Abstract
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.
Highlights
The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Analysis of ribulose 5-phosphate levels suggested that formation of the PRK/GAPDH/ CP12 complex was essential to separate the activities of the Calvin-Benson cycle from the oxidative pentose phosphate pathway (OPPP)
Higher plants differ from cyanobacteria in that are some Calvin-Benson cycle enzymes reductively activated in the light but, plastidic Glc-6-P dehydrogenase (G6PDH; EC 1.1.1.49), the first enzyme of the OPPP, is reductively inactivated (Wenderoth et al, 1997; Kruger and von Schaewen, 2003; Nee et al, 2009)
Summary
The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Higher plants differ from cyanobacteria in that are some Calvin-Benson cycle enzymes reductively activated in the light but, plastidic Glc-6-P dehydrogenase (G6PDH; EC 1.1.1.49), the first enzyme of the OPPP, is reductively inactivated (Wenderoth et al, 1997; Kruger and von Schaewen, 2003; Nee et al, 2009). Recent evidence has shown that there is heterogeneity in PRK and GAPDH regulation by redox changes and by CP12 in algal species (Maberly et al, 2010) and in PRK and GAPDH protein complex profiles in higher plant species (Howard et al, 2011) These reports indicate that the importance of CP12 and of protein aggregation to the regulation of metabolism is likely to vary between species
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