Mechanisms and regulation of organic acid accumulation in plant vacuoles

In fleshy fruits, organic acids are the main source of fruit acidity and play an important role in regulating osmotic pressure, pH homeostasis, stress resistance, and fruit quality. The transport of organic acids from the cytosol to the vacuole and their storage are complex processes. A large number of transporters carry organic acids from the cytosol to the vacuole with the assistance of various proton pumps and enzymes. However, much remains to be explored regarding the vacuolar transport mechanism of organic acids as well as the substances involved and their association. In this review, recent advances in the vacuolar transport mechanism of organic acids in plants are summarized from the perspectives of transporters, channels, proton pumps, and upstream regulators to better understand the complex regulatory networks involved in fruit acid formation.

An active RNA transport mechanism into plant vacuoles

RNA degradation inside the plant vacuole by the ribonuclease RNS2 is essential for maintaining nucleotide concentrations and cellular homeostasis via the nucleotide salvage pathway. However, the mechanisms by which RNA is transported into the vacuole are not well understood. While selective macroautophagy may contribute to this transport, macroautophagy-independent transport pathways also exist. Here we demonstrate a mechanism for direct RNA transport into vacuoles that is active in purified vacuoles and is ATP hydrolysis-dependent. We identify the RNA helicase SKI2 as a factor required for this transport pathway, as ski2 mutant vacuoles are defective in transport. ski2 mutants have an increased autophagy phenotype that can be rescued by exogenous addition of nucleosides, consistent with a function in nucleotide salvage. This newly-described transport mechanism is therefore critical for RNA degradation, recycling and cytoplasmic nucleotide homeostasis.

Multiple functions of the vacuole in plant growth and fruit quality

Vacuoles are organelles in plant cells that play pivotal roles in growth and developmental regulation. The main functions of vacuoles include maintaining cell acidity and turgor pressure, regulating the storage and transport of substances, controlling the transport and localization of key proteins through the endocytic and lysosomal-vacuolar transport pathways, and responding to biotic and abiotic stresses. Further, proteins localized either in the tonoplast (vacuolar membrane) or inside the vacuole lumen are critical for fruit quality. In this review, we summarize and discuss some of the emerging functions and regulatory mechanisms associated with plant vacuoles, including vacuole biogenesis, vacuole functions in plant growth and development, fruit quality, and plant-microbe interaction, as well as some innovative research technology that has driven advances in the field. Together, the functions of plant vacuoles are important for plant growth and fruit quality. The investigation of vacuole functions in plants is of great scientific significance and has potential applications in agriculture.

Light at the end of the tunnel: FRAP assay reveals that plant vacuoles start as a tubular network

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.

A Review of Plant Vacuoles: Formation, Located Proteins, and Functions

Vacuoles, cellular membrane-bound organelles, are the largest compartments of cells, occupying up to 90% of the volume of plant cells. Vacuoles are formed by the biosynthetic and endocytotic pathways. In plants, the vacuole is crucial for growth and development and has a variety of functions, including storage and transport, intracellular environmental stability, and response to injury. Depending on the cell type and growth conditions, the size of vacuoles is highly dynamic. Different types of cell vacuoles store different substances, such as alkaloids, protein enzymes, inorganic salts, sugars, etc., and play important roles in multiple signaling pathways. Here, we summarize vacuole formation, types, vacuole-located proteins, and functions.

The Plant Specific Insert as an Unconventional Signal in the Route to Plant Vacuoles

In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSIs domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

A novel in vitro assay reveals SNARE topology and the role of Ykt6 in autophagosome fusion with vacuoles

Autophagy is a catabolic pathway that delivers intracellular material to the mammalian lysosomes or the yeast and plant vacuoles. The final step in this process is the fusion of autophagosomes with vacuoles, which requires SNARE proteins, the homotypic vacuole fusion and protein sorting tethering complex, the RAB7-like Ypt7 GTPase, and its guanine nucleotide exchange factor, Mon1-Ccz1. Where these different components are located and function during fusion, however, remains to be fully understood. Here, we present a novel in vitro assay to monitor fusion of intact and functional autophagosomes with vacuoles. This process requires ATP, physiological temperature, and the entire fusion machinery to tether and fuse autophagosomes with vacuoles. Importantly, we uncover Ykt6 as the autophagosomal SNARE. Our assay and findings thus provide the tools to dissect autophagosome completion and fusion in a test tube.

New insight into bulb dynamics in the vacuolar lumen of Arabidopsis cells

Plant vacuoles are multifunctional organelles with dynamic and transient membranous structures, such as trans-vacuolar strands and bulbs. Bulbs are highly mobile structures that travel along trans-vacuolar strands. A candidate effector protein from Melampsora larici-populina (Mlp124357) fused with the enhanced green fluorescent protein (eGFP) was used to investigate the properties of central vacuoles and their bulbs. We discovered the coexistence of two bulb populations in Arabidopsis cells. In addition to previously-described bulbs, which present even marker protein distribution on the bulb surface, we discerned bulbs displaying irregular fusion protein distribution. Using fluorescence recovery after photobleaching (FRAP), we also demonstrated that bulbs do not exchange proteins with the tonoplast once they are formed. These results show that more than one type of bulb may co-exist in the same cell and provide evidence of micro-domains on the bulb surface. They also reveal that proteins do not flow freely from the tonoplast to the bulb membrane, giving new insight into the biology of tonoplast-derived substructures.