Antisense suppression of the small chloroplast protein CP12 in tobacco alters carbon partitioning and severely restricts growth.

The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants.

Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

Thioredoxin-mediated reversible dissociation of a stromal multiprotein complex in response to changes in light availability.

A Calvin cycle multiprotein complex including phosphoribulokinase (PRK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a small protein, CP12, has previously been identified. In this article, we have studied this complex in leaves and have shown that dissociation and reassociation of the PRK/GAPDH/CP12 complex occurs in a time frame of minutes, allowing for rapid regulation of enzyme activity. Furthermore, we have shown that the extent of formation and dissociation of the PRK/GAPDH/CP12 complex correlates with the quantity of light. These data provide evidence linking the status of this complex with the rapid and subtle regulation of GAPDH and PRK activities in response to fluctuations in light availability. We have also demonstrated that dissociation of this complex depends on electron transport chain activity and that the major factor involved in the dissociation of the pea complex was thioredoxin f. We show here that both PRK and GAPDH are present in the reduced form in leaves in the dark, but are inactive, demonstrating the role of the PRK/GAPDH/CP12 complex in deactivating these enzymes in response to reductions in light intensity. Based on our data, we propose a model for thioredoxin f-mediated activation of PRK and GAPDH by two mechanisms: directly through reduction of disulfide bonds within these enzymes and indirectly by mediating the breakdown of the complex in response to changes in light intensity.

Antisense suppression of the small chloroplast protein CP12 in tobacco: a transcriptional viewpoint.

The chloroplast protein CP12 forms a multi-enzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. Recently we reported on the importance of CP12 in vivo to higher plant metabolism using antisense suppression of CP12 in tobacco (Nicotiana tabacum). Our results indicated that while only minor changes in photosynthetic carbon fixation and in PRK and GAPDH activities were observed, striking changes in growth rates and morphology were seen. In this article we present data on the transcriptional changes observed in one of the antisense lines and we discuss the major findings in light of the metabolic phenotype described.

Oligonucleotide arrays: information from replication and spatial structure.

MOTIVATION: The introduction of oligonucleotide DNA arrays has resulted in much debate concerning appropriate models for the measurement of gene expression. By contrast, little account has been taken of the possibility of identifying the physical imperfections in the raw data. RESULTS: This paper demonstrates that, with the use of replicates and an awareness of the spatial structure, deficiencies in the data can be identified, the possibility of their correction can be ascertained and correction can be effected (by use of local scaling) where possible. The procedures were motivated by data from replicates of Arabidopsis thaliana using the GeneChip ATH1-121501 microarray. Similar problems are illustrated for GeneChip Human Genome U133 arrays and for the newer and larger GeneChip Wheat Genome microarray. AVAILABILITY: R code is freely available on request.

Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development.

Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of an Arabidopsis (Arabidopsis thaliana) cDNA in tobacco (Nicotiana tabacum) plants. In plants with increased SBPase activity, photosynthetic rates were increased, higher levels of Suc and starch accumulated during the photoperiod, and an increase in leaf area and biomass of up to 30% was also evident. Light saturated photosynthesis increased with increasing SBPase activity and analysis of CO2 response curves revealed that this increase in photosynthesis could be attributed to an increase in ribulose 1,5-bisphosphate regenerative capacity. Seedlings with increased SBPase activity had an increased leaf area at the 4 to 5 leaf stage when compared to wild-type plants, and chlorophyll fluorescence imaging of these young plants revealed a higher photosynthetic capacity at the whole plant level. Measurements of photosynthesis, made under growth conditions integrated over the day, showed that mature plants with increased SBPase activity fixed 6% to 12% more carbon than equivalent wild-type leaves, with the young leaves having the highest rates. In this paper, we have shown that photosynthetic capacity per unit area and plant yield can be increased by overexpressing a single native plant enzyme, SBPase, and that this gives an advantage to the growth of these plants from an early phase of vegetative growth. This work has also shown that it is not necessary to bypass the normal regulatory control of SBPase, exerted by conditions in the stroma, to achieve improvements in carbon fixation.

Responses of primary and secondary metabolism to sugar accumulation revealed by microarray expression analysis of the Arabidopsis mutant, pho3.

The Arabidopsis mutant pho3 accumulates sucrose and other carbohydrates to high levels, providing a means of investigating the genomic response to sucrose accumulation using microarray analysis. Wild-type and mutant plants were grown in soil to the mature rosette stage for the analysis of gene expression using the Affymetrix ATH1 chip, containing more than 22,500 probe sets. Small, but significant, decreases were observed in the expression of many genes encoding enzymes and regulatory proteins involved in primary carbon assimilation, suggesting that, in mature leaves of Arabidopsis, there is limited feedback regulation on gene expression by sugars. The study revealed a striking increase in the expression of the plastid glucose 6-phosphate/phosphate translocator, characteristically expressed only in heterotrophic tissues. This indicated a change in the nature of metabolite exchange between the plastid and the cytosol in the pho3 mutant. The expression of enzymes of starch synthesis also increased significantly. Very large increases were observed in the expression of transcription factors and enzymes involved in anthocyanin biosynthesis. This finding reinforces the emerging picture of an important role for primary metabolism in regulating secondary metabolism.