To gain insight into the synthesis and functions of enzymes of starch metabolism in leaves of Arabidopsis L. Heynth, Affymetrix microarrays were used to analyze the transcriptome throughout the diurnal cycle. Under the conditions employed, transitory leaf starch is degraded progressively during a 12-h dark period, and then accumulates during the following 12-h light period. Transcripts encoding enzymes of starch synthesis changed relatively little in amount over 24 h except for two starch synthases, granule bound starch synthase and starch synthase II, which increased appreciably during the transition from dark to light. The increase in RNA encoding granule-bound starch synthase may reflect the extensive destruction of starch granules in the dark. Transcripts encoding several enzymes putatively involved in starch breakdown showed a coordinated decline in the dark followed by rapid accumulation in the light. Despite marked changes in their transcript levels, the amounts of some enzymes of starch metabolism do not change appreciably through the diurnal cycle. Posttranscriptional regulation is essential in the maintenance of amounts of enzymes and the control of their activities in vivo. Even though the relationships between transcript levels, enzyme activity, and diurnal metabolism of starch metabolism are complex, the presence of some distinctive diurnal patterns of transcripts for enzymes known to be involved in starch metabolism facilitates the identification of other proteins that may participate in this process.Starch is the major form in which carbon is stored in plants, the major source of calories in the human diet, and an important industrial commodity (Jobling, 2004). However, our understanding of the nature and regulation of the pathways of starch synthesis and degradation is incomplete. The Arabidopsis genome sequence, together with extensive functional genomics resources, is facilitating investigations to better understand starch metabolism in plants (Zeeman et al., 2002;Smith et al., 2003). Starch is synthesized in many organs of Arabidopsis, including leaves, flowers, developing seeds, and root caps, and the structure and composition of starch isolated from leaves are similar to that from crop plants (Zeeman et al., 2002). Use of leaves enables both synthesis and degradation to be studied within a 24-h period independently of changes in plant development. The two processes are integrated with each other, and their rates are related to the duration of day and night. Characteristic changes in the content of sugars and maltooligosaccharides are also observed throughout the diurnal cycle, showing a complex integration of starch and sugar metabolism. The Arabidopsis leaf thus provides an excellent model system with which to elucidate the pathways and regulatory mechanisms of starch synthesis and breakdown in the plastids of living cells (Zeeman et al., 2002;Smith et al., 2003).The Arabidopsis genome sequence reveals many genes encoding enzymes that may be involved in starch synthesis and degradation, and...

The multilineage differentiation capacity of mouse embryonic stem (ES) cells offers a potential testing platform for gene products that mediate mammalian lineage determination and cellular specialization. Identification of such differentiation regulators is crucial to harnessing ES cells for pharmaceutical discovery and cell therapy. Here we describe the use of episomal expression technology for functional evaluation of cDNA clones during ES-cell differentiation in vitro. Several candidate cDNAs identified by subtractive cloning and expression profiling were introduced into ES cells in episomal expression constructs. Subsequent differentiation revealed that the Wnt antagonist Sfrp2 stimulates production of neural progenitors. The significance of this observation was substantiated by forced expression of Wnt-1 and treatment with lithium chloride, both of which inhibit neural differentiation. These findings reveal the importance of Wnt signaling in regulating ES-cell lineage diversification. More generally, this study establishes a path for rapid and direct validation of candidate genes in ES cells.

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.