Functions of the calmodulin-related TCH3 and TCH4-xyloglucan endotransglycosylase in Arabidopsis plants

[摘要] Plants sense their environment and respond through changes in their development and physiology. Mechanical perturbation, darkness, temperature shocks, and exogenous hormones upregulate the expression of the TCH genes in Arabidopsis. The objective of this work was to determine the functions of the TCH3 and TCH4 proteins. TCH3, a calmodulin-related protein with six potential Ca$sp{2+}$ binding sites, binds Ca$sp{2+}$, as evidenced by its mobility shift on an SDS-polyacrylamide gel in the presence of Ca$sp{2+}.$ TCH4, a protein related in sequence to the enzyme xyloglucan endotransglycosylase (XET), has XET activity. The TCH4 XET prefers nonfucosylated oligosaccharide acceptor substrates, has a pH optimum of 6 to 6.5, and has unusually high activity at low temperatures, with an optimum of 12 to 18$spcirc$C. To address the role of these proteins in Arabidopsis development and responses to the environment, plants were generated with altered expression of TCH3 or TCH4. These plants were downregulated through antisense RNA, a T-DNA insertion in the gene, or cosuppression. Plants were upregulated by a strong, constitutive promoter driving an exogenous copy of the gene. The plants developed normally and did not show any detectable changes in cellular organization, cell wall integrity, or mechanical strength. Several assays were developed to test the responses of the transgenics to environmental stimuli and hormones. The transgenics responded normally to wind, heat, darkness/low light, gravity, mechanical obstruction, osmotic stress, auxin, and brassinosteroids. However, preliminary evidence suggests that the TCH4 transgenics may respond differentially to cold. Plants with reduced TCH4 levels were more sensitive to low temperatures, and plants overexpressing TCH4 were more hardy at low temperatures. The lack of a pronounced mutant phenotype in the transgenic plants suggests genetic redundancy. Nevertheless, under specific conditions, the TCH3 and TCH4 proteins may have essential functions in Arabidopsis.

[发布机构] Rice University

[效力级别] biology