Modulation Of Primary Root Touch Responses In Arabidopsis By Apyrases And Extracellular ATP

Skewing is induced by touch stimuli the roots experience as they grow along the surface. Touch stimuli also induce the release of extracellular ATP(eATP) into plants’ extracellular matrix, and two apyrases(NTPDases) in Arabidopsis, APY1 and APY2, can help regulate the concentration of eATP. Despite these previous studies on the functions of APY1 and APY2, the general importance of these enzymes in the response of plant roots to stimuli is not well understood. In the present thesis, we explore the potential functions of apyrases and eATP in Arabidopsis primary roots by caracterrizing and comparing the primary roots growth and development.Fisrt, we report that APY1 and APY2 play an important role in the control of root skewing in Arabidopsis.The primary roots of seedlings overexpressing APY1 show less skewing than wild-type plants. Plants suppressed in their expression of APY1 show more skewing than wild-type plants. Increasing the agar concentration to 1.5% caused increased skewing as indicated by statistically significant higher HGI values for all the roots tested except for the APY1-overexpressing roots, which still grew mostly straight down. The roots overexpressing APY1 had increased primary root length compared to wild type, and both apy1 and apy2 single-knockout mutants show reduced primary root length compared to wild type.To further confirm the function of apyrase in the regulation of root skewing, we examined the behavior of RNAi mutants(R2-4A) in which APY2 is knocked out and APY1 suppression is induced by 4μM estradiol. When the estradioltreated R2-4A mutants were grown on vertical or tilted agar surfaces, skewing was increased compared to estradiol-treated wild-type roots as indicated by significantly higher HGI values.The R2-4A mutant roots that were not induced by estradiol had the same HGI value as Ws wild-type and APY2 KO roots.Correspondingly, chemical inhibition of apyrase activity increased skewing in mutants and wild-type roots.We applied two chemical apyrase inhibitors, apyrase inhibitor 4 and NGXT 1913, application of either of these apyrase inhibitors induced a statistically significant increase in the HGI and an observable increase in root skewing to the right in comparison to wild-type control roots grown on control plates. When the estradiol-induced R2-4A mutants were grown on apyrase inhibitors apyrase inhibitor 4 and NGXT 1913, right skewing was increased compared to estradiol-treated wild-type roots and to non-induced R2-4A mutants, as indicated by significantly higher HGI.Studies have indicated that right-slanting root growth is accompanied by a characteristic rotation of the root tip, which can be traced to the cell file rotation(CFR) of the primary root epidermis. We assayed the CFR in roots of APY1 overexpressing and apy1 single knockout seedlings and found that apy1 knockout roots had an increased left-handed epidermal CFR while the APY1 OE roots showed decreased left-handed epidermal CFR compared to wild-type roots.In addition, we demonstrate that exogenous application of ATP or ATPγS also increased skewing in wild-type roots, which could be blocked by co-incubation with a purinergic receptor antagonist(PPADS). In dose-response assays testing root skewing of the primary roots of wild-type and apyrase mutant seedlings of Arabidopsis with applied ATP, treatment with 1mM ATP increased the HGI value for APY1 KO and APY2 OE roots, while treatment with 2mM and 3mM ATP increased HGI values for roots of all the genotypes tested, treatment with 100μM ATPγS induced an increase in root skewing similar to 2mM ATP. To test the effects of compounds that could be derived from ATP hydrolysis, primary root skewing was examined in response to comparable concentrations of ADP, AMP and inorganic phosphate(K2HPO4), we found that the increased skewing in response to ATP and ADP was not due to phosphate release.The strong right skewing and cell file rotation phenotypes of apy1 mutant highly resemble those previous reported cytoskeleton-associated mutants grown on agar surface. Pharmacological studies show that by applied two microtubule target agents, significantly increased the skewing phenotypes of all test genotypes, especially the APY1 overexpressing line APY1 OE, which show altered sensitivity to microtubule associated drugs relative to wild type as regard to the right skewing and epidermal cell file rotation phenotypes: microtubule stabilizing agent Taxol could induce APY1 OE primary roots strong right skewing and left handed cell file rotation, while the microtubule depolymerizing agent Propyzamide could caused severely radial cell swelling in the primary roots of APY1 OE seedlings. These results indicated that the apyrase in Arabidopsis may have microtubule associated functions. In addition, in vivo microtubule visualization revealed apyrases inhibitors altered the skewing phenotypes may have effect on the cortical microtubule arrays in the primary roots of MBD-GFP seedlings from the apex to the distal end of elongation zone, confocal images show that the apyrase inhibitors treated seedlings have lot of fluorescence spots. We also examined the actin filament arrays in FABD-GFP seedling, neither the root skewing phenotypes nor actin filaments arrays is affected by the same concentrations of apyrase inhibitors.Taken together the results indicate that APY1/APY2 and eATP play important roles in the signalings steps that mediate primary roots touch responses in agar surface.