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CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants.

ABSTRACT: CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

SUBMITTER: Wedel N

PROVIDER: S-EPMC23388 | biostudies-literature | 1997 Sep

REPOSITORIES: biostudies-literature

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CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants.

Wedel N N Soll J J Paap B K BK

Proceedings of the National Academy of Sciences of the United States of America 19970901 19

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1. 13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding ...[more]

PMID: 9294236

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Project description:CP12 is a small nuclear encoded chloroplast protein that has been shown to form a complex with the Calvin cycle enzymes, PRK and GADPH. We have taken an antisense approach in order to address the importance of this regulatory protein on carbon assimilation. Transgenic Nicotiana tabacum cv. Samsun with reduced levels of the CP12 protein, produced by leaf disc transformation with a full length tobacco CP12 cDNA, regulated by the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens-mediated transformation and selection on kanamycin produced primary transformants whose progeny segregate for severity of the antisense CP12 phenotype. Because of the impact of the antisense reductions in CP12 on carbon assimilation, these plants have abnormal morphology of leaves, floral organs, greatly reduced fertility and markedly slower growth rates. Our hypothesis is that the phenotype is exposing the regulatory nature of the CP12 protein. Intermediates leaving the Calvin cycle are essential for the synthesis of hormones and perturbation of hormone balances is a potential source of the morphological and development effects observed in the CP12 antisense plants. Our objective is to understand the CP12 phenotype by identifying changes in gene expression occurring at key stages in the life cycle of the CP12 antisense plants. CP12 antisense and wild type control plants for microarray analysis were germinated in a growth chamber in agar medium with 1% sucrose and 1/2 strength MS; with a 16 h light/8 h dark cycle at 25°C. At 20 days plants were transferred to a controlled environment greenhouse and grown in Fisons Levington F2 compost at 25°C, with light levels above 500 umol m-2 s-1 ;using supplementary lighting in a 16 h light/8 h dark cycle. 3 independent sets of plants (both wild type and CP12 antisense) were grown for preparation of three independent biological replicate RNA samples. RNA was extracted according to the TIGR protocol from plants in the early in all samples. Keywords: Direct comparison

Dataset Information

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants.

Publications

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants.

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