Genetic analysis of the regulation of TCH gene expression, Final Report

The Arabidopsis TCH genes, originally isolated as a consequence of their upregulation in response to the mechanical stimulus of touch, are also upregulated by a variety of seemingly disparate environmental and hormonal stimuli. To gain insight into the complexities of TCH gene regulation, a number of approaches were taken. Regulatory elements responsible for regulation were identified and characteristics of the regulation were evaluated. Reporter genes were used to monitor expression localization and dynamics. Microarray analyses of genome-wide expression behavior indicated that touch-inducible gene expression is more widespread than generally appreciated. Identification of all touch-regulated genes shed light on the types of cellular processes that may be altered in response to mechanical stress perturbations. Expression of the TCH2 gene, also called CML24, encoding a calmodulin (CaM)-like (CML) protein, was evaluated. CML24 shares over 40% amino acid sequence identity with CaM, has 4 EF hands and undergoes a Ca2+-dependent change in migration rate through denaturing gel electrophoresis, indicating that CML24 binds Ca2+ and, as a consequence, undergoes conformational changes. CML24 expression occurs in all major organs and is induced from 2- to 15-fold in plants subjected to touch, darkness, heat, cold, hydrogen peroxide, abscisic acid (ABA) and indole-3-acetic acid. The putative CML24 regulatory region confers reporter expression at sites of predicted mechanical stress, in regions undergoing growth, in vascular tissues and various floral organs and in stomata, trichomes and hydathodes. CML24 underexpressing transgenics are resistant to ABA inhibition of germination and seedling growth, defective in long-day induction of flowering, and have enhanced tolerance to CoCl2, molybdic acid, ZnSO4 and MgCl2. These data present evidence that CML24 encodes a potential Ca2+ sensor that may function to enable responses to ABA, day length and presence of various salts. Further investigation of CML24 function and regulation led to the finding that CML24 has a critical role in nitric oxide regulation. Distinct tilling mutant alleles demonstrated that CML24 can act as a switch in the response to day length perception. Because of potential redundancy with the related CML23 gene, CML23 T-DNA insertion mutants were identified and characterized. Together, CML23 and CML24 impact the autonomous regulatory pathway of the transition to flowering. Nitric oxide levels are elevated in cml23/cml24 double mutants. Therefore, CML23 and CML24 are potential calcium sensors regulate nitric oxide accumulation. In collaboration with Drs. McCann and Carpita, fourier transform infrared spectroscopy (FTIR) was used to assess, verify and classify wall architectural changes that occur as a result of single XTH insertion mutations. Thirty-four homozygous mutant lines of Arabidopsis representing 21 members of the xyloglucan endotransglucosylase/hydrolase gene family provided a set of mutants to characterize. Kohonen networks classified cell wall architectures of xth mutant lines and previously characterized cell wall mutants. The xth mutants were found to have chemical changes in their cell walls not detectable as phenotypic growth and development changes, consistent with the existence of feed-back loops that modify wall composition in response to a life-long deficiency of a cell wall enzyme. To gain insight into the potential physiological relevance of the distinct members of the XTH family, GUS reporter fusion genes were constructed, and plants expressing these transgenes were characterized to reveal spatial and temporal patterns of expression. In addition, Genevestigator sources were mined for comprehensive and comparative XTH expression regulation analysis. These data revealed that the Arabidopsis XTHs are likely expressed in every developmental stage from seed germination through flowering. All organs showed XTH::GUS expression and most, if not all, are found to express multiple XTH::GUS genes suggesting that XTHs may contribute to morphogenesis at every developmental stage and in every plant organ. Different XTHs have remarkably diverse and distinct expression patterns indicating that paralogous genes have evolved differential expression regulation perhaps contributing to the maintenance of the large gene family. Extensive overlap in XTH expression patterns is evident; thus, XTHs may act combinatorially in determining wall properties of specific tissues or organs. Knowledge of gene-specific expression among family members yields evidence of where and when gene products may function and provides insights to guide rational approaches to investigate function through reverse genetics