R11G, T14A, S16A, Y17D and P19A have CO2/O2specificity factors (τ values) of 40, 35, 18, 39 and 44, respectively, compared with that of 44 for wild-type recombinant enzyme whereas P20A has full carboxylase activity and a τ value of 55.

Import of Modified Dl Protein and its Assembly into Photosystem II by Isolated Chloroplasts

A portion of the rbcS gene that encoded the transit peptide and 20 amino acid residues of the N-terminal region of the small subunit of ribulosebisphosphate carboxylase/oxygenase was fused to the 5' end of the psbA gene which encodes the Dl protein of PSII reaction center. The chimeric gene was expressed in vitro as a 42-kDa protein, which was imported into chloroplasts isolated from pea leaves. The imported protein was processed such that the transit peptide was lost in the stroma, the resultant protein was translocated into thylakoid membranes, and the C-terminal peptide was then removed to yield a mature protein with an N-terminal extension originated from the small subunit. The mature protein appeared to be assembled into the PSII core complex, resembling the native Dl protein in terms of protein structure and topology within the membrane. Our observations indicate that the structure of the precursor to the Dl protein includes information for the proper assembly of the protein into the PSII core complex.

Changes in ethylene and polyamines in relation to mRNA levels of the large and small subunits of ribulose bisphosphate carboxylase/oxygenase in ozone‐stressed potato foliage

ABSTRACTPotato plants (Solanum tuberosum L. cv‘Norland’) were subjected to acute and chronic exposures of ozone (O3). Periodically following or during the exposures, foliage of different physiological ages were sampled for tissue analysis. Measurement of the following parameters were included: ethylene, 1‐aminocyclopropane‐1‐carboxylic acid (ACC), N‐malonyl 1‐aminocyclopropane‐1‐carboxylic acid (MACC), and ACC oxidase activity, putrescine, spermidine, and spermine levels, and ornithine decarboxylase (ODC) activity, and levels of mRNA transcripts for the large and small subunits of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) (rbcL and rbcS, respectively). Ozone increased emission of ethylene and the concentration and activity of all associated metabolites and enzymes. Putrescine titre increased in response to O3 as did ODC activity. Some increases in spermidine were also detected. Ozone decreased the levels of mRNA for rbcL and rbcS, with the latter transcript exhibiting greater sensitivity. After acute exposures were terminated, the rbcL and rbcS transcript in younger leaves returned to levels of nonstressed plants; effects were less likely to be reversed in older tissue. The potential relationship between the changes in rbcL and rbcS and ethylene and polyamines are discussed in the context of O3 induction of accelerated senescence.

Maintenance of Chloroplast Components during Chromoplast Differentiation in the Tomato Mutant Green Flesh.

During ripening of tomato (Lycopersicon esculentum) fruit, chloroplasts develop into chromoplasts. The chloroplast-chromoplast transition is marked by the accumulation of carotenoids and the disappearance of chlorophyll, the degradation of the highly structured thylakoid membrane system, and a reduction in the levels of proteins and mRNAs associated with photosynthesis. In the tomato mutant green flesh (gf), detectable amounts of chlorophyll remain in the ripe, mutant fruit, giving rise to a rusty red fruit color and suggesting that at least chlorophyll degradation is defective in the mutant. We show here that the ultrastructure of the plastids in the ripe gf fruit maintained significant amouonts of the chloroplast thylakoid grana along with structures characteristic of tomato chromoplasts. The maintenance of chloroplast structure in the gf ripe fruit was paralleled on the molecular level by the retention of plastid photosynthetic components that normally decline significantly in ripening tomato fruits. These included the light-harvesting chlorophyll a/b-binding proteins of photosystem II, the second electron accepting plastoquinone of photosystem II binding protein, the large and small subunits of ribulose bisphosphate carboxylase/oxygenase, the 33-kD oxygen evolution protein, and cytochrome b559. Similarly, photosynthetic transcripts, cab, psbA, rbcL, rbcS, and psbE mRNAs, also accumulated to higher levels in ripening gf fruit than wild type. It is interesting that the levels of some of these transcripts, especially cab mRNA, were noticeably higher in the mature gf green fruit than in the corresponding wild-type fruit. This suggests that the onset of the effect from the gf mutation might be earlier than fruit ripening. We also observed that when chloroplast formation was blocked during the development and ripening of gf fruit, these mutant fruits were bright red and their chromoplasts were indistinguishable from those found in wild-type ripe fruits grown and ripened either in the dark or in the light. These results suggest that the lesion in gf may alleviate conditions associated with chloroplast deterioration during the chloroplast-chromoplast transition in tomato ripening but has no direct effect on chromoplast differentiation per se. The ultrastructure of gf provides unequivocal evidence that, in ripening tomato, chromoplasts indeed differentiate from preexisting chloroplasts; on the other hand, chromoplast differentiation in the dark-matured and -ripened tomato fruits indicates that chromoplast development can be a process entirely independent of the chloroplasts.

Translational Regulation of the Large and Small Subunits of Ribulose Bisphosphate Carboxylase/Oxygenase during Induction of the CO2-Concentrating Mechanism in Chlamydomonas reinhardtii

In conditions of limiting external inorganic carbon, the unicellular alga Chlamydomonas reinhardtii induces a mechanism to actively transport and accumulate inorganic carbon within the cell. A high internal inorganic carbon concentration enables the cell to photosynthesize efficiently with little oxygen inhibition, even in conditions of limiting external inorganic carbon. A correlation between limiting inorganic carbon-induced induction of the CO(2)-concentrating mechanism and decreased synthesis of the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase has been observed. Cells that had been transferred from elevated CO(2) to limiting CO(2) exhibit transient declines of label incorporation into both subunit polypeptides. We have found that this decrease in synthesis of large and small subunits results from specific and coordinated down-regulation of translation of both subunits possibly resulting, at least in part, from modification of large and small subunit transcripts.

Cloning and light‐dependent expression of the gene coding for the P‐protein of the glycine decarboxylase complex from peas

The glycine decarboxylase multienzyme complex (EC 2.1.2.10) is located in the mitochondrial matrix and catalyzes a key reaction of the photorespiratory C‐2 cycle of plants. The light‐dependent control of the synthesis of the P‐protein of this complex in pea (Pisum sativum cv. Alaska) leaves was studied using a partial (0.7 kb) cDNA clone. The size of the P‐protein mRNA detected by this probe was 3.5 kb. When the mRNA hybrid selected by this probe was translated in vitro, a protein that migrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) with an apparent molecular mass of 114 kDa was formed. This protein could be immunoprecipitated with the P‐protein‐specific antibody. The mass of the mature P‐protein on SDS‐PAGE is 100 kDa. This suggests the presence of a presequence used to direct the protein to the mitochondrial matrix. Analysis of the concentration of P‐protein mRNA showed an approximately 5‐fold increase in the abundance of this transcript in light‐grown as compared to dark‐grown pea seedlings. The time course for the production of P‐protein mRNA following exposure of dark‐grown peas to white light was compared to that for the H‐protein of glycine decarboxylase, the small subunit of ribulose bisphosphate carboxylase/oxygenase, and the chlorophyll a/b binding protein. All 4 mRNAs accumulated with similar kinetics where they remained low during the first 3 h of illumination, increased about 5‐fold during the next 8 h, and then maintained a steady state over the next 12 h.

Cytokinin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce

A pulse treatment of Norway spruce (Picea abies (L.) Karst) embryos with the cytokinin N(6)-benzyladenine induces the formation of adventitious buds from subepidermal cells in the hypocotyl and cotyledons. In addition the treatment also inhibits elongation growth, a key process during germination. In this report we demonstrate that these effects on development of the plant are associated with a suppression of the accumulation of several major chloroplast proteins during germination. These proteins include the large subunit of ribulose bisphosphate/carboxylase oxygenase, two subunits of the chloroplast ATPase, protochlorophyllide reductase and a 23000-Mr component of photosystem II. For two nuclear-encoded proteins, the small subunit of ribulose bisphosphate carboxylase/oxygenase and the light-harvesting chlorophyll a/b-binding protein, a corresponding suppression of the increase in the steady-state amounts of mRNA is recorded. The suppression of chloroplast protein synthesis is consistant with the previously documented delay in greening that results from cytokinin treatment, but the effect is opposite to that found in other plants, where cytokinins promote the synthesis of chloroplast proteins, and stimulate chloroplast biogenesis. We believe that this difference is explained by the cytokinin primarily suppressing organ development, and a strict dependance of chloroplast biogenesis on the developmental state of the organs.

Plastid disruption in a thiamine-requiring mutant of Nicotiana sylvestris blocks accumulation of specific nuclear and plastid mRNAs

In the absence of supplemental thiamine, cotyledon leaves of the thi mutant of Nicotiana sylvestris are green, but expanding true leaves (TL) become chlorotic upon emergence and the chloroplasts fail to maintain thylakoid membrane structure. Albino TL1 is deficient in transcripts from the plastid genes psbA, psbE, and rbcL. As in carotenoid-deficient mutants of maize, albino TL1 is deficient in accumulation of the chlorophyll a/b binding protein (LHCP) and its mRNA (cab). The small subunit of ribulose bisphosphate carboxylase/oxygenase (SSu) and its mRNA (rbcS) are also deficient, but not to the same extent as cab. Application of supplemental thiamine and its subsequent removal was employed to examine patterns of gene expression during the onset of plastid disruption in older leaves. Accumulation of plastid transcripts (psbA, psbE, rbcL) is blocked shortly after the onset of chlorosis in TL6. In contrast, transcripts for cab and rbcS follow their normal course of accumulation in spite of plastid degeneration, suggesting that nuclear genes are transcribed without regard to chloroplast integrity in older leaves. The conditional nature of the thi mutation provides a valuable tool for investigating temporal patterns of intracellular communication during development of primary leaves.

The role of the N-terminus of the large subunit of ribulose-bisphosphate carboxylase investigated by construction and expression of chimaeric genes.

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N-terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N-terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild-type Rubisco of A. nidulans. In contrast, when the N-terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5-bisphosphate. This confirms that the N-terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.

Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts.

The uptake and expression by plastids isolated from dark-grown cucumber cotyledons (etioplasts) of two pUC derivatives, pCS75 and pUC9-CM, respectively carrying genes for the large small subunits of ribulose bisphosphate carboxylase/oxygenase of Anacystis nidulans or chloramphenicol acetyltransferase, is reported. Untreated etioplasts take up only 3% as much DNA as that taken up by EDTA-washed etioplasts after 2 hr of incubation with nick-translated [32P]-pCS75. The presence or absence of light does not affect DNA uptake, binding, or breakdown by etioplasts. Calcium or magnesium ions inhibit DNA uptake by 86% but enhance binding (23-200%) and breakdown (163-235%) of donor DNA by EDTA-treated etioplasts. Uncouplers that abolish membrane potential (delta psi), transmembrane proton gradient (delta pH), or both do not affect DNA uptake, binding, or breakdown by etioplast. However, both DNA uptake and binding are severely inhibited by ATP. Presumably this results from the hydrolysis of ATP, because the poorly hydrolyzable analog adenyl-5'-yl imidodiphosphate does not inhibit the uptake or binding of DNA by etioplasts. beta-Lactamase specified by the ampicillin resistance gene of pCS75 can be detected only in EDTA-treated etioplasts that have been incubated with the plasmid pCS75. After the incubation of EDTA-treated etioplasts with pCS75, immunoprecipitation using antiserum to the small subunit of ribulose bisphosphate carboxylase/oxygenase from A. nidulans reveals the synthesis of small subunits; these are smaller by 2 kDa than the cucumber small subunit encoded by the nuclear genome. Treatment of etioplasts with 10 mM EDTA shows a 10-min duration to be optimal for the expression of chloramphenicol acetyltransferase encoded by pUC9-CM. A progressive increase in the expression of this enzyme is observed with an increase in the concentration of pUC9-CM in the DNA uptake medium. The plasmid-dependent incorporation of [35S]methionine by EDTA-treated organelles declines markedly during cotyledon greening in vivo.

Specific mRNA and rRNA Levels in Greening Pea Leaves during Recovery from Iron Stress

Hydroponically grown pea seedlings (Pisum sativum L., cv Alaska) were subjected to Fe stress for 10 to 16 days to produce mature chlorotic leaves. Greening was initiated by adding Fe to the nutrient solution. The levels of chlorophylls, chloroplast, and cytoplasmic rRNAs, and specific chloroplast- and nucleus-encoded mRNAs were all significantly lower in leaves developing during iron stress than in nonstressed leaves. In plants greening after addition of Fe, nuclear transcripts encoding chlorophyll a/b-binding protein and the small subunit of ribulose bisphosphate carboxylase/oxygenase increased about 5-fold in abundance following an 18 to 24 hour lag, as did the chloroplast-encoded transcript for the large subunit of the carboxylase/oxygenase. Chloroplast rRNA showed an increase over that in continually stressed control leaves only after a 40 hour lag. The chloroplast-encoded transcript encoding the Q(B)-binding 32 kilodalton polypeptide of Photosystem II showed little change during greening. Chlorophyll itself increased gradually after a lag period of 24 hours, with an increase in chlorophyll a slightly preceding that of chlorophyll b. Kinetic considerations suggest that the changes observed represent a coordinate series of events initiated by readdition of Fe and occurring in parallel. Though accumulation of mRNA for light-harvesting, chlorophyll-a/b-binding protein might limit chlorophyll accumulation at the onset, subsequent changes in the mRNA do not parallel chlorophyll changes. All three of the mRNAs showing recovery on addition of Fe to Fe-stressed plants undergo sharp diurnal fluctuations in abundance. Such fluctuations are comparable to those in nonstressed controls (mRNA for light-harvesting protein) or considerably more pronounced (mRNAs for carboxylase large and small subunits). The carboxylase small subunit mRNA and that for light-harvesting chlorophyll-binding protein were measured under constant conditions of light and temperature. Though a rhythm in greening leaves was hard to detect, it was prominent in the Fe-sufficient controls, persisting undamped through three full cycles for both mRNAs, and hence is probably circadian.

Sequence, evolution and differential expression of the two genes encoding variant small subunits of ribulose bisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii

We have sequenced the two genes for the small subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) in Chlamydomonas reinhardtii and analyzed their expression. The two genes encode variant small subunits that differ by four amino acid residues. Both genes are expressed and each is transcribed into an RNA of distinct size. The accumulation of the two RNAs changes depending on the growth conditions, so the small subunit composition of Rubisco may be expected to differ in response to the environment. The C. reinhardtii small subunit sequence is homologous to those of vascular plants or cyanobacteria, but is longer at the amino terminus and in internal positions. The number and location of the intervening sequences in the genes from C. reinhardtii and from other plants differ. In several cases, internal length differences in the polypeptide coincide with the positions of introns in the coding sequence. Thus, changes in the exon structure of the genes during evolution may have been accompanied by substantial changes in the encoded protein. The translation and splicing signals in C. reinhardtii are similar to those of other eukaryotes, but the transcription signals are less conserved and the highly biased codon usage is very unusual.

Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition

A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632–4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica . Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO 2. The catalytic core (A 8) of spinach RuBisCO could not be isolated in the native form.