Abstract
In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate. In contrast to the better characterization of the photorespiratory pathway at a molecular level, the molecular regulation and significance of the plastidic pathway are not yet well understood. An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the conversion of 3-phosphoserine to Ser in the final step of the plastidic pathway of Ser biosynthesis, was cloned by functional complementation of an Escherichia coli serB- mutant. The 1.1-kilobase pair full-length cDNA, encoding 295 amino acids in its open reading frame, contains a putative organelle targeting presequence. Chloroplastic targeting has been demonstrated by particle gun bombardment using an N-terminal 60-amino acid green fluorescence protein fusion protein. Southern hybridization suggested the existence of a single-copy gene that mapped to chromosome 1. 3-Phosphoserine phosphatase enzyme activity was detected in vitro in the overexpressed protein in E. coli. Northern analysis revealed preferential gene expression in leaf and root tissues of light-grown plants with an approximately 1.5-fold abundance in the root compared with the leaf tissues. This indicates the possible role of the plastidic pathway in supplying Ser to non-photosynthetic tissues, in contrast to the function of the photorespiratory pathway in photosynthetic tissues. This work completes the molecular cloning and characterization of the three genes involved in the plastidic pathway of Ser biosynthesis in higher plants.
Highlights
In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate
An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the conversion of 3-phosphoserine to Ser in the final step of the plastidic pathway of Ser biosynthesis, was cloned by functional complementation of an Escherichia coli serB؊ mutant
CDNA Cloning of Arabidopsis PSP by Functional Complementation of an E. coli Mutant—The E. coli serBϪ mutant strain SK472 is deficient in PSP activity and is unable to grow on M9 minimal medium plates without Ser supplementation
Summary
3-PGA, 3-phosphoglycerate; GDC, glycine decarboxylase multienzyme complex; SHMT, serine hydroxymethyltransferase; PGDH, 3-phosphoglycerate dehydrogenase; PSAT,. An alternative pathway of Ser biosynthesis that takes place in the plastids, Ser is synthesized from 3-PGA via a series of reactions catalyzed by 3-phosphoglycerate dehydrogenase (PGDH), 3phosphoserine aminotransferase (PSAT), and 3-phosphoserine phosphatase (PSP) (Fig. 1). The genes encoding Arabidopsis PGDH and PSAT have a common feature in their mRNA expression patterns, in which they demonstrated root-preferential expression [9, 10] These results suggested that the phosphorylated pathway in plastids is regulated at the mRNA expression level, so as to ensure Ser formation in non-photosynthetic tissues, in which photorespiratory Ser biosynthesis is suppressed. Together with our previous work on Arabidopsis PGDH and PSAT, the first molecular cloning and characterization of the entire plastidic pathway of Ser biosynthesis in the plant kingdom were completed
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