Cell-wall deposition of cellulose microfibrils is essential for plant growth and development. In plant cells, cellulose synthesis is accomplished by cellulose synthase complexes located in the plasma membrane. Trafficking of the complex between endomembrane compartments and the plasma membrane is vital for cellulose biosynthesis; however, the mechanism for this process is not well understood. We here report that, in Arabidopsis thaliana, Rab-H1b, a Golgi-localized small GTPase, participates in the trafficking of CELLULOSE SYNTHASE 6 (CESA6) to the plasma membrane. Loss of Rab-H1b function resulted in altered distribution and motility of CESA6 in the plasma membrane and reduced cellulose content. Seedlings with this defect exhibited short, fragile etiolated hypocotyls. Exocytosis of CESA6 was impaired in rab-h1b cells, and endocytosis in mutant cells was significantly reduced as well. We further observed accumulation of vesicles around an abnormal Golgi apparatus having an increased number of cisternae in rab-h1b cells, suggesting a defect in cisternal homeostasis caused by Rab-H1b loss function. Our findings link Rab GTPases to cellulose biosynthesis, during hypocotyl growth, and suggest Rab-H1b is crucial for modulating the trafficking of cellulose synthase complexes between endomembrane compartments and the plasma membrane and for maintaining Golgi organization and morphology.

Hypocotyls undergo different morphogenesis according to light and dark conditions. Cortical microtubules are reoriented in response to light to coordinate cell growth status. Kinesins are microtubule-based motor proteins that are mostly responsible for transporting organelles and vesicles, although some can also regulate microtubule organizations. However, whether kinesin can be involved in microtubule reorientation and hypocotyl elongation remains to be studied. Here, we demonstrated that ARMADILLO REPEAT KINESIN2 (ARK2) negatively regulated the hypocotyl elongation of Arabidopsis thaliana. The hypocotyl cells of the ark2 null allele were longer than those of the wild type and had relatively more transversely arranged cortical microtubules. Moreover, ARK2 co-localized with cortical microtubules and facilitated the light-induced reorientation of the cortical microtubule arrays. Interestingly, ARK2 protein is stable in the light and degraded through 26S proteasome pathway in the dark. Furthermore, we proved that ARK2 could interact with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which contributed to the down-regulation of ARK2 in darkness that may benefit hypocotyl growth in the dark.

Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.