The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter.

Starch is the major storage carbohydrate in higher plants and of considerable importance for the human diet and for numerous technical applications. In addition, starch can be accumulated transiently in chloroplasts as a temporary deposit of carbohydrates during ongoing photosynthesis. This transitory starch has to be mobilized during the subsequent dark period. Mutants defective in starch mobilization are characterized by high starch contents in leaves after prolonged periods of darkness and therefore are termed starch excess (sex) mutants. Here we describe the molecular characterization of the Arabidopsis sex1 mutant that has been proposed to be defective in the export of glucose resulting from hydrolytic starch breakdown. The mutated gene in sex1 was cloned using a map-based cloning approach. By complementation of the mutant, immunological analysis, and analysis of starch phosphorylation, we show that sex1 is defective in the Arabidopsis homolog of the R1 protein and not in the hexose transporter. We propose that the SEX1 protein (R1) functions as an overall regulator of starch mobilization by controlling the phosphate content of starch.

Mutation of Arabidopsis plastid phosphoglucose isomerase affects leaf starch synthesis and floral initiation.

We isolated pgi1-1, an Arabidopsis mutant with a decreased plastid phospho-glucose (Glc) isomerase activity. While pgi1-1 mutant has a deficiency in leaf starch synthesis, it accumulates starch in root cap cells. It has been shown that a plastid transporter for hexose phosphate transports cytosolic Glc-6-P into plastids and expresses restricted mainly to the heterotrophic tissues. The decreased starch content in leaves of the pgi1-1 mutant indicates that cytosolic Glc-6-P cannot be efficiently transported into chloroplasts to complement the mutant's deficiency in chloroplastic phospho-Glc isomerase activity for starch synthesis. We cloned the Arabidopsis PGI1 gene and showed that it encodes the plastid phospho-Glc isomerase. The pgi1-1 allele was found to have a single nucleotide substitution, causing a Ser to Phe transition. While the flowering times of the Arabidopsis starch-deficient mutants pgi1, pgm1, and adg1 were similar to that of the wild type under long-day conditions, it was significantly delayed under short-day conditions. The pleiotropic phenotype of late flowering conferred by these starch metabolic mutations suggests that carbohydrate metabolism plays an important role in floral initiation.

A mutant of Arabidopsis lacking a chloroplastic isoamylase accumulates both starch and phytoglycogen.

In this study, our goal was to evaluate the role of starch debranching enzymes in the determination of the structure of amylopectin. We screened mutant populations of Arabidopsis for plants with alterations in the structure of leaf starch by using iodine staining. The leaves of two mutant lines stained reddish brown, whereas wild-type leaves stained brownish black, indicating that a more highly branched polyglucan than amylopectin was present. The mutants were allelic, and the mutation mapped to position 18.8 on chromosome 1. One mutant line lacked the transcript for a gene with sequence similarity to higher plant debranching enzymes, and both mutants lacked a chloroplastic starch-hydrolyzing enzyme. This enzyme was identified as a debranching enzyme of the isoamylase type. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch in this mutant line when compared with the wild type and in the accumulation of the highly branched water-soluble polysaccharide phytoglycogen. Both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure.

Characterization of ADG1, an Arabidopsis locus encoding for ADPG pyrophosphorylase small subunit, demonstrates that the presence of the small subunit is required for large subunit stability.

Two mutants of Arabidopsis have been isolated that affect ADPG pyrophosphorylase (ADGase) activity. Previously, it has been shown that ADG2 encodes the large subunit of ADGase. This study characterizes the adg1 mutant phenotype and ADG1 gene structure. RNA blot analyses indicate that the adg1-1 mutant accumulates transcripts encoding both the large and small subunits of ADGase, while the adg1-2 mutant accumulates only large subunit transcripts. RFLP analysis and complementation of adg1 mutants with the ADGase small subunit gene demonstrate that ADG1 encodes the small subunit. Sequence analysis indicates that adg1-1 represents a missense mutation within the gene. Western blot analysis confirms that adg1 mutants contain neither the large nor the small subunit proteins, suggesting that the presence of functional small subunits is required for large subunit stability.

adg2-1 represents a missense mutation in the ADPG pyrophosphorylase large subunit gene of Arabidopsis thaliana.

Arabidopsis mutants affecting ADPG pyrophosphorylase (ADGase) activity can be divided into two complementation groups, adg1 and adg2. Previous biochemical studies of adg2-1 mutant indicated that mutant plants do not accumulate ADGase large subunit protein and that ADGase small subunits assemble as homotetramers. This suggested that the ADG2 gene may encode the large subunit of ADGase. In this paper, it is shown that adg2-1 mutant plants accumulate near wild-type levels of transcripts encoding both the large and small subunits of ADGase. However, by RFLP analysis and complementation of adg2-1 with the ADGase large subunit gene, we show that the adg2-1 mutant does represent a mutation of the ADGase large subunit gene. Sequence analysis of the adg2-1 allele revealed a missense mutation. The results therefore suggest either that the missense mutation affects the stability of the ADGase large subunit protein or that it prevents assembly of the large subunit into holoenzyme.

Characterization of a maize beta-amylase cDNA clone and its expression during seed germination.

A maize (Zea mays L.) cDNA clone (pZMB2) encoding beta-amylase was isolated from a cDNA library prepared from the aleurone RNA of germinating kernels. The cDNA encodes a predicted product of 488 amino acids with significant similarity to known beta-amylases from barley (Hordeum vulgare), rye (Secale cereale), and rice (Oryza sativa). Glycine-rich repeats found in the carboxyl terminus of the endosperm-specific beta-amylase of barley and rye are absent from the maize gene product. The N-terminal sequence of the first 20 amino acids of a beta-amylase peptide derived from purified protein is identical to the 5th through 24th amino acids of the predicted cDNA product, indicating the absence of a conventional signal peptide in the maize protein. Recombinant inbred mapping data indicate that the cDNA clone is single-copy gene that maps to chromosome 7L at position 83 centimorgans. Northern blot analysis and in vitro translation-immunoprecipitation data indicate that the maize beta-amylase is synthesized de novo in the aleurone cells but not in the scutellum during seed germination.

Phytohormone-regulated beta-amylase gene expression in rice.

The expression of beta-amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination beta-amylase is synthesized de novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the beta-amylase activity, while ABA inhibits the beta-amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of beta-amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.