Rubisco content and photosynthesis of leaves at different positions in transgenic rice with an overexpression of RBCS

As ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) activity limits light-saturated photosynthesis under present atmospheric condition, the effects of an overexpression of RBCS on Rubisco content and photosynthesis were examined in the leaves at different positions in rice ( Oryza sativa L.). Rubisco content in the transformant was significantly greater in the uppermost, fully expanded leaves but decreased to levels similar to those in wild-type plants in the lower leaves. The mRNA levels of total RBCS and rbcL in these leaves were much less than those in the expanding leaves, where Rubisco synthesis is active, suggesting commensurately low level of synthesis. Although the activation state of Rubisco was lower in the uppermost, fully expanded leaves of the transformant, it recovered to its full level in the lower leaves. As a result, the photosynthetic rate did not differ in leaves at the same position between the transformant and the wild type. Similarly, whole plant biomass did not differ between these genotypes. Thus, we conclude that although the overexpression of RBCS led to an enhancement of Rubisco protein content in the uppermost, fully expanded leaves, it does not result in increased photosynthetic rates or plant biomass, because of an apparent down-regulation in its activation state.     

Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination

Transgenic Flaveria bidentis (a C4 species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO2 assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO2 assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H2O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO2 partial pressures. Carbon isotope discrimination was measured concurrently with CO2 and H2O exchange on leaves of the control plants and T1 progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C4 species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO2 concentration and CO2 leakage from the bundle sheath; however, some down-regulation of the C4 cycle also occurred.     

Differential Involvement of the Circadian Clock in the Expression of Genes Required for Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Synthesis,Assembly, and Activation in Arabidopsis thaliana

We have investigated the role of the circadian clock in the regulation of expression of genes required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) synthesis, assembly, and activation. Circadian oscillations in RCA (the gene encoding Rubisco activase) and RBCS (the gene encoding Rubisco small subunit) mRNA accumulation, with peak abundance occurring soon after dawn, occur in Arabidopsis thaliana grown in a light-dark (LD) photoperiod. These oscillations persist in plants that have been transferred from LD to either continuous darkness (DD) or continuous light (LL). In contrast, CPN60[alpha] (the gene encoding [alpha]-chaperonin) and CPN60[beta] (the gene encoding [beta]-chaperonin) mRNA abundance oscillates in a diurnal, but not in a circadian, fashion. Although rapid damping of the circadian oscillation in RCA mRNA abundance is observed in Arabidopsis that have been grown in LD and then transferred to DD for 2 d, the circadian oscillations in RCA and RBCS mRNA abundance persist for at least five continuous cycles in LL, demonstrating the robustness of the circadian oscillator.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase deficiency delays senescence of ribulose-1,5-bisphosphate carboxylase/oxygenase but progressively impairs its catalysis during tobacco leaf development.

Transgenic tobacco (Nicotiana tabacum L. cv W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) activase grew more slowly than wild-type plants in a CO2-enriched atmosphere, but eventually attained the same height and number of leaves. Compared with the wild type, the anti-activase plants had reduced CO2 assimilation rates, normal contents of chlorophyll and soluble leaf protein, and much higher Rubisco contents, particularly in older leaves. Activase deficiency greatly delayed the usual developmental decline in Rubisco content seen in wild-type leaves. This effect was much less obvious in another transgenic tobacco with an antisense gene directed against chloroplast-located glyceraldehyde-3-phosphate dehydrogenase, which also had reduced photosynthetic rates and delayed development. Although Rubisco carbamylation was reduced in the anti-activase plants, the reduction was not sufficient to explain the reduced photosynthetic rate of older anti-activase leaves. Instead, up to a 10-fold reduction in the catalytic turnover rate of carbamylated Rubisco in vivo appeared to be the main cause. Slower catalytic turnover by carbamylated Rubisco was particularly obvious in high-CO2-grown leaves but was also detectable in air-grown leaves. Rubisco activity measured immediately after rapid extraction of anti-activase leaves was not much less than that predicted from its degree of carbamylation, ruling out slow release of an inhibitor from carbamylated sites as a major cause of the phenomenon. Nor could substrate scarcity or product inhibition account for the impairment. We conclude that activase must have a role in vivo, direct or indirect, in promoting the activity of carbamylated Rubisco in addition to its role in promoting carbamylation.     

Reductions of Rubisco activase by antisense RNA in the C4 plant Flaveria bidentis reduces Rubisco carbamylation and leaf photosynthesis

To function, the catalytic sites of Rubisco (EC 4.1.1.39) need to be activated by the reversible carbamylation of a lysine residue within the sites followed by rapid binding of magnesium. The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme is thought to aid the release of sugar phosphate inhibitors from Rubisco's catalytic sites, thereby influencing carbamylation. In C3 species, Rubisco operates in a low CO2 environment, which is suboptimal for both catalysis and carbamylation. In C4 plants, Rubisco is located in the bundle sheath cells and operates in a high CO2 atmosphere close to saturation. To explore the role of Rubisco activase in C4 photosynthesis, activase levels were reduced in Flaveria bidentis, a C4 dicot, by transformation with an antisense gene directed against the mRNA for Rubisco activase. Four primary transformants with very low activase levels were recovered. These plants and several of their segregating T1 progeny required high CO2 (>1 kPa) for growth. They had very low CO2 assimilation rates at high light and ambient CO2, and only 10% to 15% of Rubisco sites were carbamylated at both ambient and very high CO2. The amount of Rubisco was similar to that of wild-type plants. Experiments with the T1 progeny of these four primary transformants showed that CO2 assimilation rate and Rubisco carbamylation were severely reduced in plants with less than 30% of wild-type levels of activase. We conclude that activase activity is essential for the operation of the C4 photosynthetic pathway.     

Cosuppression of RBCS3B in Arabidopsis leads to severe photoinhibition caused by ROS accumulation

Key message

Cosuppression of an Arabidopsis Rubisco small subunit gene RBCS3B at Arabidopsis resulted in albino or pale green phenotypes which were caused by ROS accumulation

Abstract

As the most abundant protein on Earth, Rubisco has received much attention in the past decades. Even so, its function is still not understood thoroughly. In this paper, four Arabidopsis transgenic lines (RBCS3B-7, 18, 33, and 35) with albino or pale green phenotypes were obtained by transformation with a construct driving expression of sense RBCS3B, a Rubisco small subunit gene. The phenotypes produced in these transgenic lines were found to be caused by cosuppression. Among these lines, RBCS3B-7 displayed the most severe phenotypes including reduced height, developmental arrest and plant mortality before flowering when grown under normal light on soil. Chloroplast numbers in mesophyll cells were decreased compared to WT, and stacked thylakoids of chloroplasts were broken down gradually in RBCS3B-7 throughout development. In addition, the RBCS3B-7 line was light sensitive, and PSII activity measurement revealed that RBCS3B-7 suffered severe photoinhibition, even under normal light. We found that photoinhibition was due to accumulation of ROS, which accelerated photodamage of PSII and inhibited the repair of PSII in RBCS3B-7.     

Decreased Rubisco activity leads to dramatic changes of nitrate metabolism,amino acid metabolism and the levels of phenylpropanoids and nicotine in tobacco antisense RBCS transformants

Tobacco transformants that express an antisense RBCS construct were used to investigate the consequences of a lesion in photosynthetic carbon metabolism for nitrogen metabolism and secondary metabolism. The results show that an inhibition of photosynthesis and decrease in sugar levels leads to a general inhibition of nitrogen metabolism, and dramatic changes in the levels of secondary metabolites. The response was particularly clear in plants that received excess nitrogen. In these conditions, a decrease of Rubisco activity led to an inhibition of nitrate reductase activity, accumulation of nitrate, a decrease of amino acid levels that was larger than the decrease of sugars, and a large decrease of chlorogenic acid and of nicotine, which are the major carbon- and nitrogen-rich secondary metabolites in tobacco leaves, respectively. Similar changes were seen when nitrogen-replete wild-type tobacco was grown in low light. The inhibition of nitrogen metabolism was partly masked when wild-type plants and antisense RBCS transformants were compared in marginal or in limiting nitrogen, because the lower growth rate of the transformants alleviated the nitrogen deficiency, leading to an increase of amino acids. In these conditions, chlorogenic acid always decreased but the decrease of nicotine was ameliorated or reversed. When the changes in internal pools are compared across all the genotypes and growth conditions, two conclusions emerge. First, decreased levels of primary metabolites lead to a dramatic decrease in the levels of secondary metabolites. Second, changes of the amino acid : sugar ratio are accompanied by changes of the nicotine:chlorogenic acid ratio.     

Relationships between quantum yield for CO2 assimilation, activity of key enzymes and CO2 leakiness in Amaranthus cruentus, a C4 dicot, grown in high or low light

In C(4) photosynthesis, a part of CO(2) fixed by phosphoenolpyruvate carboxylase (PEPC) leaks from the bundle-sheath cells. Because the CO(2) leak wastes ATP consumed in the C(4) cycle, the leak may decrease the efficiency of CO(2) assimilation. To examine this possibility, we studied the light dependence of CO(2) leakiness (phi), estimated by the concurrent measurements of gas exchange and carbon isotope discrimination, initial activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and pyruvate, orthophosphate dikinase (PPDK), the phosphorylation state of PEPC and the CO(2) assimilation rate using leaves of Amaranthus cruentus (NAD-malic enzyme subtype, dicot) plants grown in high light (HL) and low light (LL). phi was constant at photon flux densities (PFDs) >200 micromol m(-2) s(-1) and was around 0.3. At PFDs <150 micromol m(-2) s(-1), phi increased markedly as PFD decreased. At 40 micromol m(-2) s(-1), phi was 0.76 in HL and 0.55 in LL leaves, indicating that the efficiency of CO(2) assimilation at low PFD was greater in LL leaves. The activities of Rubisco and PPDK, and the phosphorylated state of PEPC all decreased as PFD decreased. Theoretical calculations with a mathematical model clearly showed that the increase in phi with decreasing PFD contributed to the decrease in the CO(2) assimilation rate. It was also shown that the 'conventional' quantum yield of photosynthesis obtained by fitting the straight line to the light response curve of the CO(2) assimilation rate at the low PFD region is seriously overestimated. Ecological implications of the increase in phi in LL are discussed.     

Manipulation of Rubisco: the amount,activity, function and regulation

Genetic modification to increase the specificity of Rubisco for CO(2) relative to O(2) and to increase the catalytic rate of Rubisco in crop plants would have great agronomic importance. The availability of three-dimensional structures of Rubisco at atomic resolution and the characterization of site-directed mutants have greatly enhanced the understanding of the catalytic mechanism of Rubisco. Considerable progress has been made in identifying natural variation in the catalytic properties of Rubisco from different species and in developing the tools for introducing both novel and foreign Rubisco genes into plants. The additional complexities of assembling copies of the two distinct polypeptide subunits of Rubisco into a functional holoenzyme in vivo (requiring sufficient expression, post-translational modification, interaction with chaperonins, and interaction with Rubisco activase) remain a major challenge. The consequences of changing the amount of Rubisco present in leaves have been investigated by the use of antisense constructs. The manipulation of genes encoding Rubisco activase has provided a means to investigate the regulation of Rubisco activity.     

Molecular basis of the ribulose-1,5-bisphosphate carboxylase/oxygenase activase mutation in Arabidopsis thaliana is a guanine-to-adenine transition at the 5'-splice junction of intron 3.

Analysis of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase gene and gene products from Arabidopsis thaliana wild-type plants and the Rubisco activase-deficient mutant strain showed that the rca mutation caused GT to be changed to AT at the 5'-splice junction of intron 3 in the six-intron pre-mRNA. Northern blot analysis, genomic and cDNA sequencing, and primer extension analysis indicated that the mutation causes inefficient and incomplete splicing of the pre-mRNA, resulting in the accumulation of three aberrant mRNAs. One mutant mRNA was identical with wild-type mRNA except that it included intron 3, a second mRNA comprised intron 3 and exons 4 through 7, and the third mRNA contained exons 1 through 3. The G-to-A transition is consistent with the known mechanism of mutagenesis by ethyl methanesulfonate, the mutagen used to create the Rubisco activase-deficient strain.     

The catalytic properties of hybrid Rubisco comprising tobacco small and sunflower large subunits mirror the kinetically equivalent source Rubiscos and can support tobacco growth

Plastomic replacement of the tobacco (Nicotiana tabacum) Rubisco large subunit gene (rbcL) with that from sunflower (Helianthus annuus; rbcL(S)) produced tobacco(Rst) transformants that produced a hybrid Rubisco consisting of sunflower large and tobacco small subunits (L(s)S(t)). The tobacco(Rst) plants required CO(2) (0.5% v/v) supplementation to grow autotrophically from seed despite the substrate saturated carboxylation rate, K(m), for CO(2) and CO(2)/O(2) selectivity of the L(s)S(t) enzyme mirroring the kinetically equivalent tobacco and sunflower Rubiscos. Consequently, at the onset of exponential growth when the source strength and leaf L(s)S(t) content were sufficient, tobacco(Rst) plants grew to maturity without CO(2) supplementation. When grown under a high pCO(2), the tobacco(Rst) seedlings grew slower than tobacco and exhibited unique growth phenotypes: Juvenile plants formed clusters of 10 to 20 structurally simple oblanceolate leaves, developed multiple apical meristems, and the mature leaves displayed marginal curling and dimpling. Depending on developmental stage, the L(s)S(t) content in tobacco(Rst) leaves was 4- to 7-fold less than tobacco, and gas exchange coupled with chlorophyll fluorescence showed that at 2 mbar pCO(2) and growth illumination CO(2) assimilation in mature tobacco(Rst) leaves remained limited by Rubisco activity and its rate (approximately 11 micromol m(-2) s(-1)) was half that of tobacco controls. (35)S-methionine labeling showed the stability of assembled L(s)S(t) was similar to tobacco Rubisco and measurements of light transient CO(2) assimilation rates showed L(s)S(t) was adequately regulated by tobacco Rubisco activase. We conclude limitations to tobacco(Rst) growth primarily stem from reduced rbcL(S) mRNA levels and the translation and/or assembly of sunflower large with the tobacco small subunits that restricted L(s)S(t) synthesis.     

The Early-Acting Peroxin PEX19 Is Redundantly Encoded,Farnesylated, and Essential for Viability in Arabidopsis thaliana

Peroxisomes are single-membrane bound organelles that are essential for normal development in plants and animals. In mammals and yeast, the peroxin (PEX) proteins PEX3 and PEX19 facilitate the early steps of peroxisome membrane protein (PMP) insertion and pre-peroxisome budding from the endoplasmic reticulum. The PEX3 membrane protein acts as a docking site for PEX19, a cytosolic chaperone for PMPs that delivers PMPs to the endoplasmic reticulum or peroxisomal membrane. PEX19 is farnesylated in yeast and mammals, and we used immunoblotting with prenylation mutants to show that PEX19 also is fully farnesylated in wild-type Arabidopsis thaliana plants. We examined insertional alleles disrupting either of the two Arabidopsis PEX19 isoforms, PEX19A or PEX19B, and detected similar levels of PEX19 protein in the pex19a-1 mutant and wild type; however, PEX19 protein was nearly undetectable in the pex19b-1 mutant. Despite the reduction in PEX19 levels in pex19b-1, both pex19a-1 and pex19b-1 single mutants lacked notable peroxisomal β-oxidation defects and displayed normal levels and localization of peroxisomal matrix and membrane proteins. The pex19a-1 pex19b-1 double mutant was embryo lethal, indicating a redundantly encoded critical role for PEX19 during embryogenesis. Expressing YFP-tagged versions of either PEX19 isoform rescued this lethality, confirming that PEX19A and PEX19B act redundantly in Arabidopsis. We observed that pex19b-1 enhanced peroxisome-related defects of a subset of peroxin-defective mutants, supporting a role for PEX19 in peroxisome function. Together, our data indicate that Arabidopsis PEX19 promotes peroxisome function and is essential for viability.     

Description and applications of a rapid and sensitive non-radioactive microplate-based assay for maximum and initial activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase

D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the first step in photosynthetic carbon assimilation and represents the largest sink for nitrogen in plants. Improvement of its kinetic properties or the efficiency with which it is used in planta would benefit photosynthesis, nitrogen and water use efficiency, and yield. This paper presents a new non-radioactive microplate-based assay, which determines the product [3-phosphoglycerate (3-PGA)] in an enzymic cycle between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase. High sensitivity permits use of highly diluted extracts, and a short reaction time to avoid problems due to fall-off. Throughput was several hundreds of samples per person per day. Sensitivity and convenience compared favourably with radioisotopic assays, which were previously used to assay Rubisco. Its use is illustrated in three applications. (1) Maximal and initial activities and the K(m) for ribulose-1,5-bisphosphate were determined in raw extracts of leaves from several species. Similar values were obtained from those in the literature. (2) Diurnal changes were compared in rosettes of wild-type (WT) Arabidopsis and the starchless pgm mutant. Despite these dramatic differences in carbon metabolism, Rubisco activity and activation were similar in both genotypes. (3) A preliminary association mapping study was performed with 118 Arabidopsis accessions, using 183 markers that probably cover approximately 3-8% of the total genome. At a P-value < 0.005, two, two and no quantitative trait loci (QTL) were found for Rubisco maximal activity, initial activity and activation state, respectively. Inspection of the genomic regions that span these markers revealed these QTL involved genes not previously implicated in the regulation of Rubisco expression or activity.