The root-hair pattern of Arabidopsis is determined through a regulatory circuit composed of transcription factor genes. The homeobox gene GLABRA2 ( GL2 ) has been considered a key component, acting farthest downstream in this regulation. GL2 modified to include a transactivating function caused epidermal cells to develop ectopic root hairs or root hair–like structures. With this system, the phospholipase Dζ1 gene ( AtPLD ζ 1 ) was identified as a direct target of GL2. Inducible expression of AtPLD ζ 1 promoted ectopic root-hair initiation. We conclude that GL2 exerts its regulatory effect on root-hair development through modulation of phospholipid signaling.

Opening up Vps34 protein complexes: During intracellular membrane trafficking, large protein complexes regulate and adapt the activity of signal transducer enzymes such as the class III phosphatidylinositol 3-kinase Vps34. These large enzyme complexes are present in all eukaryotic cells, having widespread importance in neurodegeneration, aging, and cancer; however, a structural understanding has been lacking. Rostislavleva et al. provide atomic-resolution insights into the structures of the Vps34-containing protein complexes required for autophagy, endocytic sorting, and cytokinesis. The V-shaped complexes can undergo opening motions, which allows them to adapt to and phosphorylate membranes. Science , this issue p. 10.1126/science.aac7365

While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3–green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a–GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

Yohei Ohashi, Shirley Tremel, Roger L. Williams