Plant ESCRT protein ALIX coordinates with retromer complex in regulating receptor-mediated sorting of soluble vacuolar proteins

Vacuolar proteins play essential roles in plant physiology and development, but the factors and the machinery regulating their vesicle trafficking through the endomembrane compartments remain largely unknown. We and others have recently identified an evolutionarily conserved plant endosomal sorting complex required for transport (ESCRT)-associated protein apoptosis-linked gene-2 interacting protein X (ALIX), which plays canonical functions in the biogenesis of the multivesicular body/prevacuolar compartment (MVB/PVC) and in the sorting of ubiquitinated membrane proteins. In this study, we elucidate the roles and underlying mechanism of ALIX in regulating vacuolar transport of soluble proteins, beyond its conventional ESCRT function in eukaryotic cells. We show that ALIX colocalizes and physically interacts with the retromer core subunits Vps26 and Vps29 in planta. Moreover, double-mutant analysis reveals the genetic interaction of ALIX with Vps26 and Vps29 for regulating trafficking of soluble vacuolar proteins. Interestingly, depletion of ALIX perturbs membrane recruitment of Vps26 and Vps29 and alters the endosomal localization of vacuolar sorting receptors (VSRs). Taken together, ALIX functions as a unique retromer core subcomplex regulator by orchestrating receptor-mediated vacuolar sorting of soluble proteins.Peer reviewe

Advances in omics research on peanut response to biotic stresses

Peanut growth, development, and eventual production are constrained by biotic and abiotic stresses resulting in serious economic losses. To understand the response and tolerance mechanism of peanut to biotic and abiotic stresses, high-throughput Omics approaches have been applied in peanut research. Integrated Omics approaches are essential for elucidating the temporal and spatial changes that occur in peanut facing different stresses. The integration of functional genomics with other Omics highlights the relationships between peanut genomes and phenotypes under specific stress conditions. In this review, we focus on research on peanut biotic stresses. Here we review the primary types of biotic stresses that threaten sustainable peanut production, the multi-Omics technologies for peanut research and breeding, and the recent advances in various peanut Omics under biotic stresses, including genomics, transcriptomics, proteomics, metabolomics, miRNAomics, epigenomics and phenomics, for identification of biotic stress-related genes, proteins, metabolites and their networks as well as the development of potential traits. We also discuss the challenges, opportunities, and future directions for peanut Omics under biotic stresses, aiming sustainable food production. The Omics knowledge is instrumental for improving peanut tolerance to cope with various biotic stresses and for meeting the food demands of the exponentially growing global population

ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies

Abstract Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ringlike structures (1-2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP-ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP-ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP-ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells

alpha 2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth

[EN] COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (alpha-, beta-, beta' -, gamma-, delta-, epsilon- and xi-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of gamma and delta, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of alpha 2-COP, an Arabidopsis alpha-COP isoform. The alpha 2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24 delta 5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The alpha 2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the alpha 2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the alpha 2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of alpha 2-COP affects the expression of secretory pathway genes.Antibody against alpha-COP was kindly provided by Prof. F. Wieland. This work was supported by the Ministerio de Economia y Competitividad (grant number. BFU2012-33883 to FA and MJM; grant numbers BIO2011-26302 and BIO2014-55946-P to MP), the Generalitat Valenciana (grant numbers ISIC/2013/004 and GVACOMP2014-202 to FA and MJM) and the Research Grants Council (grant number AoE/M-05/12 to LJ). NP and CB were recipients of a fellowship from Ministerio de Educacion (FPU program). We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants and the microscopy and genomics section of SCSIE (University of Valencia).Gimeno-Ferrer, F.; Pastor-Cantizano, N.; Bernat-Silvestre, C.; Selvi-Martinez, P.; Vera Sirera, FJ.; Gao, C.; Perez Amador, MA.... (2017). alpha 2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth. Journal of Experimental Botany. 68(3):391-401. https://doi.org/10.1093/jxb/erw446S39140168

ESCRT‐dependent vacuolar sorting and degradation of the auxin biosynthetic enzyme YUC1 flavin monooxygenase

YUC flavin monooxygenases catalyze the rate-limiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP-YUC1. GFP-YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP-YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP-YUC1 was ubiquitinated and in this form GFP-YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP-YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes

Real-Time Detection of Caspase-3-Like Protease Activation in Vivo Using Fluorescence Resonance Energy Transfer during Plant Programmed Cell Death Induced by Ultraviolet C Overexposure1[C]

Caspase-like proteases have been demonstrated to be involved in plant programmed cell death (PCD). Here, the time scale of caspase-3-like protease activation was investigated in single living plant cells undergoing PCD induced by ultraviolet C (UV-C) overexposure. The real-time detection of caspase-3-like protease activation was achieved by measuring the degree of fluorescence resonance energy transfer (FRET) within a recombinant substrate containing enhanced cyan fluorescent protein (ECFP) linked by a peptide possessing the caspase-3 cleavage sequence, DEVD, to enhanced yellow fluorescent protein (EYFP; i.e. ECFP-DEVD-EYFP). Microscopic observations demonstrated that the ECFP-DEVD-EYFP fusion protein could be expressed correctly and the FRET from ECFP to EYFP could be found in transfected Arabidopsis (Arabidopsis thaliana) protoplasts. At 30 min after exposure to UV-C, caspase-3-like protease activation indicated by the decrease in FRET ratio occurred, taking about 1 h to reach completion in single living protoplasts. Mutation in the DEVD tag or a caspase-3 inhibitor could prevent the changes in FRET ratio induced by UV-C treatment, confirming that the decrease in FRET ratio was due to the cleavage of fusion protein as a result of caspase-3-like protease activation. This activation was further confirmed by in vitro caspase-3 substrate assay and western-blot analysis, showing the occurrence of cleavage in ECFP-DEVD-EYFP protein but not in the protein with a mutant DEVD tag. In summary, these results represent direct evidence for the activation of caspase-3-like protease in UV-C-induced PCD, and the FRET technique is a powerful tool for monitoring key events of PCD in living cells in real time