Functions Of CML24 In Primary Root Mechanoresponses In Arabidopsis

As plant roots grow though the soil, they encounter a variety of mechanostimuli, including lateral pressure, friction by soil particles, and impedance by physical barriers, and gravity. Appropriate perception and response to these mechanostimuli substantially contribute to the adaption and survival of plants to local soil environment.Past researches have demonstrated that the expression of a vast collection of Arabidopsis genes can be significantly influenced by mechanostimuli. Among these genes are the four original Arabidopsis TCH genes (touch inducible genes). TCH1~3 encode calmodulin (CaM) and calmodulin-like (CML) proteins, which suggested potential involvement of calcium signal and potential calcium signal sensors in Arabidopsis mechanoresponses. Recent evidence implicates calcium signal as an early trigger of the complex Arabidopsis mechanotransduction pathways. However, whether TCH1~3 genes really take mechano-related functions in Arabidopsis, and if yes, what kind of functions do they take remain unknown. In the present thesis, we used a reverse genetic approach and explore the potential functions of the TCH1~3 in Arabidopsis primary root mechanoresponses by characterizing and comparing the primary root mechanoresponses between TCH1~3 mutants and wild type (Col-0) at organal, cellular and subcellular levels. The results indicated that TCH2 (also named CML24) may have a role in thigmoresponse of Arabidopsis primary root via regulation of cortical microtubule orientation, and that normal function of CML24 is important in adaption of Arabidopsis primary roots to the variation of mechanical condition in soil environment. Our relusts provided direct evidences for the hypothetical functions of CML24 in Arabidopsis mechanoresposnes, implicated directions for further researches to comprehensively elucidate the functions of CML24, and contributed important supplement to the current understanding of the roles of calcium communication in plant mechanoresponses. Following are the main studies of the present thesis:Because of gravitropism, root tips of Arabidopsis grown on backward tilted agar surfaces are constantly stimulated by touch. Thus, we screened the TCH gene(s) that may function in primary root mechanoresponses by characterizing and comparing the primary root growth behaviors on agar surfaces between TCH1~3 mutants and wild type (Col-0) at the beginning of this thesis work. The results indicated that primary roots of all the TCH1~3 mutants tested showed normal waving growth and gravitropism. However, primary roots of two point-mutant alleles of TCH2, cml24-2 and cml24-4, exhibited reduced primary root length, skewing growth and epidermal cell file rotation with preferential directions. The skewing and epidermal cell file rotation phenotypes of cml24 mutants were dependent on agar surface touch, suggesting that cml24 mutants may have defects in primary root thigmoresponse.The skewing and epidermal cell file rotation phenotypes of cml24 mutants highly resemble those of reported microtubule-associated mutants grown under same condition. Pharmacological studies by supplementing the agar growth medium with microtubule-targeted agents of different concentrations revealed that cml24 mutants have altered sensitivity to microtubule-targeted agents relative to wild type as regard to the skewing and epidermal cell file rotation phenotypes, which suggested that CML24 may have microtubule-related functions. Characterization of primary root growth behaviors of GFP-MBD/cml24 homozygous seedlings on agar surfaces provided genetic evidence to support this possibility. GFP-MBD transgene and cml24 mutations showed non-additive effect on primary root growth behaviors on agar surfaces. In addition, in vivo microtubule visualization revealed agar surface-dependent difference in cortical microtubule orientation of epidermal cells in primary root elongation zone between GFP-MBD (Col-0) and GFP-MBD/cml24-4. This difference was absent in liquid growth condition, which consisted with a hypothesis that CML24 may have a role in thigmoresponse of Arabidopsis primary roots via regulation of cortical microtubule orientation.Stacking agar medium layers with different concentrations of agar and embedding glass barriers into growth medium provided efficient approaches for mimicking the variation of penetration resistance in natural soil environment. Thus, to further confirm the defective thigmoresponse of cml24 mutant primary roots and their functional consequences in growth responses to changing mechanical conditions in soil environments, we conducted hard-agar layer penetration and glass barrier response assay in primary roots of wild type and cml24 mutants. Primary roots of cml24 mutants showed reduced penetration ratio from a softer agar layer to a harder agar layer compared to those of wild type, which suggested that primary roots of cml24 mutants were more sensitive to increasing in penetration resistance of growth environment than those of wild type. Furthermore, primary roots of cml24 mutants that did not penetrate the harder agar layer, showed coiling growth with biased directions on the interfaces of the harder agar layer, while those of wild type showed wavy meandering growth. Primary roots of cml24 mutants were also impaired in forming the'step-like'growth response upon counteracting a glass barrier in front of their growth pathway, which was reproducibly observed in wild-type primary roots. These results implicate the importance of normal CML24 function in adaption of Arabidopsis primary roots to the variation of mechanical condition in soil environment.Based on the hypothetical role of CML24 as a potential calcium signal sensor, the real-time calcium signal patterns in primary roots of wild type and cml24 mutants induced by mechanical bending were quantitatively measured, and it was found that the primary roots of YC3.6/cml24 homozygous seedlings were able to generate a similar two-peak calcium signal in response to mechanical bending as YC3.6(Col-0) primary roots did, which supported the classic hypothesis that"the functions of CML24 in Arabidopsis mechanoresponse may mainly be located downstream of calcium signal". However, we also found that the resting level of cytosolic Ca2+ concentration in primary roots of YC3.6/cml24-2 was higher than those of YC3.6, the second peaks of bending-induced calcium signals in primary roots of both YC3.6/cml24-2 and YC3.6/cml24-4 sustained longer than those of YC3.6, and that the relative increased amplitude of the second peak in primary roots of YC3.6/cml24-4 was higher than those of YC3.6. Thus, we hypothesized that CML24 may also have a role in regulation of cytosolic Ca2+ homeostasis.