Arabidopsis PAT 10-dependent Tonoplast Association Of A CBL-CIPK Signaling Module Dampens ABA Signaling During Stomatal Movement

Opening and closure of stomata, each consisting of two guard cells and being vividly described as "mouths of terrestrial plants”,are critical for the uptake of CO2,water transpiration, responses to pathogens and to harsh environmental conditions such as drought. Multiple environmental cues regulate stomatal movement, among which the phytohormone ABA is critical Thus, to reveal their molecular mechanism in regulation of stomatal movements will provide theoretical guidance of plant tolerance and will guide our agricultural production.We used the Arabidopsis as plant materials, reporting that PROTEIN S-Acyl TRANSFERASE 10(PAT10) mediates the tonoplast association of CBLs in guard cells,which in turn recruit CIPKs to form a signaling module. The two-component calcium sensing module negatively mediates ABA signaling by inhibiting vacuolar potassium efflux and thus preventing fast vacuolar convolution Early events of ABA signaling include Ca,a influx and [Ca2+]cyt elevation, for which positive feedback through CPKs have been demonstrated but negative feedback is obscure. Our results can fill in this gap,enhancing the recognition about ABA pathway. The main results and conclusions presented in this thesis are as follows:(1) PAT10 loss-of-function resulted in ABA hypersensitive stomatal movement.PATIO mutant displayed pleiotropic developmental defects compared with the wild type under soil growth condition Indeed, the null mutant for PATIO, pat10, was hypersensitive to ABA-induced stomatal closure. We specifically expressed PAT10 in guard cells and got a similar result with pat10 in the response of ABA induced stomatal closure.(2) The CBL2 CBL3 double mutant mimics pat10 in ABA-hypersensitive stomata]movement.There is a paper reported that the tonoplast localization depending on S-acylation of PATIO. We performed fluorescence co-localization in guard cells and membrane ing experiments to verify those results. The phenotypic resemblance of pat10 and cbl2 cbl3 is consistent with the fect that the tonop last association of CBL2 was abolished in pat10 guard cells. Furthermore, cbl2 cbl3 was hypersensitive to ABA-induced stomatal closure. These results suggested that PATIO-mediated tonoplast CBL2 and CBL3 are negative regulators of ABA response during stomatal movement.(3) Functional loss of PATIO or CBL2 CBL3 resulted in hypersensitivity to ABA-induced Ca2+influx and [Ca2+]cyt increase during stomatal movement.We first performed patch-clamp analysis to determine Ca2+ currents at the guard cells' protoplast plasma membrane. Both pat10 and cbl2 cbl3 were comparable to wild type without ABA treatment whereas hypersensitive to ABA-induced Ca2+ influx. To determine whether [Ca2+]cyt was elevated in guard cells ofpat10 and cbl2 cbl3 upon ABA treatment, we introduced cytoplasmic aequorin into the mutants and observed the higher[Ca2+]cyt. The simultaneous application of LaCl3 suppressed the ABA hypersensitive stomatal closure and [Ca2+]cyt increase of pat10 and cbl2 cbl3. EGTA suppressed the ABA hypersensitive stomatal closure as well. This result further demonstrated that mutations at PATIO or CBL2 CBL3 resulted in hypersensitivity in Ca2+-mediated ABA signaling during stomatal movement.(4) Mutations at PATIO and CBL2/CBL3 resulted in hypersensitivity to ABA-induced vacuolar convolution.We introduced a GFP-translational fusion of the inositol transporter INT1, a tonoplast associated protein, into pat10 and cbl2 cbl3. ABA treatment induced a rapid vacuolar convolution in wild type, while in conparison, vacuolar convolutibn upon ABA treatment was more thorough in pat10 and cbl2 cbl3, resulting in a significantly more convoluted vacuolar structure. vverexpression of NHX2, a K+/H+ anti-transporter protein,could rescue the ABA induced significant vascular convolution which implied the efflux of K+out of vacuoles plays a key role in vacuolar convolution of pat10and cbl2 cbl3.(5) Two tonoplast CIPKs are parts of the CBL-CIPK module during stomatal movement.The calcium-dependent interaction of CBLs with the NAF motif relieves the self-inhibition and activates the CIPKs. Here we identified CIPK9 and CIPK17 which could interact with CBL2 and CBL3 on the to nop last. cipk9 and cipk17 were hypersensitive to ABA induced stomatal closure. Above all, CIPK9 and CIPK17 play a role at the downstream of PAT10-CBL2/3 complex, negatively regulating ABA pathway in stomatal movement.