Pumping up the volume - vacuole biogenesis in Arabidopsis thaliana.

Plant architecture follows the need to collect CO2, solar energy, water and mineral nutrients via large surface areas. It is by the presence of a central vacuole that fills much of the cell volume that plants manage to grow at low metabolic cost. In addition vacuoles buffer the fluctuating supply of essential nutrients and help to detoxify the cytosol when plants are challenged by harmful molecules. Despite their large size and multiple important functions, our knowledge of vacuole biogenesis and the machinery underlying their amazing dynamics is still fragmentary. In this review, we try to reconcile past and present models for vacuole biogenesis with the current knowledge of multiple parallel vacuolar trafficking pathways and the molecular machineries driving membrane fusion and organelle shape.

Dot/Icm-Translocated Proteins Important for Biogenesis of the Coxiella burnetii-Containing Vacuole Identified by Screening of an Effector Mutant Sublibrary.

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary for C. burnetii virulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identify C. burnetii Nine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies in Coxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations in cbu0414 (coxH1), cbu0513, cbu0978 (cem3), cbu1387 (cem6), cbu1524 (caeA), cbu1752, or cbu2028 resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. The cbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain of C. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by the cbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire of C. burnetii factors that contribute to CCV biogenesis.

The DC1-domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis.

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain-containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine- and histidine-rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre-vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 - a member of the SNARE vesicle-associated protein family and previously related to a sterol-binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.

Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation.

Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 domain-containing protein2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.

The I-BAR protein Ivy1 is an effector of the Rab7 GTPase Ypt7 involved in vacuole membrane homeostasis.

Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show that Ivy1, the yeast homolog of mammalian missing-in-metastasis (MIM), is a vacuolar effector of Ypt7-GTP and interacts with the EGO/ragulator complex, an activator of the target of rapamycin kinase complex 1 (TORC1) on vacuoles. Loss of Ivy1 does not affect EGO vacuolar localization and function. In combination with the deletion of individual subunits of the V-ATPase, however, we observed reduced TORC1 activity and massive enlargement of the vacuole surface. Consistent with this, Ivy1 localizes to invaginations at the vacuole surface and on liposomes in a phosphoinositide- and Ypt7-GTP-controlled manner, which suggests a role in microautophagy. Our data, thus, reveal that Ivy1 is a novel regulator of vacuole membrane homeostasis with connections to TORC1 signaling.

AtVps11 is essential for vacuole biogenesis in embryo and participates in pollen tube growth in Arabidopsis.

Vacuoles are multiple functional and essential organelles in plants. Studies in Saccharomyces cerevisiae had identified a tethering factor HOPS (Homotypic Fusion and Vacuolar Protein Sorting) complex that plays a critical role in vacuole biogenesis. The HOPS complex consists of four core subunits (Vps11, Vps16, Vps18 and Vps33) and two special subunits (Vps39 and Vps41). All these subunits were found in Arabidopsis, and our knowledge of the function of Arabidopsis HOPS complex are still limited. In this study, we investigated the function of AtVps11 gene in Arabidopsis, we found that vps11/- lead to embryo lethal, vacuole biogenesis in embryo was impaired. Furthermore, pollen tube growth was arrested by vps11 mutation, however, no obvious vacuole biogenesis defects were found in vps11 pollen tube. Our study indicated that in Arabidopsis, Vps11 is required for vacuole biogenesis in embryo, which is essential for embryogenesis. It also plays a role in pollen tube growth but looks not required for vacuole biogenesis in pollen tube.

ENV7 and YCK3, which encode vacuolar membrane protein kinases, genetically interact to impact cell fitness and vacuole morphology.

Saccharomyces cerevisiae vacuoles serve as a model for membrane fusion and fission. Yck3, a vacuolar membrane kinase, has been implicated in regulation of vacuole fusion. Recently, we established Env7 as another vacuolar membrane protein kinase with similar but nonredundant function to Yck3. Here, we report that native Env7 localizes to the vacuole independent of Yck3, where as its phosphorylation is YCK3 dependent. We also show that env7Δyck3Δ double mutant exhibits severely compromised fitness, altered cell size and bud vacuoles, and F-class vacuolar morphology. Our results establish negative genetic interactions between ENV7 and YCK3 and suggest cooperative roles for the two conserved genes in regulation of membrane dynamics. Such genetic buffering supports a critical role for membrane flux in global cell fitness.

Bioinformatic Analysis for Identifying Transcription Factors Involved in Protein Secretion and Vacuole Formation.

Protein secretion and vacuole formation are vital processes in plant cells, playing crucial roles in various aspects of plant development, growth, and stress responses. Multiple regulators have been uncovered to be involved in these processes. In animal cells, the transcription factor TFEB has been extensively studied and its role in lysosomal biogenesis is well understood. However, the transcription factors governing protein secretion and vacuole formation in plants remain largely unexplored. In recent years, an increasing number of bioinformatics databases and tools have been developed, facilitating computational prediction and analysis of the function of genes or proteins in specific cellular processes. Leveraging these resources, this chapter aims to provide practical guidance on how to effectively utilize these existing databases and tools for the analysis of key transcription factors involved in regulating protein secretion and vacuole formation in plants, with a particular focus on Arabidopsis and other higher plants. The findings from this analysis can serve as a valuable resource for future experimental investigations and the development of targeted strategies to manipulate protein secretion and vacuole formation in plants.

RNA HELICASE 32 is essential for female gametophyte development in Arabidopsis.

The normal progression of mitotic cycles and synchronized development within female reproductive organs are pivotal for sexual reproduction in plants. Nevertheless, our understanding of the genetic regulation governing mitotic cycles during the haploid phase of higher plants remains limited. In this study, we characterized RNA HELICASE 32 (RH32), which plays an essential role in female gametogenesis in Arabidopsis. The rh32 heterozygous mutant was semi-sterile, whereas the homozygous mutant was nonviable. The rh32 mutant allele could be transmitted through the male gametophyte, but not the female gametophyte. Phenotypic analysis revealed impaired mitotic progression, synchronization, and cell specification in rh32 female gametophytes, causing the arrest of embryo sacs. In the delayed pollination test, none of the retarded embryo sacs developed into functional female gametophytes, and the vast majority of rh32 female gametophytes were defective in the formation of the large central vacuole. RH32 is strongly expressed in the embryo sac. Knock-down of RH32 resulted in the accumulation of unprocessed 18 S pre-rRNA, implying that RH32 is involved in ribosome synthesis. Based on these findings, we propose that RH32 plays a role in ribosome synthesis, which is critical for multiple processes in female gametophyte development.

Vacuole Biogenesis in Plants: How Many Vacuoles, How Many Models?

Vacuoles are the largest membrane-bounded organelles and have essential roles in plant growth and development, but several important questions on the biogenesis and dynamics of lytic vacuoles (LVs) remain. Here, we summarize and discuss recent research and models of vacuole formation, and propose, with testable hypotheses, that besides inherited vacuoles, plant cells can also synthesize LVs de novo from multiple organelles and routes in response to growth and development or external factors. Therefore, LVs may be further classified into different subgroups and/or populations with different pH, cargos, and functions, among which multivesicular body (MVB)-derived small vacuoles are the main source for central vacuole formation in arabidopsis root cortical cells.

Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation.

Reprogramming cells to study vacuolar development.

During vegetative and embryonic developmental transitions, plant cells are massively reorganized to support the activities that will take place during the subsequent developmental phase. Studying cellular and subcellular changes that occur during these short transitional periods can sometimes present challenges, especially when dealing with Arabidopsis thaliana embryo and seed tissues. As a complementary approach, cellular reprogramming can be used as a tool to study these cellular changes in another, more easily accessible, tissue type. To reprogram cells, genetic manipulation of particular regulatory factors that play critical roles in establishing or repressing the seed developmental program can be used to bring about a change of cell fate. During different developmental phases, vacuoles assume different functions and morphologies to respond to the changing needs of the cell. Lytic vacuoles (LVs) and protein storage vacuoles (PSVs) are the two main vacuole types found in flowering plants such as Arabidopsis. Although both are morphologically distinct and carry out unique functions, they also share some similar activities. As the co-existence of the two vacuole types is short-lived in plant cells, how they replace each other has been a long-standing curiosity. To study the LV to PSV transition, LEAFY COTYLEDON2, a key transcriptional regulator of seed development, was overexpressed in vegetative cells to activate the seed developmental program. At the cellular level, Arabidopsis leaf LVs were observed to convert to PSV-like organelles. This presents the opportunity for further research to elucidate the mechanism of LV to PSV transitions. Overall, this example demonstrates the potential usefulness of cellular reprogramming as a method to study cellular processes that occur during developmental transitions.