The Molecular Mechanism Of Microtubule-associated Protein WDL3Regulating Hypocotyl Elongation In Arabidopsis

The microtubule cytoskeleton is involved in plant growth, development, and morphogenesis. In plant cells highly dynamic microtubules form various microtubule arrays, which are controlled by microtubule-associated proteins.Studies have showed that microtubules play a crucial role in hypocotyl cell growth. Their orientation and dynamics are regulated by light, plant hormones, and other factors. Light significantly inhibits hypocotyl cell elongation. Dark-grown seedlings exhibit elongated and etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit hypocotyl growth in the light.In Arabidopsis there are seven members in microtubule-associated protein WAVE-DAMPENED2-LIKE (WDL) family. WDL3is one of the members, but its physiological function and its role in microtubule regulation are unclear. Here, we demonstrated that WDL3functions in hypocotyl cell elongation, and its protein level is regulated by an ubiquitin-26S proteasome-dependent pathway in response to light.We analyzed the characters of WDL3related to microtubules. Cosedimentation and transient expression assays showed that WDL3directly bound to microtubules both in vitro and in vivo. Further experiment results revealed that WDL3bundled and stabilized microtubules in vitro, indicating that WDL3is a microtubule-stabilizing factor. This was further confirmed by the observations of microtubules in cells. Cortical microtubules in the cells of WDL3-overexpressing seedlings had stronger tolerance to microtubule-disrupting drug oryzalin, compared to that in wild-type cells.Observations showed that WDL3RNA interference (RNAi) Arabidopsis seedlings had much longer hypocotyls than wild type when grown in the light. On the other hand, WDL3overexpression resulted in overall shortening of hypocotyl cells and the stabilization of cortical microtubules in the light. Analysis of microtubule dynamics showed that the catastrophe frequency was increased in WDL3RNAi cells, but decreased in WDL3-overexpressing cells, compared with that in wild-type cells. After exposure of the etiolated seedlings to light, cortical microtubules in hypocotyl cells transferred from a transverse array into oblique and longitudinal patterns. This cortical microtubule reorganization was hindered in WDL3RNAi cells, but accelerated in WDL3-overexpressing cells. These results demonstrated that WDL3is involved in light-regulated cortical microtubule reorganization. More importantly, the analysis of WDL3protein level showed that WDL3protein was abundant in the light, but was degraded in the dark. Pharmacological experiment demonstrated that the degradation of WDL3protein was through the26S proteasome pathway. In addition, overexpression of WDL3inhibited etiolated hypocotyl growth of regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) seedlings in the dark, but not wild-type seedlings.Our study indicated that an ubiquitin-26S proteasome-dependent mechanism regulates the protein level of WDL3in response to light to modulate hypocotyl cell elongation. This study proposes a new pathway involved in light-regulated hypocotyl cell elongation, and provides a new concept for elucidating the mechanisms underlining the regulation of hypocotyl growth.