Molecular and genetic dissection of sugar signal transduction pathway in Arabidopsis thaliana

While sugars are important signaling molecules for the control of plant growth and development, little is known about the molecular mechanisms underlying sugar sensing and signal transduction in plants. To date, many glucose insensitive mutants have been found to be alleles of abscisic acid (ABA) biosynthetic or perception mutants. This scenario implies that traditional mutant screens may preferentially identify indirect effects rather than components directly involved in sugar response. On the basis of the information on the previous mutant screens, steady-state mRNA analysis, and global transcription profiling, I hypothesized that sugar signals might be transmitted through an ABA-independent signal transduction pathway. I have established a mutant screen system using marker genes that are very sensitive to glucose. ASN1 ( asparagine synthetase1) is completely repressed by low levels of exogenous glucose (<0.1%) in less than 3 h and is derepressed by removing exogenous glucose from the culture medium. Glucose repression of ASN1 also involves protein phosphorylation but not de novo protein synthesis. In addition, hexokinase appears to be required for the glucose repression of ASN1. Most critically, ASN1 repression by glucose is not affected by ABA under the conditions tested.; To carry out a screen for sugar response mutants with altered expression of ASN1, wild-type Arabidopsis plants have been transformed with reporter constructs carrying the ASN1 promoter fused with luciferase or green fluorescent protein genes. Homozygous transgenic plants containing the reporter gene have been generated. Significant quantities of EMS (ethyl methane sulphonate) mutagenized M2 seeds from three selected lines were obtained.; Recent DNA microarray analyses in our lab have shown that a wide range of genes are regulated by glucose, such as those associated with carbohydrate metabolism, transcriptional regulation, and metabolite transport. Among those, I found the expression of two putative ACT (Aspartokinase, Chorismate mutase, and prephenate dehydrogenases (TyrA)) domain-containing protein kinase genes (ACTPK) was highly affected by glucose. Functional characterization has been accomplished to determine the sugar response and temporal and spatial expression of ACTPK. The ACTPK3 transcript was completely abolished by low levels of exogenous glucose (0.1%, w/v) in 3 h. Interestingly, steady-state mRNA analysis results showed that ACTPK3 is likely controlled by the circadian clock that is epistatic of sugar response. The glucose repression of ACTPK3 required sugar transport, sugar phosphorylation, hexokinase, de novo protein synthesis, and protein dephosphorylation. Furthermore, the ACTPK3 repression by glucose was not modulated by ABA. (Abstract shortened by UMI.)...