rbcS Gene Research Articles

Ultrastructural organization of chloroplasts and mitochondria of transgenic tomato plants expressing the FeSOD1 gene from Arabidopsis thaliana (L.) Heynh. under salt stress

Ultrastructural organization of chloroplasts and mitochondria of transgenic tomato plants expressing the FeSOD1 gene from Arabidopsis thaliana (L.) Heynh. with the pea rbcS gene leader sequence under chloride and sulfate salinity has been studied. Dark respiration of transgenic tomato regenerants under stress conditions has been estimated. It has been established that expression of the FeSOD1 gene in transgenic tomato plants under salinity leads to a decrease in damage of ultrastructure organization of plastids and mitochondria and affects the size and shape of their inclusions, suggesting a protective effect of this gene under salinity. The obtained experimental data show the effectiveness of using a genetic engineering strategy of redirection and subsequent accumulation of cytoplasmic Fe-dependent SOD from Arabidopsis thaliana (L.) Heynh. in chloroplasts for increasing the tolerance of crops to salt stress.

Targeted quantitative analysis of a diurnal RuBisCO subunit expression and translation profile in Chlamydomonas reinhardtii introducing a novel Mass Western approach

RuBisCO catalyzes the rate-limiting step of CO2 fixation in photosynthesis. Hypothetical mechanisms for the regulation of rbcL and rbcS gene expression assume that both large (LSU) and small (SSU) RuBisCO subunit proteins (RSUs) are present in equimolar amounts to fit the 1:1 subunit stoichiometry of the holoenzyme. However, the actual quantities of the RSUs have never been determined in any photosynthetic organism. In this study the absolute amount of rbc transcripts and RSUs was quantified in Chlamydomonas reinhardtii grown during a diurnal light/dark cycle. A novel approach utilizing more reliable protein stoichiometry quantification is introduced. The rbcL:rbcS transcript and protein ratios were both 5:1 on average during the diurnal time course, indicating that SSU is the limiting factor for the assembly of the holoenzyme. The oscillation of the RSUs was 9h out of phase relative to the transcripts. The amount of rbc transcripts was at its maximum in the dark while that of RSUs was at its maximum in the light phase suggesting that translation of the rbc transcripts is activated by light as previously hypothesized. A possible post-translational regulation that might be involved in the accumulation of a 37-kDa N-terminal LSU fragment during the light phase is discussed. A novel MS based approach enabling the exact stoichiometric analysis and absolute quantification of protein complexes is presented in this article. The application of this method revealed new insights in RuBisCO subunit dynamics.

The response of rbcL, rbcS and rca genes in cucumber (Cucumis sativus L.) to growth and induction light intensity

Low light is one of the most important factors affecting cucumber growth in winter solar greenhouses in northern China. Whether induction light intensity has an effect on expressions of rbcL, rbcS and rca genes in cucumber leaves grown under low light for long periods of time remains unclear. Here, two genotypes of cucumber were used to determine the effects of growth and induction light levels on photosynthetic induction. It was found that regardless of growth and induction light intensity, the transcript levels of rbcL, rbcS and rca increased quickly once the plant was exposed to light. The variations of the photosynthesis (P N), stomatal conductance (g s), actual photosystem II efficiency (ΦPS II) and Rubisco activity were similar to transcript levels. Plants grown at a photon flux density (PFD) of 100 μmol m−2 s−1 had lower P N, and initial and total Rubisco activity than those grown at 600 μmol m−2 s−1, regardless of induction PFD; for plants grown at the same PFD, P N, g s and Rubisco activation rate induced under PFD of 100 μmol m−2 s−1 were lower than those under 600 μmol m−2 s−1. Rubisco, especially RCA, played a great role on photosynthesis during photosynthetic induction. Compared with Jinyan No. 2, Deltastar may possess potential low-light tolerance with a lower decrease of P N, and initial and total Rubisco activities when grown and induced at 100 μmol m−2 s−1 than 600 μmol m−2 s−1. Moreover, the mechanism of Rubisco activase in regulating Rubisco during photosynthetic induction is also discussed.

Cytonuclear evolution of rubisco in four allopolyploid lineages.

Allopolyploidization in plants entails the merger of two divergent nuclear genomes, typically with only one set (usually maternal) of parental plastidial and mitochondrial genomes and with an altered cytonuclear stoichiometry. Thus, we might expect cytonuclear coevolution to be an important dimension of allopolyploid evolution. Here, we investigate cytonuclear coordination for the key chloroplast protein rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), which is composed of nuclear-encoded, small subunits (SSUs) and plastid-encoded, large subunits. By studying gene composition and diversity as well as gene expression in four model allopolyploid lineages, Arabidopsis, Arachis, Brassica, and Nicotiana, we demonstrate that paralogous nuclear-encoded rbcS genes within diploids are subject to homogenization via gene conversion and that such concerted evolution via gene conversion characterizes duplicated genes (homoeologs) at the polyploid level. Many gene conversions in the polyploids are intergenomic with respect to the diploid progenitor genomes, occur in functional domains of the homoeologous SSUs, and are directionally biased, such that the maternal amino acid states are favored. This consistent preferential maternal-to-paternal gene conversion is mirrored at the transcriptional level, with a uniform transcriptional bias of the maternal-like rbcS homoeologs. These data, repeated among multiple diverse angiosperm genera for an important photosynthetic enzyme, suggest that cytonuclear coevolution may be mediated by intergenomic gene conversion and altered transcription of duplicated, now homoeologous nuclear genes.

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on a HAB causing Alexandrium tamarense

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on Alexandrium tamarense were measured through studying the algicidal procedure, nuclear damage and transcription of related genes. Medium components were optimized to improve algicidal activity, and characteristics of algicidal extracts were determined. Transmission electron microscope analysis revealed that the cell structure was broken. Cell membrane integrity destruction and nuclear structure degradation were monitored using confocal laser scanning microscope, and the rbcS, hsp and proliferating cell nuclear antigen (PCNA) gene expressions were studied. Results showed that 1.0% tryptone, 0.4% glucose and 0.8% MgCl2 were the optimal nutrient sources. The algicidal extracts were heat and pH stable, non-protein and less than 1kD. Cell membrane and nuclear structure integrity were lost, and the transcription of the rbcS and PCNA genes were significantly inhibited and there was up-regulation of hsp gene expression during the exposure procedure. The algicidal extracts destroyed the cell membrane and nuclear structure integrity, inhibited related gene expression and, eventually, lead to the inhibition of algal growth. All the results may elaborate firstly the cell death process and nuclear damage in A. tamarense which was induced by algicidal extracts, and the algicidal extracts could be potentially used as bacterial control of HABs in future.

Delayed expression of SAGs correlates with longevity in CMS wheat plants compared to its fertile plants.

Reproductive sinks regulate monocarpic senescence in crop plants. Monocarpic senescence was studied in wheat fertile (cv. HW 2041) and its isonuclear cytoplasmic male sterile (CMS) line. CMS plants exhibited slower rate of senescence accompanied by longer green leaf area duration and slower deceleration in chlorophyll, protein content, PN and rubisco content coupled with lower protease activities than fertile (F) plants. CMS plants also exhibited lower ROS levels and less membrane damage than F plants. CMS plants maintained better antioxidant defense, less oxidative damage in chloroplast and higher transcript levels of both rbcL and rbcS genes during senescence than F plants. F plants exhibited early induction and higher expression of SAGs like serine and cysteine proteases, glutamine synthetases GS1 and GS2, WRKY53 transcription factor and decline in transcript levels of CAT1 and CAT2 genes than CMS plants. Hence, using genetically fertile and its CMS line of wheat it is confirmed that delayed senescence in the absence of reproductive sinks is linked with slower protein oxidation, rubisco degradation and delayed activation of SAGs. Better antioxidant defense in chloroplasts at later stages of senescence was able to mitigate the deleterious effects of ROS in CMS plants. We propose that delayed increase in ROS in cytoplasmic male sterile wheat plants resulted in delayed activation of WRKY53, SAGs and the associated biochemical changes than fertile plants.

Exploring mechanisms linked to differentiation and function of dimorphic chloroplasts in the single cell C4 species Bienertia sinuspersici.

BackgroundIn the model single-cell C4 plant Bienertia sinuspersici, chloroplast- and nuclear-encoded photosynthetic enzymes, characteristically confined to either bundle sheath or mesophyll cells in Kranz-type C4 leaves, all occur together within individual leaf chlorenchyma cells. Intracellular separation of dimorphic chloroplasts and key enzymes within central and peripheral compartments allow for C4 carbon fixation analogous to NAD-malic enzyme (NAD-ME) Kranz type species. Several methods were used to investigate dimorphic chloroplast differentiation in B. sinuspersici.ResultsConfocal analysis revealed that Rubisco-containing chloroplasts in the central compartment chloroplasts (CCC) contained more photosystem II proteins than the peripheral compartment chloroplasts (PCC) which contain pyruvate,Pi dikinase (PPDK), a pattern analogous to the cell type-specific chloroplasts of many Kranz type NAD-ME species. Transient expression analysis using GFP fusion constructs containing various lengths of a B. sinuspersici Rubisco small subunit (RbcS) gene and the transit peptide of PPDK revealed that their import was not specific to either chloroplast type. Immunolocalization showed the rbcL-specific mRNA binding protein RLSB to be selectively localized to the CCC in B. sinuspersici, and to Rubisco-containing BS chloroplasts in the closely related Kranz species Suaeda taxifolia. Comparative fluorescence analyses were made using redox-sensitive and insensitive GFP forms, as well comparative staining using the peroxidase indicator 3,3-diaminobenzidine (DAB), which demonstrated differences in stromal redox potential, with the CCC having a more negative potential than the PCC.ConclusionsBoth CCC RLSB localization and the differential chloroplast redox state are suggested to have a role in post-transcriptional rbcL expression.

Cloning and differential expression analysis of a newrbcSgene fromLemna gibba

A novel Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit gene (named ssu4d) was cloned from Lemna gibba by a novel chromosome walking technology. The full-length of ssu4d cDNA (named ssu4dc), contained a 522 bp open reading frame encoding a protein of 174 amino acids. Sequence analysis of ssu4dc and ssu4d showed that ssu4d contained an intron between +355 nt to +1125 nt downstream of transcriptional iniative site. ssu4dc contained 54 bp of 5′ untranslated region (UTR), and an open reading frame of 174 amino acids consisting of a chloroplast transit peptide with 57 amino acids and a mature protein of 117 amino acids. The deduced amino acid sequence of ssu4dc shared 95–96% identity with L. gibba RbcS protein. Real time-PCR analysis showed differential expression of individual rbcS genes in light-grown Lemna gibba. And the levels of SSU4dc mRNA was regulated by the action of phytochrome, there was variability in the amount of expression of SSU4dc RNA comprared to the SSU1 and SSU5B from Lemna gibba.

Unusual Small Subunit That Is Not Expressed in Photosynthetic Cells Alters the Catalytic Properties of Rubisco in Rice

Rubisco small subunits (RbcSs) are encoded by a nuclear multigene family in plants. Five RbcS genes, OsRbcS1, OsRbcS2, OsRbcS3, OsRbcS4, and OsRbcS5, have been identified in rice (Oryza sativa). Among them, the amino acid sequence of OsRbcS1 differs notably from those of other rice RbcSs. Phylogenetic analysis showed that OsRbcS1 is genetically distant from other rice RbcS genes and more closely related to RbcS from a fern and two woody plants. Reverse transcription-PCR and promoter β-glucuronidase analyses revealed that OsRbcS1 was not expressed in leaf blade, a major photosynthetic organ in rice, but was expressed in leaf sheath, culm, anther, and root central cylinder. In leaf blade of transgenic rice overexpressing OsRbcS1 and leaf sheath of nontransgenic rice, OsRbcS1 was incorporated into the Rubisco holoenzyme. Incorporation of OsRbcS1 into Rubisco increased the catalytic turnover rate and Km for CO2 of the enzyme and slightly decreased the specificity for CO2, indicating that the catalytic properties were shifted to those of a high-activity type Rubisco. The CO2 assimilation rate at low CO2 partial pressure was decreased in overexpression lines but was not changed under ambient and high CO2 partial pressure compared with nontransgenic rice. Although the Rubisco content was increased, Rubisco activation state was decreased in overexpression lines. These results indicate that the catalytic properties of Rubisco can be altered by ectopic expression of OsRbcS1, with substantial effects on photosynthetic performance in rice. We believe this is the first demonstration of organ-specific expression of individual members of the RbcS gene family resulting in marked effects on Rubisco catalytic activity.

Rootstocks improve cucumber photosynthesis through nitrogen metabolism regulation under salt stress

We examined the growth, photosynthetic parameters, initial and total ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, the relative expression of rbcL, rbcS, and rca gene, and nitrogen metabolism of cucumber (Cucumis sativus L. cv. Jinchun No.2, CS) plants grafted onto figleaf gourd (Cucurbita ficifolia Bouche, CF) and pumpkin (Cucurbita moschata Duch. cv. Chaojiquanwang, CM) rootstocks. Growth inhibition under salt stress (90 mM NaCl) was characterized by the irreversible inhibition of CO2 assimilation in the cucumber plants grafted onto cucumber rootstocks (CS/CS). In contrast, this effect was significantly alleviated by grafting the cucumber plants onto the CF and CM roots (CS/CF, CS/CM). Under NaCl stress, the CS/CF and CS/CM plants exhibited higher photosynthetic activity, higher initial and total Rubisco activity, and higher Rubisco-related gene expression than the CS/CS plants. Salinity resulted in a lesser increase in nitrate content and decrease in free amino acid content in the CS/CF and the CS/CM plants compared with the CS/CS plants. Accordingly, the activity of nitrate reductase, glutamine synthetase, and glutamate synthase decreased significantly, especially in the CS/CS plants. These results suggest that grafting cucumber plants onto salt-tolerant rootstocks enhances Rubisco activity and the expression of Rubisco-related genes by effectively accelerating nitrate transformation into amino acids under NaCl stress, thereby improving the photosynthetic performance of cucumber leaves.

Structural and Transcriptional Characterization of rbcS Genes of Cotton (Gossypium hirsutum)

Ribulose bis-carboxylase/oxygenase small subunit genes (rbcS) are found to be highly expressed in most plant species. Promoters of rbcS genes have been isolated, characterized, and used to enhance expression of transgenes in transgenic lines. In this study, we have identified five different members of the rbcS gene family in cotton (Gossypium hirsutum). All five genes contain three exons and two introns. Nucleotide sequences of the rbcS genes are conserved in their coding regions, but exhibit considerable sequence divergence in both introns and in upstream and downstream flanking sequences. Based on sequence identity, these genes are grouped into two subfamilies. Promoters of all rbcS genes have been isolated, and their cis-acting motifs, exhibiting different arrangements, have been identified. All five rbcS genes are differentially expressed in leaf and boll tissues of cotton plants at different stages of plant development. Three of the five isolated rbcS genes are found to be highly expressed in analyzed tissues. Multiple transcriptional start sites of all members of the gene family have been identified in leaf and boll tissues of cotton plants. Moreover, different members of the rbcS gene family from the two progenitor diploid species of G. hirsutum, namely, Gossypium herbaceum and Gossypium raimondii, have also been isolated to identify the lineage of the five rbcS genes. In addition, a total of three candidate promoters that can be used to enhance expression of transgenes in cotton have been identified.

Availability of Rubisco small subunit up-regulates the transcript levels of large subunit for stoichiometric assembly of its holoenzyme in rice.

Rubisco is composed of eight small subunits coded for by the nuclear RBCS multigene family and eight large subunits coded for by the rbcL gene in the plastome. For synthesis of the Rubisco holoenzyme, both genes need to be expressed coordinately. To investigate this molecular mechanism, the protein synthesis of two subunits of Rubisco was characterized in transgenic rice (Oryza sativa) plants with overexpression or antisense suppression of the RBCS gene. Total RBCS and rbcL messenger RNA (mRNA) levels and RBCS and RbcL synthesis simultaneously increased in RBCS-sense plants, although the increase in total RBCS mRNA level was greater. In RBCS-antisense plants, the levels of these mRNAs and the synthesis of the corresponding proteins declined to a similar extent. The amount of RBCS synthesized was tightly correlated with rbcL mRNA level among genotypes but not associated with changes in mRNA levels of other major chloroplast-encoded photosynthetic genes. The level of rbcL mRNA, in turn, was tightly correlated with the amount of RbcL synthesized, the molar ratio of RBCS synthesis to RbcL synthesis being identical irrespective of genotype. Polysome loading of rbcL mRNA was not changed. These results demonstrate that the availability of RBCS protein up-regulates the gene expression of rbcL primarily at the transcript level in a quantitative manner for stoichiometric assembly of Rubisco holoenzyme.

Isolation and Functional Analysis of the Poplar RbcS Gene Promoter

A tissue-specific promoter, Pt-RbcS, from Populus was isolated and cloned based on alignment of AtRBCS-2B cDNA with genomic Populus sequences. Sequence analysis of Pt-RbcS revealed cis-acting regulatory elements in the promoter region, including an ATCT-motif, BoxI, GAG-motif, I-box, G-box, BoxII, GATA-motif, and TCT-motif, which are involved in light responses. In transgenic tobacco lines carrying the β-glucuronidase (GUS) gene driven by the Pt-RbcS promoter, GUS expression was detected in leaves and stems, but not in roots. Transgenic poplar lines harboring constructs carrying the GUS gene driven by truncated Pt-RbcS promoters revealed distinctive expression patterns for five different promoter constructs. The Pt-RbcS promoter was expressed preferentially in photosynthetic tissues such as leaves and stems. Moreover, deletion analysis of the 1,547 bp Pt-RbcS promoter region revealed that a 927-bp DNA segment is critical for expression of Pt-RbcS in green tissues. Overall, our study suggests that the Pt-RbcS promoter from Populus could be applied to genetically improve the photosynthetic efficiency of woody plants.

Ectopic Expression of Rubisco Subunits in Maize Mesophyll Cells Does Not Overcome Barriers to Cell Type-Specific Accumulation

In maize (Zea mays), Rubisco accumulates in bundle sheath but not mesophyll chloroplasts, but the mechanisms that underlie cell type-specific expression are poorly understood. To explore the coordinated expression of the chloroplast rbcL gene, which encodes the Rubisco large subunit (LS), and the two nuclear RBCS genes, which encode the small subunit (SS), RNA interference was used to reduce RBCS expression. This resulted in Rubisco deficiency and was correlated with translational repression of rbcL. Thus, as in C3 plants, LS synthesis depends on the presence of its assembly partner SS. To test the hypothesis that the previously documented transcriptional repression of RBCS in mesophyll cells is responsible for repressing LS synthesis in mesophyll chloroplasts, a ubiquitin promoter-driven RBCS gene was expressed in both bundle sheath and mesophyll cells. This did not lead to Rubisco accumulation in the mesophyll, suggesting that LS synthesis is impeded even in the presence of ectopic SS expression. To attempt to bypass this putative mechanism, a ubiquitin promoter-driven nuclear version of the rbcL gene was created, encoding an epitope-tagged LS that was expressed in the presence or absence of the Ubi-RBCS construct. Both transgenes were robustly expressed, and the tagged LS was readily incorporated into Rubisco complexes. However, neither immunolocalization nor biochemical approaches revealed significant accumulation of Rubisco in mesophyll cells, suggesting a continuing cell type-specific impairment of its assembly or stability. We conclude that additional cell type-specific factors limit Rubisco expression to bundle sheath chloroplasts.

The Cytonuclear Dimension of Allopolyploid Evolution: An Example from Cotton Using Rubisco

During allopolyploid speciation, two divergent nuclear genomes merge, yet only one (usually the maternal) of the two sets of progenitor organellar genomes is maintained. Rubisco (1,5-bisphosphate carboxylase/oxygenase) is composed of nuclear-encoded small subunits (SSUs) and plastome-encoded large subunits (LSUs), providing an ideal system to explore the evolutionary process of cytonuclear accommodation. Here, we take initial steps in this direction, using Gossypium allopolyploids as our model. SSU copies from divergent (5-10 My) progenitor diploids ("A" and "D" genomes) were combined at the time of polyploid formation 1-2 Ma, with the LSU encoded by the maternal A-genome parent. LSU genes from A- and D-genome diploids and AD-genome allopolyploids were sequenced, revealing several nonsynonymous substitutions and suggesting the possibility of differential selection on the nuclear-encoded rbcS partner following allopolyploid formation. Sequence data for the rbcS gene family revealed nonreciprocal homoeologous recombination between A- and D-rbcS homoeologs in all polyploid species but not in a synthetic intergenomic F1 hybrid, demonstrating "gene conversion" during allopolyploid evolution. All progenitor rbcS genes are retained and expressed in the five extant allopolyploid species, but analysis of the leaf transcriptome showed that A-homoeologs are preferentially expressed in both the allopolyploid and hybrid, consistent with the maternal origin of rbcL. Although rbcS genes from both progenitor genomes are expressed, some appear to have experienced mutations that may represent cytonuclear coevolution.

Sequence analysis and potentials of the native RbcS promoter in the development of an alternative eukaryotic expression system using green Microalga Ankistrodesmus convolutus.

The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of β-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5′-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.

Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalga Ankistrodesmus convolutus

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.

The Feasibility Analysis of PVX and TRV Vectors as the VIGS Tool for Studying the Gene Function

Abstract A method of virus-induced post-transcriptional gene silencing for studying rbcS gene function was established and optimized using tobacco rattle virus vector and potato X virus vector as materials in this study. The following analyses were conducted: phenotypic characterization of rbcS gene silenced plants, transcription levels of rbcS gene by RT-PCR, and protein levels of rbcS by the antibodies of rbcS. For feasibility assessment of the PVX and TRV vectors on the VIGS for studying gene function, the statistical analysis was first employed in which it contained the two-factor variance analysis model and F-test based upon a large amount of data of the response of photosynthetic rates, the photosynthetic active radiation intensity and CO2 concentrations generated from the rbcS silenced and control plants. Our findings suggested that TRV vector was superior to PVX vector.

Effect of individual suppression of RBCS multigene family on Rubisco contents in rice leaves

In higher plants, a small subunit of Rubisco is encoded for by an RBCS multigene family in the nuclear genome. However, it is unknown how each multigene member contributes to the accumulation of Rubisco holoenzyme. Here, four RBCS genes that are highly expressed in leaf blaedes of rice (Oryza sativa L.) were individually suppressed by RNAi, and the effects on leaf Rubisco content were examined at seedling, vegetative and reproductive stages. Rubisco contents in each transgenic line declined irrespective of growth stage, and the ratios of Rubisco-N to total N were 66-96% of wild-type levels. The mRNA levels of the suppressed RBCS genes declined significantly, whereas those of the unsuppressed ones did not change drastically. These results indicate that four RBCS genes all contribute to accumulation of Rubisco holoenzyme irrespective of growth stage and that suppression of one RBCS gene is not fully compensated by other RBCS genes. Additionally, the mRNA levels of the large subunit of Rubisco showed a change similar to that of total RBCS mRNA level irrespective of genotype and growth stage. These results suggest that gene expression of RBCS and RBCL is regulated in a coordinated manner at the transcript level in rice.

Detection of the toxic marine diatom Pseudo-nitzschia multiseries using the RuBisCO small subunit ( rbcS) gene in two real-time RNA amplification formats

The marine diatom genus Pseudo-nitzschia includes species that produce domoic acid, a neurotoxin responsible for illness and mortality in both humans and marine wildlife. Because of the expertise and time required for the microscopic discrimination of species, molecular methods that monitor environmental concentrations of Pseudo-nitzschia provide a rapid alternative for the early detection of blooms and prediction of toxin accumulation. We have developed a nucleic acid sequence-based amplification with internal control RNA (IC-NASBA) assay and a quantitative reverse transcription PCR (qRT-PCR) assay for the detection of the toxic species Pseudo-nitzschia multiseries targeting the ribulose-1,5-biphosphate carboxylase/oxygenase small subunit ( rbcS) gene. Both methods use RNA amplification and fluorescence-based real-time detection. Due to a limited rbcS sequence database, primers were designed and used to sequence this gene from 14 strains of Pseudo-nitzschia (representing six species) and 19 other marine diatoms. The IC-NASBA and qRT-PCR assays had a limit of detection of one cultured cell of P. multiseries and were linear over four and five orders of magnitude, respectively ( r 2 ≥ 0.98). Neither of the assays detected closely related organisms outside the Pseudo-nitzschia genus, and the qRT-PCR assay was specific to P. multiseries. While cross-reactivity of primers with unknown species prevented reliable detection of P. multiseries in spiked environmental samples using IC-NASBA, the qRT-PCR assay had positive detection from 10 7 cells/L to 10 3 cells/L. Nearly a 1:1 relationship was observed between predicted and calculated cell concentrations using qRT-PCR. Based on a diel expression study, the rbcS transcript copy number per cell ranged from 2.16 × 10 4 to 5.35 × 10 4, with the highest expression during early to mid photoperiod. The rbcS qRT-PCR assay is useful for the detection and enumeration of low concentrations of P. multiseries in the environment.