rbcS Transcripts Research Articles

Targeted knockdown of ribulose-1, 5-bisphosphate carboxylase-oxygenase in rice mesophyll cells

We generated antisense constructs targeting two of the five Rubisco small subunit genes (OsRBCS2 and 4) which account for between 30–40 % of the RBCS transcript abundance in leaf blades. The constructs were driven by a maize phosphoenolpyruvate carboxylase (PEPC) promoter known to have enriched expression in mesophyll cells (MCs). In the resulting lines leaf, Rubisco protein content was reduced by between 30–50 % and CO2 assimilation rate was limited under photorespiratory and non-photorespiratory conditions. A relationship between Rubisco protein content and CO2 assimilation rate was found. This was associated with a significant reduction in dry biomass accumulation and grain yield of between 37–70%. In addition to serving as a resource for reducing Rubisco accumulation in a cell-preferential manner, these lines allow us to characterize gene function and isoform specific suppression on photosynthesis and growth. Our results suggest that the knockdown of multiple genes is required to completely reduce Rubisco accumulation in MCs.

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.

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.

Normalization using ploidy and genomic DNA copy number allows absolute quantification of transcripts, proteins and metabolites in cells

BackgroundQuantification of transcripts, proteins, or metabolites is straightforward when the factor used to normalize these values remains constant between samples. However, normalization factors often vary among samples and thus must be developed for each new analytical method.ResultsWe demonstrate quantification of transcript and protein levels in Arabidopsis based on genomic DNA copy number. We extracted total nucleic acid from 3-week-old rosette leaves of wild-type Arabidopsis and the pale-green/dwarf mutant, abc4, and quantified the number of transcripts by quantitative reverse-transcription PCR using genomic DNA copy number and ploidy (as determined by cytometry) for normalization. Our data indicated that normalization using genes commonly employed as references resulted in inaccuracies in transcript levels of the genes RBC-L and RBC-S (encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase) in wild type and mutant. Normalization using genomic DNA copy number and ploidy, however, appropriately showed that the RBC-L and RBC-S transcript levels per cell in the mutant were significantly lower than that in wild type. Furthermore, quantification revealed that a cell of a 3-week-old wild-type Arabidopsis rosette leaf had an average of 7.5 × 103 transcripts of RBC-L, 9.9 × 103 transcripts of RBC-S, and 1.4 × 106 18S rRNA. We similarly analyzed the accumulation of RBC-L and LHCP (light-harvesting chlorophyll a/b protein) in wild type and mutant based on ploidy and genomic DNA copy number that was determined by direct quantitative PCR analysis of extracts using a DNA polymerase tolerant to a wide range of common PCR inhibitors. Furthermore, we estimated the number of RBC-L molecules (2.63 × 108) and chlorophyll molecules (1.85 × 109) in each cell in 3-week-old wild-type rosette leaves; these values had relatively low coefficients of variation, underscoring the reliability of our method.ConclusionGenomic DNA copy number and ploidy are useful as general normalization factors, providing an easy method for determining the number of transcripts, proteins, and metabolites in a cell.

Tocopherol deficiency in transgenic tobacco (Nicotiana tabacum L.) plants leads to accelerated senescence

alpha-Tocopherol constitutes the major lipophilic antioxidant in thylakoid membranes, which cooperates with the soluble antioxidant system to alleviate oxidative stress caused by reactive oxygen species (ROS) during oxygenic photosynthesis. Tocopherol accumulates during leaf senescence, indicating the necessity for increased redox buffer capacity in senescent leaves, and tocopherol deficiency has been shown to restrict sugar export from source leaves by inducing callose plugging in the vasculature. We have generated tocopherol-deficient tobacco plants that contain as few as 1% of wild-type (WT) tocopherol in leaves by silencing homogentisate phytyltransferase (HPT). Employing HPT : RNAi plants, we have assessed the importance of tocopherol during leaf senescence and for sugar export. Irrespective of whorl position, the content of free sugars and starch was lower in HPT : RNAi leaves than in WT during the vegetative phase, and no accumulation of callose or a reduction in sugar exudation compared to WT was evident. Based on our observations, we discuss lipid peroxidation as a potential modulator of tocopherol-mediated signalling. Furthermore, senescence was accelerated in lower leaves of HPT transgenics, as indicated by elevated GS1 and reduced rbcS transcript amounts. Oxidative stress was increased in virescent lower source leaves, suggesting that the lack of tocopherol triggers premature senescence.

Cytokinin Import Rate as a Signal for Photosynthetic Acclimation to Canopy Light Gradients

Plants growing in dense canopies are exposed to vertical light gradients and show photosynthetic acclimation at the whole-plant level, resulting in efficient photosynthetic carbon gain. We studied the role of cytokinins transported through the transpiration stream as one of probably multiple signals for photosynthetic acclimation to light gradients using both tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). We show that substantial variation in leaf transpiration parallels the light gradient in tobacco canopies and experimental reduction of the transpiration rate of a leaf, independent of light, is sufficient to reduce photosynthetic capacity in both species, as well as transcript levels of the small subunit of Rubisco (rbcS) gene in Arabidopsis. Mass spectrometric analysis of xylem sap collected from intact, transpiring tobacco plants revealed that shaded leaves import less cytokinin than leaves exposed to high light. In Arabidopsis, reduced transpiration rate of a leaf in the light is associated with lower cytokinin concentrations, including the bioactive trans-zeatin and trans-zeatin riboside, as well as reduced expression of the cytokinin-responsive genes ARR7 and ARR16. External application of cytokinin to shaded leaves rescued multiple shade effects, including rbcS transcript levels in both species, as did locally induced cytokinin overproduction in transgenic tobacco plants. From these data, we conclude that light gradients over the foliage of a plant result in reduced cytokinin activity in shaded leaves as a consequence of reduced import through the xylem and that cytokinin is involved in the regulation of whole-plant photosynthetic acclimation to light gradients in canopies.

Genotypic Variation of Rubisco Expression, Photosynthetic Electron Flow and Antioxidant Metabolism in the Chloroplasts of Chill-exposed Cucumber Plants

Genetic improvement of agronomic crops is necessary to cope with chilling stress. To identify the physiological factors responsible for this genotypic difference in chill-induced inhibition of photosynthesis, leaf CO2 assimilation, the electron flux in the chloroplast and the antioxidant metabolism in isolated chloroplasts were examined in two genotypes of cucumber (Cucumis sativus) plants with distinct chilling tolerance. Cucumber plants were exposed to 100 micromol m(-2) s(-1) at 9/7 degrees C (day/night) for 10 d and were then returned to optimal conditions for 2 d. Chilling resulted in more significant reductions in rbcL and rbcS transcripts, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) content and initial Rubisco activity, leading to higher electron flux to O2 in the chilling-sensitive genotype than in the chilling-tolerant genotype. The chilling-tolerant genotype showed lower H2O2 contents in the chloroplasts by maintaining higher H2O2-scavenging activity in the chloroplasts than in the chilling-sensitive genotype. H2O2 accumulation in chloroplast was negatively correlated with the initial Rubisco activity and photosynthetic rate.

Synthesis of Rubisco gene products is upregulated by increasing the copy number of rbcL gene in Chlamydomonas chloroplast genome, without increased accumulation of the two Rubisco subunits

In an attempt to increase the amount of Rubisco in a green alga, the Chlamydomonas reinhardtii rbcL gene connected to the aadA cassette was integrated into chloroplast inverted repeats in this organism to generate a transformant with three copies of rbcL (3L). Synthesis and accumulation of the rbcL transcript in 3L were 2.3 and 1.4 times those of the control, respectively. Although synthesis of the large subunit (LSU) in 3L was 2.6 times that in the control, accumulation of the LSU was only 1.1 times that in the control. Synthesis of rbcS transcript and the small subunit (SSU) in 3L was also upregulated to 1.7 and 1.3 times those in the control, respectively, while their accumulation was unaffected. These results suggest that an increase in rbcL gene number upregulates transcription of rbcL from chloroplast genomes and that of rbcS from a nuclear genome, but does not affect the accumulation of LSU and SSU.

Cytokinin Effects on Ribulose‐1,5‐Bisphosphate Carboxylase/Oxygenase and Nitrogen Partitioning in Rice during Ripening

High yield rice (Oryza sativa L.) cultivar Akenohoshi can realize a higher rate of photosynthesis during ripening by maintaining a higher level of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) than cv. Nipponbare. To clarify the causal factors for the difference in the levels of Rubisco in leaves between rice cultivars and the mechanisms of action of cytokinins on the maintenance of high levels of Rubisco in leaves, the level of rbcL and rbcS transcripts and the nitrogen content of leaves were investigated. The Rubisco content remained high not only in plants given additional nitrogen fertilizer (NF) but also in plants sprayed with 6-benzylaminopurine (BA). There was a close correlation between the Rubisco content and the level of rbcL and rbcS mRNAs. The maintenance of high leaf levels of Rubisco and of rbcL and rbcS mRNAs was related to the high nitrogen content in leaves, irrespective of the treatments and cultivars. The maintenance of high leaf nitrogen content was caused by an increase in nitrogen partitioning to leaves and not by an increase in nitrogen absorption by plants treated with BA. These results indicate that there are two probable effects of BA on the leaf level of Rubisco. One is the direct effect of BA inducing the synthesis of Rubisco. The other is the indirect effect of BA inducing the effective partitioning of nitrogen to leaves. Cytokinins could account for the difference in the reduction in leaf level of Rubisco during senescence between the rice cultivars.

Diurnal changes of Rubisco in response to elevated CO 2, temperature and nitrogen in wheat grown under temperature gradient tunnels

Growth at elevated CO 2 and temperature often leads to decreased Rubisco activity. We investigated the effects of increased CO 2, temperature and nitrogen on the diurnal changes in the control of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity in wheat ( Triticum aestivum L.). Spring wheat was grown at ambient and 700 μmol mol −1 CO 2, under ambient and 4 °C warmer temperatures and with two levels of nitrogen supply in field tunnels in a Mediterranean environment. At ear emergence, elevated CO 2 increased Rubisco activation, but decreased Rubisco protein and, with high nitrogen, Rubisco specific activity and had no effect on the rbcS transcript. Warmer temperatures tended to decrease the rbcS mRNA level and Rubisco protein, although the effect on Rubisco activity was small. High nitrogen decreased Rubisco activation or specific activity, depending on the CO 2 concentration. It increased Rubisco protein at the end of the night, but accelerated its diurnal loss. The main changes after anthesis were the disappearance of the decrease in Rubisco specific activity caused by elevated CO 2, an increase in this activity with above-ambient temperatures combined with high nitrogen and that high temperature and nitrogen did not affect Rubisco protein contents. This study suggests that decreased leaf protein and increased levels of a Rubisco inhibitor, rather than gene repression by sugars, are involved in acclimation to elevated CO 2. High nitrogen increases this down regulation. Changes during growth in levels of leaf metabolites and protein may alter the relative importance of levels of inhibitors and Rubisco amounts for Rubisco regulation.

Molecular characterization of a gene encoding a vegetative storage protein (CiVSP) from Cichorium intybus and its expression in the root and shoot in relation to nitrogen status and pathogen resistance

In addition to their putative role in nitrogen storage, some vegetative storage proteins (VSPs) support further roles in biotic and abiotic stress responses. Functions of the 17 kDa VSP from witloof chicory (CiVSP) in N storage and plant resistance to pathogens and its regulation by nitrogen were investigated. The N‐terminal end of this protein was sequenced and the corresponding full‐length cDNA was obtained. The expression of the CiVsp gene was studied in various organs of chicory grown under replete or limited nitrogen supply. A strong expression of CiVsp was observed in both taproot and fine roots of mature plants and seedlings. CiVsp transcripts were also detected in mature leaves, especially in veins. In senescing leaves CiVsp transcripts accumulated concomitantly to a decrease in RbcS transcript abundance and Rubisco small‐subunit degradation. CiVSP protein accumulated significantly only in the subterranean part of the plant during late stages of development. Nitrate limitation caused a reduction in CiVsp mRNA accumulation and a delay in CiVSP storage in the taproot. It is concluded that CiVSP accumulation is regulated at the transcriptional level by N external supply and that the protein is involved in long and short‐term N storage. In silico analysis indicated that CiVSP is highly homologous with several allergens and PR‐10 proteins. Moreover, CiVsp transcript and protein expression were significantly higher in Erwinia carotovora‐resistant chicory inbred lines compared with susceptible lines, suggesting its involvement in chicory resistance to pathogens attack.

Rubisco expression in rice leaves is related to genotypic variation of photosynthesis under elevated growth CO2 and temperature

ABSTRACTGenetic modifications of agronomic crops will likely be necessary to cope with global climate change. This study tested the hypotheses that genotypic differences in rice (Oryza sativa L.) leaf photosynthesis at elevated [CO2] and temperature are related to protein and gene expression of Rubisco, and that high growth temperatures under elevated [CO2] negatively affect photosystem II (PSII) photochemical efficiency. Two rice cultivars representing an indica (cv. IR72) and japonica type (cv. M103) were grown in 350 (ambient) and 700 (elevated) µmol CO2 mol−1 at 28/18, 34/24 and 40/30 °C sinusoidal maximum/minimum, day/night temperatures in outdoor, sunlit, environment‐controlled chambers. Leaf photosynthesis of IR72 favoured higher growth temperatures more than M103. Rubisco total activity and protein content were negatively affected in both genotypes by high temperatures and elevated CO2. However, at moderate to high growth temperatures, IR72 leaves averaged 71 and 39% more rbcS transcripts than M103 under ambient and elevated CO2, respectively, and likewise had greater Rubisco activity and protein content. Expression of psbA (D1 protein of PSII) in IR72 leaves increased with temperature, whereas it remained constant for M103, except for a 20% decline at 40/30 °C under elevated CO2. Even at the highest growth temperatures, PSII photochemical efficiency was not impaired in either genotype grown under either ambient or elevated CO2. Genotypic differences exist in rice for carboxylation responses to elevated CO2 and high temperatures, which may be useful in developing genotypes suited to cope with global climate changes.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose:fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light.

We describe a highly efficient two-step single-cell reverse transcriptase-polymerase chain reaction technique for analyzing gene expression at the single-cell level. Good reproducibility and a linear dose response indicated that the technique has high specificity and sensitivity for detection and quantification of rare RNA. Actin could be used as an internal standard. The expression of message for Rubisco small subunit (RbcS), chlorophyll a/b-binding protein (Cab), sucrose (Suc):fructan-6-fructosyl transferase (6-SFT), and Actin were measured in individual photosynthetic cells of the barley (Hordeum vulgare) leaf. Only Actin was found in the non-photosynthetic epidermal cells. Cab, RbcS, and 6-SFT genes were expressed at a low level in mesophyll and parenchymatous bundle sheath (BS) cells when sampled from plants held in dark for 40 h. Expression increased considerably after illumination. The amount of 6-SFT, Cab, and RbcS transcript increased more in mesophyll cells than in the parenchymatous BS cells. The difference may be caused by different chloroplast structure and posttranscriptional control in mesophyll and BS cells. When similar single-cell samples were assayed for Suc, glucose, and fructan, there was high correlation between 6-SFT gene expression and Suc and glucose concentrations. This is consistent with Suc concentration being the trigger for transcription. Together with earlier demonstrations that the mesophyll cells have a higher sugar threshold for fructan polymerization, our data may indicate separate control of transcription and enzyme activity. Values for the sugar concentrations of the individual cell types are reported.

Regulation of carbonic anhydrase gene expression in cotyledons of cotton (Gossypium hirsutum L.) seedlings during post-germinative growth.

Recently, plastidial carbonic anhydrase (CA) cDNA clones encoding functional carbonic anhydrase enzymes were isolated from a 48 h dark-grown cotton seedling (cotyledons) cDNA library (Hoang et al., Plant Cell Physiol. 40: 1999). Here we examined the levels of relative transcript abundance and enzyme activities in cotyledons at different developmental stages and under different environmental conditions (i.e. altering CO2 and light conditions), during post-germinative seedling growth. Relative CA transcript levels and total CA enzyme activity in cotyledons of cotton seedlings increased from 18 h to 72 h of post-germinative growth in the dark, although somewhat later than the glyoxylate cycle enzyme, MS. When 24 h old seedlings were exposed to light for an additional 24 h, CA activity in greening cotyledons increased about 2-fold (compared with controls kept in the dark), whereas relative CA transcript levels were essentially the same. Removal of seed coats from cotyledons of 24 h old seedlings dramatically increased relative CA transcript abundance (measured 24 h later) in the dark, but did not influence CA enzyme activity. Manipulation of external CO2 environments (zero, ambient, or high) modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activity. On the other hand, regardless of the external CO2 conditions, cotyledons of seedlings exposed to light exhibited increased CA activity, concomitant with increased Rubisco activity and increased chlorophyll content. Collectively, our data suggest that steady-state levels of CA and rbcS transcripts are increased in response to environmental CO2 conditions, while CA (and Rubisco) enzyme activities are likely modulated at the post-transcriptional level following exposure of seedlings to light, and in parallel with development of functional chloroplasts.

Screening for high CO2 responsive mutants of Arabidopsis

As atmospheric levels of CO2 continue to rise, there is significant interest in the mechanisms by which plants can respond to these changes. To identify specific genes involved in high CO2 acclimation, we have been screening for Arabidopsis mutants that exhibit a non-wild type response when exposed to elevated (1000 ppm) CO2 concentrations. Two classes of mutants have been characterized. The first, designated as CO2 non-responsive (cnr), do not display the typical stress responses of increased anthocyanin production, leaf cupping, and stunted growth found in wild type plants exposed to similar high CO2 levels. The second group of mutants are described as CO2 hyper-responsive (chr) in that their stress responses are more severe than wild type plants. Characterization of these mutants has involved an analysis of growth and morphology, photosynthetic capacity, foliar carbohydrate levels, and photosynthetic gene (ca1, cab, rbcS) transcript abundance. The relationship between the high CO2 phenotypes and growth in the presence of high levels of glucose/sucrose will also be described. Molecular characterization of two cnr mutants isolated from T-DNA tagged populations has identified two different mechanisms by which Arabidopsis is able to minimize a stress response to high level of CO2.

Soybean photosynthesis, Rubisco, and carbohydrate enzymes function at supraoptimal temperatures in elevated CO 2

Summary Soybean (Glycine max L. Merr. cv. Bragg) was grown season-long in eight sunlit, controlled–environment chambers at two daytime [CO2] of 350 (ambient) and 700 (elevated) µmol mol –1 . Dry bulb day/night maximum/minimum air temperatures, which followed a continuously and diurnally varying, near sine-wave control set point that operated between maximum (daytime, at 1500 EST) and minimum (nighttime, at 0700 EST) values, were controlled at 28/18 and 40/30 uC for the ambient-CO2 plants, and at 28/18, 32/22, 36/26, 40/30, 44/34 and 48/38 uC for the elevated-CO2 plants. The objective was to assess the upper threshold tolerance of photosynthesis and carbohydrate metabolism with increasing temperatures at elevated [CO2], as it is predicted that air temperatures could rise as much as 4–6 uC within the 21st century with a doubling of atmospheric [CO2]. Leaf photosynthesis measured at growth [CO2] and temperature was greater for elevated-CO2 plants and was highest at 32/22 uC, but markedly declined at temperatures above 40/30 uC. Growth temperatures from 28/18 to 40/30 uC had little effect on midday total activity and protein content of Rubisco, while higher temperatures substantially reduced them. Conversely, midday Rubisco rbcS transcript abundance declined with increasing temperatures from 28/18 to 48/38 uC. Elevated-CO2 plants exceeded the ambientCO2 plants in most aspects of carbohydrate metabolism. Under elevated [CO2], midday activities of ADPG pyrophosphorylase and sucrose-P synthase and invertase paralleled net increases in starch and sucrose contents, respectively. They were highest at 36/26–40/30 uC, but declined at higher or lower growth temperatures. Thus, in the absence of other climatic stresses, soybean photosynthesis and carbohydrate metabolism would perform well under rising atmospheric [CO2] and temperature predicted for the 21st century.

The role of inorganic phosphate in the development of freezing tolerance and the acclimatization of photosynthesis to low temperature is revealed by the pho mutants of Arabidopsis thaliana.

Low temperature inhibits sucrose synthesis, leading to a phosphate-limitation of photosynthesis. We have used the Arabidopsis pho1-2 and pho2-1 mutants with decreased and increased shoot phosphate, respectively, to investigate whether low phosphate triggers cold acclimatization of photosynthetic carbon metabolism. Wild-type Arabidopsis, pho1-2 and pho2-1 were grown at 23 degrees C and transferred to 5 degrees C to investigate acclimatization in pre-existing leaves and in new leaves developing at 5 degrees C. The development of frost tolerance and the accumulation of proline and sugars was unaltered or improved in pho1-2, and impaired in pho2-1. Sucrose phosphate synthase and cytoplasmic fructose-1,6-bisphosphatase activity and protein increase after transfer to 5 degrees C. This increase was accentuated in pho1-2 and attenuated in pho2-1. RBCS and LHCB2 transcript levels decrease in pre-formed wild-type leaves after transfer to 5 degrees C and recover in new leaves that develop at 5 degrees C. The initial decrease was attenuated in pho1-2, and accentuated in pho2-1, where the recovery in new leaves was also suppressed. Rubisco activity increased in wild-type leaves that developed at 5 degrees C. This increase was accentuated in pho1-2 and absent in pho2-1. NADP-glyceraldehyde-3-phosphate dehydrogenase, plastidic fructose-1,6-bisphosphatase and aldolase activity increase relative to phosphoglycerate kinase, transketolase and phosphoribulokinase in wild-type leaves at 5 degrees C. This shift was accentuated in pho1-2 and reversed in pho2-1. Transcript levels for COR genes increase transiently 1 day after transfer to 5 degrees C but were very low in leaves that developed at 5 degrees C in wild-type Arabidopsis, pho1-2 and pho2-1. We conclude that low phosphate plays an important role in triggering cold acclimatization of leaves, leading in particular to an increase of Rubisco expression, changes in other Calvin cycle enzymes to minimize sequestration of phosphate in metabolites, and increased expression of sucrose biosynthesis enzymes.

RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing.

Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transgene. In each instance, homology-based, RNA-directed methylation was associated with silencing. When the GFP-transcribed region was targeted, PTGS affected both transgene and viral RNA levels. When methylation was targeted to a promoter region, transgene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) by inoculation with potato virus X-rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP-transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transgene associated with systemic spread of a gene-silencing signal from the infiltrated part of the plant. This transgene methylation was not affected when systemic PTGS was blocked by suppressors of silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epigenetic model of PTGS in which transgene methylation is associated with an RNA-DNA interaction that ensures that PTGS is maintained.

C0 2 Enrichment Delays a Rapid, Drought-Induced Decrease in Rubisco Small Subunit Transcript Abundance

Rice (Oryza sativa L. cv. IR-72) was grown in sunlit chambers at 350 and 700 gmol CO2 mol-1 under conditions of continuous flooding (control) or drought which was imposed at panicle initiation, to evaluate the effects of C02 enrichment and soil water deficit on photosynthesis and Rubisco gene expression. Leaf and canopy photosynthetic rates were enhanced by high [CO2] but reduced by drought. High [CO2] and severe drought both reduced rbcS transcript abundance, along with the activity, activation and protein content of Rubisco, but the Km (CO2) was not affected. The transition from moderate to severe drought caused a rapid decline, within 24 h, in the rbcS transcript abundance. High [CO2], however, delayed the adverse effects of severe drought on rbcS transcript abundance and activities of Rubisco, and permitted photosynthesis to continue for an extra day during the drought-stress cycle.

Changes in growth CO2 result in rapid adjustments of ribulose-1, 5-bisphosphate Carboxylase/Oxygenase small subunit gene expression in expanding and mature leaves of rice

The accumulation of soluble carbohydrates resulting from growth under elevated CO2 may potentially signal the repression of gene activity for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS). To test this hypothesis we grew rice (Oryza sativa L.) under ambient (350 &mgr;L L-1) and high (700 &mgr;L L-1) CO2 in outdoor, sunlit, environment-controlled chambers and performed a cross-switching of growth CO2 concentration at the late-vegetative phase. Within 24 h, plants switched to high CO2 showed a 15% and 23% decrease in rbcS mRNA, whereas plants switched to ambient CO2 increased 27% and 11% in expanding and mature leaves, respectively. Ribulose-1,5-bisphosphate carboxylase/oxygenase total activity and protein content 8 d after the switch increased up to 27% and 20%, respectively, in plants switched to ambient CO2, but changed very little in plants switched to high CO2. Plants maintained at high CO2 showed greater carbohydrate pool sizes and lower rbcS transcript levels than plants kept at ambient CO2. However, after switching growth CO2 concentration, there was not a simple correlation between carbohydrate and rbcS transcript levels. We conclude that although carbohydrates may be important in the regulation of rbcS expression, changes in total pool size alone could not predict the rapid changes in expression that we observed.