COP1 Mediates Degradation Of Microtubule-associated Protein WDL3 In Regulating Arabidopsis Hypocotyl Elongation

Light plays a crucial role in plant growth and development,and negatively regulates plant hypocotyl elongation through its signaling pathway.Microtubule-associated proteins(MAPs)control the organization,stability,and dynamics of microtubules functioning in plant growth and plant cell morphogenesis.Previous studies have shown that light regulates the arrangement of the microtubule,however,whether the microtubule is involved in light signaling-mediated cell growth and the underlying mechanisms are still largely unknown.CONSTITUTIVE PHOTOMORPHOGENIC 1(COP 1),a well-known E3 ubiquitin ligase,functions as a central regulator of plant growth and photomorphogenic development in plants.In the dark,COP1 targets a subset of nuclear-localized photomorphogenesis-promoting factors for degradation,negatively regulating the seedling photomorphogenesis.Dark-grown cop1 mutant seedlings display the characteristics of light-grown seedlings,including short hypocotyls.In darkness,COP1 protein is mostly localized in the nucleus;however,some COP1 is also detectable outside the nucleus although the biological significance of this distribution remains largely unclear.A recent study showed that COP1 activity is crucial for microtubule destabilization,which is involved in darkness-and abscisic acid-induced stomatal closure,but the underlying molecular mechanisms of how COP1 regulated the microtubules remain largely unknown.Previous studies have shown that microtubule-associated protein WDL3 participated in the control of hypocotyl cell elongation,and WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark.However,the molecular mechanism of this degradation was unclear.In this study,we first examined whether COP1 could physically interact with WDL3.In vitro GST pull-down assay showed that GST-WDL3 could successfully pull down COP1.The firefly luciferase complementation imaging(LCI)assay and coimmunoprecipitation(co-IP)assay showed that COP1 interacts with WDL3 in vivo,and further analysis revealed that the interaction is at the cortical microtubules in a dark-dependent manner.Moreover,our biochemical data showed that COP1 directly ubiquitinates WDL3 in vitro.Genetic evidences further showed that WDL3 protein is degraded in WT seedlings but is abundant in the cop1 mutants in the dark.Decreasing WDL3 expression changed the microtubule organization and stability,and partially suppressed the short hypocotyl phenotype of etiolated copl-6 seedlings.These results together demonstrated that WDL3 is targeted by COP1 for degradation in the dark.In Arabidopsis there are eight members in microtubule-associated protein WAVE-DAMPENED 2(WVD2)/WVD2-LIKE(WDL)family,with a conserved TPX2 domain at the C terminal.Previous studies have shown that other members of the family did not degrade in the dark except WDL3,suggesting that there are special structural features in WDL3 protein.In this study,we performed the yeast two-hybrid and firefly LCI assays,and showed that the N terminus(1-195)but not the C terminus(196-339)of WDL3 interacted with COP1 in vitro and in vivo.In addition,COP1 directly ubiquitinates WDL3 N terminus(1-195)in vitro,suggesting that the N terminus is important for WDL3 degradation.Based on the above results,our study reveals a function of COP1 in the cytoplasm to mediate hypocotyl cell elongation by regulating the abundance of WDL3 in a dark-dependent manner.These results of this study propose a new pathway of posttranscriptional regulation of MAPs to control hypocotyl cell elongation,and uncover a cytoplasmic substrate of COP1 that functions as a microtubule-associated protein in mediating hypocotyl cell elongation.