rbcS Gene Research Articles

The influence of HCO3-, K+ and HSO3 - onRUBISCO large-subunit (rbcL) and small-subunit (rbcS) genes expression

KHCO3 and NaHSO3 were sprayed on the leaves of cucumber, and HCO3-, K+, HSO3- in liquor can accelerate the expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) large-subunit (rbcL) and RUBISCO small-subunit (rbcS) genes. These resultsproved that, mRNA content in the leaves of rbcL and rbcS was increased and the carboxylation activity of RUBISCO was also improved obviously, the content of RUBISCO has not increased but decreased. According to the analysis, we obtain that K+ ,HCO3-, HSO3- could affect the expression of rbcL and rbcS in a transcription way. Key words: RUBISCO large-subunit (rbcL), RUBISCO small-subunit (rbcS) expression,RUBISCO.

Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus

An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

A Gene Duplication/Loss Event in the Ribulose-1,5-Bisphosphate-Carboxylase/Oxygenase (Rubisco) Small Subunit Gene Family among Accessions of Arabidopsis thaliana

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), the most abundant protein in nature, catalyzes the assimilation of CO(2) (worldwide about 10(11) t each year) by carboxylation of ribulose-1,5-bisphosphate. It is a hexadecamer consisting of eight large and eight small subunits. Although the Rubisco large subunit (rbcL) is encoded by a single gene on the multicopy chloroplast genome, the Rubisco small subunits (rbcS) are encoded by a family of nuclear genes. In Arabidopsis thaliana, the rbcS gene family comprises four members, that is, rbcS-1a, rbcS-1b, rbcS-2b, and rbcS-3b. We sequenced all Rubisco genes in 26 worldwide distributed A. thaliana accessions. In three of these accessions, we detected a gene duplication/loss event, where rbcS-1b was lost and substituted by a duplicate of rbcS-2b (called rbcS-2b*). By screening 74 additional accessions using a specific polymerase chain reaction assay, we detected five additional accessions with this duplication/loss event. In summary, we found the gene duplication/loss in 8 of 100 A. thaliana accessions, namely, Bch, Bu, Bur, Cvi, Fei, Lm, Sha, and Sorbo. We sequenced an about 1-kb promoter region for all Rubisco genes as well. This analysis revealed that the gene duplication/loss event was associated with promoter alterations (two insertions of 450 and 850 bp, one deletion of 730 bp) in rbcS-2b and a promoter deletion (2.3 kb) in rbcS-2b* in all eight affected accessions. The substitution of rbcS-1b by a duplicate of rbcS-2b (i.e., rbcS-2b*) might be caused by gene conversion. All four Rubisco genes evolve under purifying selection, as expected for central genes of the highly conserved photosystem of green plants. We inferred a single positive selected site, a tyrosine to aspartic acid substitution at position 72 in rbcS-1b. Exactly the same substitution compromises carboxylase activity in the cyanobacterium Anacystis nidulans. In A. thaliana, this substitution is associated with an inferred recombination. Functional implications of the substitution remain to be evaluated.

RBCS1 expression in coffee: Coffea orthologs, Coffea arabica homeologs, and expression variability between genotypes and under drought stress

BackgroundIn higher plants, the inhibition of photosynthetic capacity under drought is attributable to stomatal and non-stomatal (i.e., photochemical and biochemical) effects. In particular, a disruption of photosynthetic metabolism and Rubisco regulation can be observed. Several studies reported reduced expression of the RBCS genes, which encode the Rubisco small subunit, under water stress.ResultsExpression of the RBCS1 gene was analysed in the allopolyploid context of C. arabica, which originates from a natural cross between the C. canephora and C. eugenioides species. Our study revealed the existence of two homeologous RBCS1 genes in C. arabica: one carried by the C. canephora sub-genome (called CaCc) and the other carried by the C. eugenioides sub-genome (called CaCe). Using specific primer pairs for each homeolog, expression studies revealed that CaCe was expressed in C. eugenioides and C. arabica but was undetectable in C. canephora. On the other hand, CaCc was expressed in C. canephora but almost completely silenced in non-introgressed ("pure") genotypes of C. arabica. However, enhanced CaCc expression was observed in most C. arabica cultivars with introgressed C. canephora genome. In addition, total RBCS1 expression was higher for C. arabica cultivars that had recently introgressed C. canephora genome than for "pure" cultivars. For both species, water stress led to an important decrease in the abundance of RBCS1 transcripts. This was observed for plants grown in either greenhouse or field conditions under severe or moderate drought. However, this reduction of RBCS1 gene expression was not accompanied by a decrease in the corresponding protein in the leaves of C. canephora subjected to water withdrawal. In that case, the amount of RBCS1 was even higher under drought than under unstressed (irrigated) conditions, which suggests great stability of RBCS1 under adverse water conditions. On the other hand, for C. arabica, high nocturnal expression of RBCS1 could also explain the accumulation of the RBCS1 protein under water stress. Altogether, the results presented here suggest that the content of RBCS was not responsible for the loss of photosynthetic capacity that is commonly observed in water-stressed coffee plants.ConclusionWe showed that the CaCe homeolog was expressed in C. eugenioides and non-introgressed ("pure") genotypes of C. arabica but that it was undetectable in C. canephora. On the other hand, the CaCc homeolog was expressed in C. canephora but highly repressed in C. arabica. Expression of the CaCc homeolog was enhanced in C. arabica cultivars that experienced recent introgression with C. canephora. For both C. canephora and C. arabica species, total RBCS1 gene expression was highly reduced with WS. Unexpectedly, the accumulation of RBCS1 protein was observed in the leaves of C. canephora under WS, possibly coming from nocturnal RBCS1 expression. These results suggest that the increase in the amount of RBCS1 protein could contribute to the antioxidative function of photorespiration in water-stressed coffee plants.

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

SUMMARYThe usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.

Changes in Rubisco Kinetics during the Evolution of C4 Photosynthesis in Flaveria (Asteraceae) Are Associated with Positive Selection on Genes Encoding the Enzyme

Rubisco, the primary photosynthetic carboxylase, evolved 3-4 billion years ago in an anaerobic, high CO(2) atmosphere. The combined effect of low CO(2) and high O(2) levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO(2) and O(2), leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C(4) photosynthesis largely solves this problem by facilitating a high CO(2)/O(2) ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcat(CO2) and lower CO(2)/O(2) specificity (S(C/O)) is well supported, but the structural basis for the differences between C(3) and C(4) Rubiscos is not clear. Flaveria (Asteraceae) includes C(3), C(3)-C(4) intermediate, and C(4) species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C(3) to C(4) photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C(4) Rubiscos from the ancestral C(3) species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic "C(4)" SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus.

Characterization of SSU5C promoter of a rbcS gene from duckweed (Lemna gibba)

Photosynthesis-associated nuclear genes are able to respond to multiple environmental and developmental signals. Studies have shown that light signals coordinate with hormone signaling pathways to control photomorphogenesis. A small subunit of ribulose-1,5 bisphosphate carboxylase/oxygenase (rbcS) gene promoter was cloned from duckweed (Lemna gibba). Sequence analysis revealed this promoter is different from the previously reported rbcs promoters and is named SSU5C. Analysis of T1 transgenic tobacco plants with a reporter gene under the control of the SSU5C promoter revealed that this promoter is tissue-specific and is positively regulated by red light. Promoter deletion analysis confirmed a region from position -152 to -49 relative to the start of transcription containing boxes X, Y and Z, and is identified to be critical for phytochrome responses. Further functional analysis of constructs of box-X, Y, Z, which was respectively fused to the basal SSU5C promoter, defined boxes X, Y and Z alone are able to direct phytochrome-regulated expression, indicating that boxes Y and Z are different from those of the SSU5B promoters in L. gibba. This promoter may be used for plant gene expression in a tissue-specific manner.

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits: ENGINEERED rbcS cDNA FOR EXPRESSION IN CHLAMYDOMONAS

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO(2) fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO(2)/O(2) specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO(2)/O(2) specificity but a lower carboxylation V(max) than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO(2)/O(2) specificity and retain near normal V(max) values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO(2) is concentrated for optimal photosynthesis.

Mechanisms underlying the impacts of ozone on photosynthetic performance

The present study examined the effects of ozone on the photosynthetic capacity of two ‘Cos’ lettuce ( Lactuca sativa L.) varieties grown widely in Greece. The role of Rubisco and Rubisco activase in mediating the O 3-enhanced decline in carboxylation efficiency was explored. The transcript and protein abundances of Rubisco activase plus Rubisco large and small subunits were monitored over the leaf life-span in plants subject to ozone levels similar to those experienced regularly during the spring and summer time in Southern Europe (100 ppb ozone 8 h d −1). Ozone exposure led to a decline in net CO 2 fixation (i.e. a decline in the maximum light- and CO 2-saturated rate of assimilation, i.e. A max), a reduction in carboxylation efficiency ( V cmax) and a decline in the maximum modelled capacity for RuBP regeneration ( J max). The decline in carboxylation efficiency induced by ozone was linked to a decrease in the amount of Rubisco LSU and SSU protein, an effect resulting–at least in part–from a decrease in the transcript abundance of rbcS1 and to a lesser extent of rbcL. The work outlined in this paper shows that members of the rbcS gene family respond differentially to ozone-induced oxidative stress.

Highly Conserved Small Subunit Residues Influence Rubisco Large Subunit Catalysis

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus

Brassinosteroids (BRs) are a new group of plant growth substances that promote plant growth and productivity. We showed in this study that improved growth of cucumber (Cucumis sativus) plants after treatment with 24-epibrassinolide (EBR), an active BR, was associated with increased CO(2) assimilation and quantum yield of PSII (Phi(PSII)). Treatment of brassinazole (Brz), a specific inhibitor for BR biosynthesis, reduced plant growth and at the same time decreased CO(2) assimilation and Phi(PSII). Thus, the growth-promoting activity of BRs can be, at least partly, attributed to enhanced plant photosynthesis. To understand how BRs enhance photosynthesis, we have analyzed the effects of EBR and Brz on a number of photosynthetic parameters and their affecting factors, including the contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Northern and Western blotting demonstrated that EBR upregulated, while Brz downregulated, the expressions of rbcL, rbcS and other photosynthetic genes. In addition, EBR had a positive effect on the activation of Rubisco based on increased maximum Rubisco carboxylation rates (V (c,max)), total Rubisco activity and, to a greater extent, initial Rubisco activity. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments suggested a role of RCA in BR-regulated activation state of Rubisco. Enhanced expression of genes encoding other Calvin cycle genes after EBR treatment may also play a positive role in RuBP regeneration (J (max)), thereby increasing maximum carboxylation rate of Rubisco (V (c,max)). Thus, BRs promote photosynthesis and growth by positively regulating synthesis and activation of a variety of photosynthetic enzymes including Rubisco in cucumber.

Evidence for a role of hexokinases as conserved glucose sensors in both monocot and dicot plant species

The role of the hexokinases (HXKs) as glucose (Glc) sensors has been mainly demonstrated for Arabidopsis (Arabidopsis thaliana) HXK1 (AtHXK1) but has yet to be shown in other plant species. In our recent publication, we reported that two rice (Oryza sativa) HXKs, OsHXK5 and OsHXK6, also function as Glc sensors. These two enzymes harbor both mitochondrial targeting peptides (mTPs) and nuclear localization signals (NLSs), and we confirmed their dual-targeting ability to nuclei and mitochondria using GFP fusion experiments. Consistently, it has been previously known that AtHXK1 is predominantly associated with mitochondria but is also present in nuclei in vivo at appreciable levels. Notably, the expression of OsHXK5, OsHXK6, or their catalytically inactive mutant alleles complemented the Arabidopsis glucose insensitive2 (gin2) mutant. In addition, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the Rubisco small subunit (RbcS) gene in response to glucose treatment. Our results thus provided evidence that OsHXK5 and OsHXK6 can function as glucose sensors in rice. Hence, the available current data suggest that the role of the HXKs as Glc sensors may be conserved in both monocot and dicot plant species, and that the nuclear localization of AtHXK1, OsHXK5 and OsHXK6 may be critical for Glc sensing and signaling.

Isolation and characterization of the rbcS genes from a sterile mutant of Ulva pertusa (Ulvales, Chlorophyta) and transient gene expression using the rbcS gene promoter

We isolated two different genomic DNAs (UprbcS1 and UprbcS2) encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and portions of the 5′- and 3′-flanking regions from sterile Ulva pertusa Kjellman. The UprbcS1 and UprbcS2 genes had three introns in the coding region. Each predicted UprbcS polypeptide was a 180-amino-acid (AA) residue including a 38-AA transit peptide, although the 104th AA residue was replaced. The nucleotide sequences of UprbcS cDNAs isolated from a cDNA library corresponded to that of the UprbcS1 gene, suggesting that the UprbcS1 gene was predominantly expressed in sterile U. pertusa compared to UprbcS2. Southern blot analysis showed that each UprbcS gene was a single-copy gene in the sterile U. pertusa genome. Northern hybridization indicated that the expression of UprbcS was induced and repressed by dark and light treatments, respectively. When sterile U. pertusa cells were transformed with an expression vector containing the UprbcS1 promoter and terminator sequences fused with the green fluorescent protein (GFP) gene, GFP fluorescence was observed in the cells transformed. These results suggest that the UprbcS1 gene promoter is light regulated and highly active in the sterile U. pertusa cells and is available for genetic transformation system in the alga.

Rubisco Oligomers Composed of Linked Small and Large Subunits Assemble in Tobacco Plastids and Have Higher Affinities for CO2 and O2

Manipulation of Rubisco within higher plants is complicated by the different genomic locations of the large (L; rbcL) and small (S; RbcS) subunit genes. Although rbcL can be accurately modified by plastome transformation, directed genetic manipulation of the multiple nuclear-encoded RbcS genes is more challenging. Here we demonstrate the viability of linking the S and L subunits of tobacco (Nicotiana tabacum) Rubisco using a flexible 40-amino acid tether. By replacing the rbcL in tobacco plastids with an artificial gene coding for a S40L fusion peptide, we found that the fusions readily assemble into catalytic (S40L)8 and (S40L)16 oligomers that are devoid of unlinked S subunits. While there was little or no change in CO2/O2 specificity or carboxylation rate of the Rubisco oligomers, their Kms for CO2 and O2 were reduced 10% to 20% and 45%, respectively. In young maturing leaves of the plastome transformants (called ANtS40L), the S40L-Rubisco levels were approximately 20% that of wild-type controls despite turnover of the S40L-Rubisco oligomers being only slightly enhanced relative to wild type. The reduced Rubisco content in ANtS40L leaves is partly attributed to problems with folding and assembly of the S40L peptides in tobacco plastids that relegate approximately 30% to 50% of the S40L pool to the insoluble protein fraction. Leaf CO2-assimilation rates in ANtS40L at varying pCO2 corresponded with the kinetics and reduced content of the Rubisco oligomers. This fusion strategy provides a novel platform to begin simultaneously engineering Rubisco L and S subunits in tobacco plastids.

Cloning and Sequencing of a Full-Length cDNA Encoding the RuBPCase Small Subunit ( RbcS) in Tea ( Camellia sinensis)

This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit ( RbcS) from tea plant [ Camellia sinensis (L.) O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP (cDNA amplified fragment length polymorphism), we have isolated some transcript-derived fragments (TDFs) occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp (EF011075) cDNA was obtained via rapid amplification of cDNA ends (RACE). It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.

Strategies to facilitate transgene expression in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has been identified as a promising organism for the production of recombinant proteins. While during the last years important improvements have been developed for the production of proteins within the chloroplast, the expression levels of transgenes from the nuclear genome were too low to be of biotechnological importance. In this study, we integrated endogenous intronic sequences into the expression cassette to enhance the expression of transgenes in the nucleus. The insertion of one or more copies of intron sequences from the Chlamydomonas RBCS2 gene resulted in increased expression levels of a Renilla-luciferase gene used as a reporter. Although any of the three RBCS2 introns alone had a positive effect on expression, their integration in their physiological number and order created an over-proportional stimulating effect observed in all transformants. The secretion of the luciferase protein into the medium was achieved by using the export sequence of the Chlamydomonas ARS2 gene in a cell wall deficient strain and Renilla-luciferase could be successfully concentrated with the help of attached C-terminal protein tags. Similarly, a codon adapted gene variant for human erythropoietin (crEpo) was expressed as a protein of commercial relevance. Extracellular erythropoietin produced in Chlamydomonas showed a molecular mass of 33 kDa probably resulting from post-translational modifications. Both, the increased expression levels of transgenes by integration of introns and the isolation of recombinant proteins from the culture medium are important steps towards an extended biotechnological use of this alga.

Heterologous expression and initial characterization of recombinant RbcX protein from Thermosynechococcus elongatus BP-1 and the role of RbcX in RuBisCO assembly.

In the cyanobacterial RuBisCO operon from Thermosynechococcus elongatus the rbcX gene is juxtaposed and cotranscribed with the rbcL and rbcS genes which encode large and small RuBisCO subunits, respectively. It has been suggested that the rbcX position is not random and that the RbcX protein could be a chaperone for RuBisCO. In this study, the RbcX protein from T. elongatus was overexpressed, purified and preliminary functional studies were conducted. The recombinant protein purified from Escherichia coli extracts was predominantly present in a soluble fraction in a dimeric form. Coexpression experiments have demonstrated that RbcX can mediate RbcL dimer (L(2)) formation, and that it is essential for the L(8) core complex assembly. This is the first characterization of the RbcX protein from a thermophilic organism.

Botryticides affect grapevine leaf photosynthesis without inducing defense mechanisms

The effects of the two botryticides, fludioxonil (fdx) and fenhexamid (fhd), were investigated on grapevine leaves (Vitis vinifera L. cv. Pinot noir) following photosynthesis and defense mechanisms. Treatments were carried out in vineyard at the end of flowering. Phytotoxicity of both fungicides was evaluated by measuring variations of leaf photosynthetic parameters and correlated expression of photosynthesis-related genes. Results demonstrated that similar decrease in photosynthesis was caused by fdx and fhd applications. Moreover, the mechanism leading to photosynthesis alteration seems to be the same for both fungicides. Stomatal limitation to photosynthetic gas exchange did not change following treatments indicating that inhibition of photosynthesis was mostly attributed to non-stomatal factors. Nevertheless, fungicides-induced depression of photosynthesis was related neither to a decrease in Rubisco carboxylation efficiency and in the capacity for regeneration of ribulose 1,5-bisphosphate nor to loss in PSII activity. However, fdx and fhd treatments generated repression of genes encoding proteins involved in the photosynthetic process. Indeed, decreased photosynthesis was coupled with repression of PsbP subunit of photosystem II (psbP1), chlorophyll a/b binding protein of photosystem I (cab) and Rubisco small subunit (rbcS) genes. A repression of these genes may participate in the photosynthesis alteration. To our knowledge, this is the first study of photosynthesis-related gene expression following fungicide stress. In the meantime, defense responses were followed by measuring chitinase activity and expression of varied defense-related genes encoding proteins involved in phenylpropanoid synthesis (PAL) or octadecanoid synthesis (LOX), as well as pathogenesis-related protein (Chi4C). No induction of defense was observed in botryticides-treated leaves. To conclude, the photosynthesis is affected without any triggering of plant defense responses.

Unique and Overlapping Expression Patterns among Members of Photosynthesis-Associated Nuclear Gene Families in Arabidopsis

Light provides crucial positional information in plant development, and the morphogenetic processes that are orchestrated by light signals are triggered by changes of gene expression in response to variations in light parameters. Control of expression of members of the RbcS and Lhc families of photosynthesis-associated nuclear genes by light cues is a paradigm for light-regulated gene transcription, but high-resolution expression profiles for these gene families are lacking. In this study, we have investigated expression patterns of members of the RbcS and Lhc gene families in Arabidopsis (Arabidopsis thaliana) at the cellular level during undisturbed development and upon controlled interference of the light environment. Members of the RbcS and Lhc gene families are expressed in specialized territories, including root tip, leaf adaxial, abaxial, and epidermal domains, and with distinct chronologies, identifying successive stages of leaf mesophyll ontogeny. Defined spatial and temporal overlap of gene expression fields suggest that the light-harvesting and photosynthetic apparatus may have a different polypeptide composition in different cells and that such composition could change over time even within the same cell.

An Optimized, Chemically Regulated Gene Expression System for Chlamydomonas

Background Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoter is repressed by copper and induced by nickel ions. However, induction by nickel is weak in some strains, poorly reversible by chelating agents like EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas growth. Removal of these bottlenecks will encourage the wide use of this promoter as a chemically regulated gene expression system.MethodologyUsing a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to improve the strength and reversibility of CYC6 promoter induction. Use of the first intron of the RBCS2 gene or of a modified TAP medium increases the strength of CYC6 induction up to 20-fold. In the modified medium, induction is also obtained after addition of specific copper chelators, like TETA. At low concentrations (up to 10 µM) TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50 µM). Neither TETA nor Ni show toxicity effects at the concentrations used. Unlike induction by Ni, induction by TETA is completely reversible by micromolar copper concentrations, thus resulting in a transient “wave” in luciferase activity, which can be repeated in subsequent growth cycles.ConclusionsWe have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled “waves” in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growth media, is a reliable and economically sustainable system for the temporally controlled expression of foreign genes in Chlamydomonas.