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

ABSTRACT: 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.

SUBMITTER: Howard TP

PROVIDER: S-EPMC3337198 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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

Howard Thomas P TP Upton Graham J G GJ Lloyd Julie C JC Raines Christine A CA

Plant signaling & behavior 20111201 12

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 chang ...[more]

PMID: 22112458

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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

Project description:A tobacco chloroplast hypothetical open reading frame 4 (YCF4) has been reported as a non-essential assembly factor for photosynthesis based on an incomplete knockout of YCF4, just 93 of 184 amino acids from the N-terminus were knocked out. On the other hand, we removed the complete sequence of YCF4 from tobacco chloroplasts and observed that ΔYCF4 plants were unable to survive photoautotrophically as their growth was hampered in the absence of an external carbon supply, clearly showing that the YCF4 is essential for photosynthesis. Initially, the aadA gene was introduced into the tobacco plastome replacing the complete YCF4 gene through homologous recombination events. The replacement of YCF4 with aadA was confirmed by PCR and Southern blot analysis in ΔYCF4 plants. Homoplasmic ΔYCF4 plants had a light green phenotype, and the leaves became pale yellow as the plants grew older. The structure of chloroplasts of ΔYCF4 mutants of light green phenotype was studied using a transmission electron microscope (TEM), and the micrographs demonstrated structural anomalies in the chloroplasts; including shape, size, and grana stacking compared to the wild-type plants. Further, transcriptome analysis revealed that the expression of PSI, PSII, and ribosomal genes remained unchanged in ∆YCF4 plants. On the other hand, transcriptome levels of rbcL (Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit), LHC (Light-Harvesting Complex), and ATP Synthase (atpB and atpL) decreased, indicating that the YCF4 has the function(s) in addition to assembling the photosynthetic complex. This was confirmed by in-silico protein-protein interactions of full-length YCF4 as well as 93 and 91 of 184 amino acids from N- and C-termini of the full-length protein, which revealed that the C-terminus (91 aa) of YCF4 is important in interacting with other chloroplast proteins. These findings provide genetic support for the plastid YCF4 gene’s critical role in regulating the plastid gene expression and assembling the photosynthetic complex.

Dataset Information

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

Publications

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

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