Functional characterization of two genes of the GDSL hydrolase gene family

Abstract

Hydrolases are an important class of enzymes in living organisms. They include GDSL hydrolase, a family of hydrolytic enzymes with a broad range of substrates whose members are characterized by a distinct GDSL amino acid motif. In Arabidopsis thaliana, the GDSL hydrolase family contains 100 members, but the majority of the respective genes remain uncharacterized. Thus, the aim of this study is to characterize two homologous members of GDSL hydrolase, CGM3 and CGM4 (”Contains GDSL Motif”). The full-length CGM3 and CGM4 proteins were localized in the apoplast. Although CGM3 and CGM4 share high sequence similarity, CGM3 was expressed in imbibed seeds, and CGM4 in the early lineage of stomatal development. However, in the absence of CGM4, CGM3 was expressed in the early stomatal lineage cells, suggesting that these homologous genes are, at least in part, functionally redundant. This thesis explores the function of the homologous genes CGM3 and CGM4, based on the results of anatomical, physiological, biophysical and biochemical experiments using cgm3cgm4 T-DNA insertion lines. A comparison of the stomatal patterning and index of cgm3cgm4 and wild type showed no obvious differences, but the ultrastructure of the stomata included an apparently thicker stomatal ledge in cgm3cgm4 than in the wild type. Physiological experiments showed that, in response to various abiotic and biotic stimuli, cgm3cgm4 plants had a tendency of a decreased width to length ratio of the stomatal pore (aperture index) and a transpiration rate lower than that of wild type plants. A transcriptome analysis showed no obvious differences in ABA-induced gene expression between cgm3cgm4 and wild type plants. According to a biophysical analysis (atomic force microscopy) of the stiffness at the stomatal pore area, there was a tendency towards an increased stiffness in the stomata of the cgm3cgm4 mutants but not in those of the wild type. Additionally, a biochemical analysis of the cell wall composition of the mutant plants suggested a reduction in the abundance of polyphenol derivatives compared to the wild type. These results indicated that the cgm3cgm4 plants have a mechanical defect in the opening-closing dynamics of their mature stomata, independent of ABA signaling. A mature stoma consists of two guard cells surrounding the stomatal pore. During the developmental process, stomatal precursors undergo symmetric cell division, giving rise to guard cells. At a later stage of development, the stomatal pore is formed between the guard cells. The guard cell wall facing the pore is a crucial site for the establishment of a typical, unevenly thickened, cell wall architecture and stomatal ledge, which influences the opening-closing dynamics of stomata. The research described in this thesis provides that the GDSL hydrolase genes CGM3 and CGM4 play roles in cell wall biogenesis, influencing stomatal opening-closing dynamics.