The Arabidopsis thaliana genome encodes three ␣-amylase-like proteins (AtAMY1, AtAMY2, and AtAMY3). Only AtAMY3 has a predicted N-terminal transit peptide for plastidial localization. AtAMY3 is an unusually large ␣-amylase (93.5 kDa) with the C-terminal half showing similarity to other known ␣-amylases. When expressed in Escherichia coli, both the whole AtAMY3 protein and the C-terminal half alone show ␣-amylase activity. We show that AtAMY3 is localized in chloroplasts. The starch-excess mutant of Arabidopsis sex4, previously shown to have reduced plastidial ␣-amylase activity, is deficient in AtAMY3 protein. Unexpectedly, T-DNA knock-out mutants of AtAMY3 have the same diurnal pattern of transitory starch metabolism as the wild type. These results show that AtAMY3 is not required for transitory starch breakdown and that the starch-excess phenotype of the sex4 mutant is not caused simply by deficiency of AtAMY3 protein. Knockout mutants in the predicted non-plastidial ␣-amylases AtAMY1 and AtAMY2 were also isolated, and these displayed normal starch breakdown in the dark as expected for extraplastidial amylases. Furthermore, all three AtAMY double knock-out mutant combinations and the triple knock-out degraded their leaf starch normally. We conclude that ␣-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves.

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.Most plants synthesize starch in their chloroplasts during photosynthesis and degrade it during the subsequent night. The regulation of transitory starch metabolism in photosynthetic tissues is clearly different from the long-term, reserve starch metabolism in non-photosynthetic tissues (Caspar, 1994). Many mutations that affect the starch of certain cereal seeds, potato, pea, and Chlamydomonas reinhardtii have been isolated and characterized (Hannah, 1997). Although studies of these mutants have greatly added to our knowledge of starch metabolism, these mutants studied are relatively specific for the reserve and reproductive organs, and do not affect starch metabolism in the vegetative parts of plants. Mutants that affect starch metabolism in the vegetative parts of Arabidopsis would be useful to extend our understanding on starch metabolism and its role in the plant.Previously, several nuclear-encoded, recessive mutants of Arabidopsis, pgm1, adg1, and adg2, were isolated and characterized for their low starch content or lack of starch in leaves (Caspar et al

A recessive Arabidopsis mutation, carbohydrate accumulation mutantl (caml), which maps to position 22.8 on chromosome 3, was identified by screening leaves of ethyl methanesulfonate-mutagenized M2 plants stained with iodine for altered starch content. lncreased starch content in leaves of the c a m l mutant was observed at the onset of flowering. This mutant also had a delayed floral initiation phenotype with more rosette leaves than the parenta1 line. In addition, activities of several enzymes associated with starch metabolism were altered in the c a m l mutant. The late-flowering mutant gigantea (gi) also manifested an elevated starch leve1 in leaves. However, not all late-flowering mutants had increased leaf starch content. Double mutants c a m l a d g l (for AJP-glucose pyrophosphorylase), c a m l pgm (for -phosphoglucomutase), and g i p g m had no observable starch in leaves but showed the late-flowering phenotype, demonstrating tThat the elevated starch content is not the cause of late floral initiation. The pleiotropic effects of c a m l and gi suggest that they may play regulatory roles in starch metabolism and floral initiation. These data suggest that starch accumulation and floral initiation may share a common regulatory pathway.

SummaryTwo 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.

Starch synthesis and degradation require the participation of many enzymes, occur in both photosynthetic and nonphotosynthetic tissues, and are subject to environmental and developmental regulation. We examine the distribution of starch in vegetative tissues of Arabidopsis (Arabidopsis thaliana) and the expression of genes encoding core enzymes for starch synthesis. Starch is accumulated in plastids of epidermal, mesophyll, vascular, and root cap cells but not in root proper cells. We also identify cells that can synthesize starch heterotrophically in albino mutants. Starch synthesis in leaves is regulated by developmental stage and light. Expression of gene promoter-β-glucuronidase fusion constructs in transgenic seedlings shows that starch synthesis genes are transcriptionally active in cells with starch synthesis and are inactive in root proper cells except the plastidial phosphoglucose isomerase. In addition, ADG2 (for ADPG PYROPHOSPHORYLASE2) is not required for starch synthesis in root cap cells. Expression profile analysis reveals that starch metabolism genes can be clustered into two sets based on their tissue-specific expression patterns. Starch distribution and expression pattern of core starch synthesis genes are common in Arabidopsis and rice (Oryza sativa), suggesting that the regulatory mechanism for starch metabolism genes may be conserved evolutionarily. We conclude that starch synthesis in Arabidopsis is achieved by spatial coexpression of core starch metabolism genes regulated by their promoter activities and is fine-tuned by cell-specific endogenous and environmental controls.