SignificanceCell-fate determination and cellular behavior in plants rely mainly on positional information and intercellular communication. A plethora of cues are perceived by surface receptors and integrated into an adequate cellular output. Here, we show that the small receptor-like protein RLP44 acts as an intermediary to connect the receptors for two well-known signaling molecules, brassinosteroid and phytosulfokine, to control cell fate in the root vasculature. Furthermore, we show that the brassinosteroid receptor has functions that are independent from the responses to its hormone ligands and reveal that phytosulfokine signaling promotes procambial cell identity. These results provide a mechanistic framework for the integration of multiple signaling pathways at the plasma membrane by shifting associations of receptors in multiprotein complexes.
SignificancePlant vacuoles play unique roles such as storage and coloring, in addition to lysosomal/vacuolar functions shared by eukaryotes: degradation and recycling of waste. To fulfill these complex and specialized functions, plant vacuolar trafficking occurs through multiple, uniquely regulated transport pathways. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are involved in lysosomal/vacuolar trafficking in nonplant systems, although they also exist in plants. However, it remains almost entirely unknown how these tethering complexes regulate the unique aspects of plant vacuolar transport. Here, we show that HOPS and CORVET mediate distinct vacuolar trafficking pathways in coordination with different sets of soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins and RAB GTPase. Our findings provide further evidence for the unique evolutionary diversification of the vacuolar transport system in plants.
The vacuole is an essential organelle in plant cells, and its dynamic nature is important for plant growth and development. Homotypic membrane fusion is required for vacuole biogenesis, pollen germination, stomata opening, and gravity perception. Known components of the vacuole fusion machinery in eukaryotes include SNARE proteins, Rab GTPases, phosphoinositides, and the homotypic fusion and vacuolar protein sorting (HOPS) tethering complex. HOPS function is not well characterized in plants, but roles in embryogenesis and pollen tube elongation have been reported. Here, we show that HOPS subunits VPS33 and VPS41 accumulate in late endosomes and that VPS41, but not VPS33, accumulates in the tonoplast via a wortmannin-sensitive process. VPS41 and VPS33 proteins bind to liposomes, but this binding is inhibited by phosphatidylinosiltol-3-phosphate [PtdIns(3)P] and PtdIns(3,5)P, which implicates a nonconserved mechanism for HOPS recruitment in plants. Inducible knockdown of VPS41 resulted in dramatic vacuole fragmentation phenotypes and demonstrated a critical role for HOPS in vacuole fusion. Furthermore, we provide evidence for genetic interactions between VPS41 and VTI11 SNARE that regulate vacuole fusion, and the requirement of a functional SNARE complex for normal VPS41 and VPS33 localization. Finally, we provide evidence to support VPS33 and SYP22 at the initial stage for HOPS-SNARE interactions, which is similar to other eukaryotes. These results highlight both conserved and specific mechanisms for HOPS recruitment and function during vacuole fusion in plants.
The V-ATPase is a versatile proton-pump found in a range of endomembrane compartments yet the mechanisms governing its differential targeting remain to be determined. In Arabidopsis, VHA-a1 targets the V-ATPase to the TGN/EE whereas VHA-a2 and VHA-a3 are localized to the tonoplast. We report here that the VHA-a1 targeting domain serves as both an ER-exit and as a TGN/EE-retention motif and is conserved among seed plants. In contrast, Marchantia encodes a single VHA-isoform that localizes to the TGN/EE and the tonoplast in Arabidopsis. Analysis of CRISPR/Cas9 generated null alleles revealed that VHA-a1 has an essential function for male gametophyte development but acts redundantly with the tonoplast isoforms during vegetative growth. We propose that in the absence of VHA-a1, VHA-a3 is partially re-routed to the TGN/EE. Our findings contribute to understanding the evolutionary origin of V-ATPase targeting and provide a striking example that differential localization does not preclude functional redundancy.
Plant vacuoles play key roles in cellular homeostasis performing catabolic and storage functions, regulating pH and ion balance 1,2. The essential role of vacuoles for plant cell viability makes them a notoriously difficult subject to study. As a consequence, there is still no consensus on the mechanism of vacuolar establishment and the source of membrane material for it. Our previous suggestion of endoplasmic reticulum (ER) being the main contributor of membrane for growing young vacuoles3 was recently challenged in a study proposing that plant vacuoles are formed de novo via homotypic fusion of multivesicular bodies (MVBs)4. Authors of this work pointed out issues that might explain our seemingly contradictory observations and we have thus carefully revaluated our hypothesis. Using the Arabidopsis thaliana root as a model, we provide a systematic overview of successive vacuolar biogenesis stages, starting from the youngest cells proximate to the quiescent center. We validate our previous conclusions by demonstrating that the vacuolar dye BCECF is fully suitable for studying the organelle morphology and provide 3D models from vacuoles of all developmental stages. We established a customized FRAP assay and proved that even at the earliest stages of biogenesis, vacuoles comprise a connected network. Together, this adds to a growing body of evidence indicating that vacuolar structures cannot originate solely from MVBs.
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