| Regulation of gibberellin (GA) levels has an important impact on plant growth, and regulation of GA biosynthesis can be used to enhance agriculturally important traits, such as plant height and fruit yield. Although the GA biosynthetic pathway has been well characterized, little is known about the regulation of upstream GA biosynthetic enzymes, including copalyl diphosphate synthase (CPS) and kaurene synthase (KS), which catalyze the first committed step in GA biosynthesis. This work reports an investigation of the regulation of the GA pathway in Arabidopsis from several perspectives: the role of light regulation of AtCPS, identification of trans-acting factors regulating AtCPS, internal regulation within the GA pathway, and expression profiling of GA-responsive genes that might mediate physiological responses to GA.; Quantitative RT-PCR was used to measure AtCPS mRNA levels in tissue from germinating seeds and seedlings grown in continuous white light or darkness. In both cases, AtCPS mRNA accumulation was higher in light-grown tissues. The timing of AtCPS up-regulation suggests that it may mediate aspects of light-induced morphology. To identify potential regulators of AtCPS, a CPS-GUS reporter construct was used in a forward genetic screen. Numerous putative mutants were identified with changes in seedling GUS activity. One previously-identified mutant affecting CPS-GUS in developing seeds was shown to be allelic to the Polycomb Group gene FIS2, and was shown to regulate multiple GA biosynthetic genes.; To examine the role of internal regulation of GA biosynthesis, accumulation of bioactive GAs and GA intermediates was measured in plants that overexpress CPS and/or KS. CPS and CPS/KS overexpressors accumulate more of the early GA intermediates ent-kaurene and kaurenoic acid, but not bioactive GAs. KS overexpressors have WT accumulation of GA and GA intermediates, suggesting that CPS is limiting for early GA biosynthesis. Microarray analysis was used to gain a global perspective of seedling-expressed GA-responsive genes and potential targets of the GA signaling proteins RGA and GAI. 108 genes were identified as GA-responsive, and one of these, the GRAS protein SCARECROW-LIKE3, was shown to be a putative positive regulator of GA response. |
