Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.

ALIX mediates endosomal trafficking and turnover of abscisic acid receptors through the ESCRT/MVB pathway, modulating abscisic acid–mediated inhibition of plant

Significance: The regulation of protein abundance of receptors and transporters at the plasma membrane is important for proper signaling in many biological pathways. The removal of plasma membrane proteins can occur via the endocytic protein degradation pathway, in which posttranslational modification by ubiquitin plays a key role. The activity of ubiquitinating and deubiquitinating enzymes can determine the ubiquitination status of a given target protein, and it has been shown that both classes of enzymes have important physiological roles. However, how these enzymes themselves are regulated at the molecular level has not yet been completely understood. In this study, we report a possible mechanism by which the deubiquitinating enzyme AMSH3 is regulated by its interacting protein, apoptosis-linked gene-2 interacting protein X (ALIX), in Arabidopsis .

Phosphate (Pi) is a macronutrient that is essential for plant life. Several regulatory components involved in phosphate homeostasis have been identified, revealing a very high complexity at the cellular and subcellular levels. Determining the phosphate content in plants is crucial to understanding this regulation, and short real-time ³³Pi uptake imaging experiments have shown Pi movement to be highly dynamic. Furthermore, gene modulation by phosphate is finely controlled by localization of this ion at the tissue as well as cellular and subcellular levels. Deciphering these regulations requires access to and quantification of the phosphate pool in the various plant compartments. This review presents the different techniques available to measure, visualize and trace phosphate in plants, with a discussion of the future prospects.

Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response.