Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.     

Chromosomal Mapping of Genes in theRBCS Family in Arabidopsis thaliana

In Arabidopsis thaliana, four genes have been identified inthe RBCS gene family, one being assigned to subfamily RBCS-Aand the other three to subfamily RBCS-B (1B, 2B and 3B). Todetermine the chromosomal location ofthese genes, hybridizationanalysis with CIC YAC high-density filters was carried out forthe RBCS-A gene, and CAPS analysis for the three RBCS-B genes,based on the finding that restriction fragment length polymorphismis present in the upstream region of the gene RBCS-3B. The RBCS-Agene was mapped at 100.8 cM from the top of chromosome 1 andthe three RBCS-B genes at 62.70 cM from the top of chromosome5.     

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.     

Analysis of Chromatin Structure in the Control Regions of the <Emphasis Type="Italic">Chlamydomonas HSP70A</Emphasis> and <Emphasis Type="Italic">RBCS2</Emphasis> Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of ~700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.     

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.     

Structural effects in vitro of the anti-inflammatory drug diclofenac on human erythrocytes and molecular models of cell membranes

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly on those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated and reported. This report presents the following evidence that diclofenac interacts with red cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that diclofenac interacted with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes it was observed that the drug induced changes different from the normal biconcave morphology of most red blood cells. This is the first time in which structural effects of diclofenac on the human erythrocyte membrane have been described.