Antisense Suppression of the Small Chloroplast Protein CP12 in Tobacco Alters Carbon Partitioning and Severely Restricts Growth  

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.

The circadian oscillator in Synechococcus elongatus controls metabolite partitioning during diurnal growth

“…6 B and C). In contrast, the repression of CP12 in tobacco resulted in accumulation of complex insoluble metabolites such as protein and cell wall components (43). In WT S. elongatus we observe increased abundance of unknowns that correlate strongly with compounds involved in fatty acid and glycerolipid biosynthesis; both these biosynthetic pathways would be important for cell wall and membrane biosynthesis in cyanobacteria.…”

“…In S. elongatus inactivation of CP12 resulted in decreased OPPP activity, in which a decrease in cellular fructose-6-phosphate could be detected directly (36). Additionally, in tobacco plants more active CP12 was associated with more starch, soluble sugars (including sucrose), and amino acids (43). The metabolic shifts observed in a ΔkaiC mutant in the morning mirror those seen when CP12 is active, including increased levels of fructose-6-phosphate, sucrose, nucleotides, and amino acids (Fig.…”

Comparative Analysis of 126 Cyanobacterial Genomes Reveals Evidence of Functional Diversity Among Homologs of the Redox-Regulated CP12 Protein

“…The C-terminal extension (CTE) of GapB consists of the GAPDH-binding module of CP12 (approximately 30 residues from the C-terminal half of CP12), including the C-terminal Cys pair. All cyanobacteria, algae, and plants contain GAPDH composed of four GapA subunits, but in higher plants, an A2B2 heterotetramer, composed of two GapA and two GapB subunits, is the major chloroplast form of GAPDH (Petersen et al, 2006;Howard et al, 2011aHoward et al, , 2011b. GapA and GapB form complexes in response to light, with the CTE playing the regulatory role of CP12 (Wedel and Soll, 1998;Scheibe et al, 2002;Trost et al, 2006).…”

“…Further studies in higher plants have proposed a role for CP12 outside of Calvin cycle regulation. CP12 is expressed in nonphotosynthetic Arabidopsis (Arabidopsis thaliana) tissues, and antisense suppression of tobacco (Nicotiana tabacum) CP12 disrupts a variety of developmental processes (Singh et al, 2008;Howard et al, 2011aHoward et al, , 2011c. Moreover, higher plant genomes encode up to three forms of CP12, which are differentially expressed, but all have been shown to bind GAPDH and PRK (Singh et al, 2008;Marri et al, 2010).…”

Conformational Selection and Folding-upon-binding of Intrinsically Disordered Protein CP12 Regulate Photosynthetic Enzymes Assembly

“…Because both GAPDH and phosphoribulokinase activities are inhibited in the complex but are fully recovered upon dissociation (10), CP12 could effectively contribute to the modulation of the Calvin-Benson cycle under the natural variable light/dark regime (13). The thioredoxin-dependent CP12 may, therefore, work as a light-sensitive redox switch in chloroplasts and was recently shown by antisense technology to be required for normal growth and development in transgenic tobacco plants (14). Moreover, in the cyanobacterium Synechococcus elongatus PCC7942, growth of CP12-knock out mutants was inhibited under light/dark cycle but not in continuous light (7).…”