The function of guard cell expressed genes in Arabidopsis thaliana

Guard cells play an essential role in controlling stomatal aperture and respond to many stimuli including changes in the concentration of CO2, light and phytohormones such as ABA. In this project, the function of Arabidopsis genes identified by a microarray screen, as being potentially expressed in guard cells was studied. Two genes encoding an Arabidopsis protein kinase APKla, and a putative pectinesterase AP Ea and their closely related genes AP K 1 band AP Eb respectively were studied. GUS reporter gene fusions were constructed for each gene and expressed in Arabidopsis to investigate expression patterns. The results indicated relatively high guard cell expression for at least three of the genes (APKla. APKlb and APEa). Homozygous knockout mutant plants were identified for each gene of the four guard cell target genes by RT-PCR of T-DNA insertion lines. Putative overexpressing plant lines were created by placing three of the genes (AP KI a, AP K 1 band AP Eb) under the control of the CaMV 355 promoter in transgenic plants. Several physiological techniques were applied to the knockout and over-expressing plant lines to assess whether each gene product had a function in stomatal aperture control or stomatal development. Stomatal conductances were measured using Infra Red Gas Analysis (IRGA) to investigate mutant responses to different light intensities. This suggested an impairment in apkl a and APKl b knockout mutants in stomatal response to light-induced opening. Photosynthetic rate and Ci were measured for apk l a and apklb knockout mutants and no differences to wild-type values were observed. Stomatal indices and densities were examined and no significant differences were found in kinase knockout mutants and over-expressing plants. Stomatal aperture responses of kinase and pectinesterase mutants and putative over-expressers to light. ABA and CO2 were measured in leaf epidermal strip bioassay experiments. The kinase knockout m utants and putative over-expressers had reduced their stomatal apertures in response to light-induced stomatal opening and showed no differences to controls in dark induced-stomatal closure experiments (apart from APKlaOE which had enhanced dark-induced closure). But the kinase knockout mutants also showed reduced stomatal opening responses to C02 free air. A variety of responses to ABA-inhibition of stomatal opening and CO2-induced closure were exhibited by the kinase mutants and putative over-expressers. Relative water content (RWC) was significantly higher in droughted kinase knockout mutant plants perhaps because of their reduced stomatal opening. It is therefore possible that the AP K 1 a and AP K I b genes may useful in produces drought tolerance plants. Pectinesterase knockout mutants and putative over-expresser lines also had reduced stomatal apertures following exposure to light and no differences to wild-type were obtained following dark adaptation (apart from APEbOE). apea-l, apeb-l and apeb- 2 exhibited insensitivity to ABA and C02 since they maintained larger stomatal apertures than controls following exposure to ABA and ambient CO2• Droughted apea mutant plants had significantly lower relative water content perhaps because of their ABA and CO2 insensitivity, whereas no significant differences were noticed between apeb mutants and control plants. Stomatal indices and densities were examined and apea and apeb stomatal densities were similar to control when plants were grown under ambient CO2 but apea mutant plants had higher stomatal densities when grown under either low or elevated C02.EThOS - Electronic Theses Online ServiceGBUnited Kingdo