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.

Functions of RMR proteins in intracellular trafficking in the moss "Physcomitrium patens" Mitten (previously "Physcomitrella patens")

In this study, I focused on a new family of receptors, called RMRs (Receptor-like Membrane RING-H2) and I tried to investigate their role in the moss Physcomitrium patens Mitten (previously Physcomitrella patens). There is some evidence that in Angiosperms, RMRs are vacuolar receptors for the neutral/storage vacuole that is a compartment where storage proteins and metabolites are accumulated during seeds development or in somatic tissues. It is distinguished from lytic vacuole which has the same functions as animal lysosomes. The five PpRMR genes have been knocked-out, yielding viable material without visible phenotype (Ayachi, 2012). A trafficking phenotype was described by Fahr (2017) who generated the construct Citrine-Cardosin (Ci-Card) composed of the fluorescent protein Citrine fused to the C-terminal vacuolar sorting determinant (ctVSD) from cardosin A (cardosin is addressed to the vacuole in higher plants —Pereira et al., 2013). The fusion protein was delivered to the central vacuole of PpWT but mistargeted in PpRMR-KO lines, indicating that the targeting of this protein to the vacuole depends on PpRMRs. The introduction of this thesis presents the plant endomembrane system, with particular attention to vacuolar transport and ubiquitylation. In the second chapter, I show the techniques used to attempt to detect PpRMRs by Western Blot: our failure may be due to a rapid degradation of these proteins, which could prevent their detection. In the third chapter, I focused on PpRMR2 involvement in ubiquitylation. We hypothesize that PpRMRs are E3 ligases because they are members of the PA-TM-RING protein family. Most of these proteins have an E3 ubiquitin ligase activity in animals (Seroogy et al., 2004; Borchers et al., 2002), for this reason, we think that plant PpRMRs could have this function as well, which could contribute to vacuolar targeting. Indeed, I could confirm that PpRMR2 has an E3 ubiquitin ligase activity. PpRMRs substrates are still unknown in moss thus we have analysed putative candidates supposing that they could be ubiquitylated by PpRMRs. We have tested this hypothesis through in vitro ubiquitylation assays, obtaining ambiguous results. In the fourth chapter, I show preliminary results about the visible phenotype of PpRMR-KO mutants: PpWT and PpRMR-KO lines displayed phenotypic differences in leafy gametophores, which were accentuated upon salt stress exposure. Lastly, I transformed the transgenic lines PpWT/Ci-Card and Pp5KO/Ci-Card with mutated versions of PpRMR2 and analysed their effect on vacuolar transport by confocal microscopy. For most of the constructions tested, the trafficking was perturbed in both lines. Only PpWT/Ci-Card expressing PpRMR2ΔSer (lacking the Serine-Rich motif) displayed a typical vacuolar pattern.

MTV proteins unveil ER- and microtubule-associated compartments in the plant vacuolar trafficking pathway

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1. Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.

Integration of two RAB5 groups during endosomal transport in plants

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells.

Distinct sets of tethering complexes, SNARE complexes, and Rab GTPases mediate membrane fusion at the vacuole in Arabidopsis

Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.