Arabidopsis MICROTUBULE DESTABILIZING PROTEIN 60 Is Involved In Ethylene Promotion Of Hypocotyl Elongation

The phytohormone ethylene plays crucial roles in regulation of plant growth in response to multiple developmental and environmental cues,including promotion of hypocotyl elongation.Numerous studies have showed that microtubule-associated proteins(MAPs)mediate the hypocotyl cell growth through regulation of the cortical microtubule orientation and dynamics.However,the mechanisms of the microtubule regulation in ethylene-induced hypocotyl elongation in response to multiple cues are largely unclear.In this study,a novel microtubule-associated protein MICROTUBULE DESTABILIZING PROTEIN 60(MDP60)from Arabidopsis thaliana is identified as a positive regulator of hypocotyl cell elongation.ChIP-qPCR,EMSA,and yeast one hybrid assays showed that the transcription factor PIF3 involved in ethylene singnal pathway targets MDP60 promoter directly.Quantitative real-time PCR analyses from ethylene-insensitive mutant ein2-5 and the PIF3 loss-of-function mutant pif3-3 indicated that ethylene upregulates MDP60 expression via the transcription factor PIF3.GUS staining shows that MDP60 is mainly expressed in light-grown hypocotyls.The effect of ethylene on the promotion of hypocotyl cell elongation is significantly suppressed in MDP60 RNAi transgenic lines and mdp60 mutant generated by using the latest CRISPR/Cas9 technology.Moreover,MDP60 overexpression significantly increases the hypocotyl length of light-grown seedlings from mutant ein2-5 and pif3-3,demonstrating that MDP60 plays a positive role in light-grown hypocotyl elongation.Since PIF3 protein is unstable under light condition,submergence treatment was performed to test the role of MDP60 on ethylene-promoted hypocotyl elongation.Ethylene production is increased and hypocotyl length is much longer in submergence-treated wild type seedlings,but not in ein2-5 and pif3-3 mutant,suggesting that ethylene signaling pathway plays an important role in submergence-induced hypocotyl elongation by stabilizing PIF3 protein.Quantitative real-time PCR shows that MDP60 expression is induced by submergence and the hypocotyl length of MDP60 RNAi seedlings are much shorter compared with the wild type after the submergence treatment.These findings suggest that MDP60 plays a positive role in ethylene signaling-induced hypocotyl cell elongation in response to multiple cues,including the submergence.In vitro co-sedimentation and immunofluorescence labeling assays show that MDP60 directly binds to microtubules.Transient assay indicates that MDP60 co-localizes with the cortical microtubules in vivo.These results demonstrate that MDP60 is a novel microtubule-associated protein.TIRFM assay shows that MDP60 regulates microtubule dynamics by directly depolymerizing microtubules.The microtubule-destabilizing activity of MDP60 was further investigated in MDP60 RNAi hypocotyl cells using the microtubule-disrupting drug oryzalin.The result shows that microtubules are less sensitive to oryzalin treatment when MDP60 expression level is decreased,demonstrating that MDP60 functions as a microtubule destabilizer.Furthermore,regulation of cortical microtubule reorientation is found to be essential for ethylene-promoted hypocotyl elongation.And reorganization of cortical microtubules in the cells of MDP60 RNAi seedlings is less sensitive to ethylene treatments.These findings indicate that ethylene signaling promotes hypocotyl cell elongation through regulation of cortical microtubule organization,which is mediated by a novel microtubule-destabilizing protein MDP60 in Arabidopsis.This study reveals a potential mechanism participateing in ethylene-induced promotion of hypocotyl cell elongation through microtubules in response to environmental and abiotic cues in Arabidopsis.