Using Arabidopsis Mesophyll Protoplasts to Study Unfolded Protein Response Signaling

Various environmental stresses or artificial reagents can trigger unfolded protein accumulation in the endoplasmic reticulum (ER) due to the folding capacity of the ER being exceeded. This is termed ER stress, and triggers the unfolded protein response (UPR). Assays for activation of the UPR in plants include Tunicamycin (Tm)- or dithiothreitol (DTT)-mediated root growth inhibition, analysis of splicing of the UPR-responsive transcription factor bZIP60 (basic Leucine Zipper Domain 60), and upregulation of relevant UPR genes. We provide here a quick and robust method to detect UPR signaling in Arabidopsis thaliana protoplasts. This assay can also be applied to other plant species for which protoplasts can be isolated

Structure and function of endosomes in plant cells

Endosomes are a heterogeneous collection of organelles that function in the sorting and delivery of internalized material from the cell surface and the transport of materials from the Golgi to the lysosome or vacuole. Plant endosomes have some unique features, with an organization distinct from that of yeast or animal cells. Two clearly defined endosomal compartments have been studied in plant cells, the trans-Golgi network (equivalent to the early endosome) and the multivesicular body (equivalent to the late endosome), with additional endosome types (recycling endosome, late prevacuolar compartment) also a possibility. A model has been proposed in which the trans-Golgi network matures into a multivesicular body, which then fuses with the vacuole to release its cargo. In addition to basic trafficking functions, endosomes in plant cells are known to function in maintenance of cell polarity by polar localization of hormone transporters and in signaling pathways after internalization of ligand-bound receptors. These signaling functions are exemplified by the BRI1 brassinosteroid hormone receptor and by receptors for pathogen elicitors that activate defense responses. After endocytosis of these receptors from the plasma membrane, endosomes act as a signaling platform, thus playing an essential role in plant growth, development and defense responses. Here we describe the key features of plant endosomes and their differences from those of other organisms and discuss the role of these organelles in cell polarity and signaling pathways

Gravitropism and Lateral Root Emergence are Dependent on the Trans-Golgi Network Protein TN01

The trans-Golgi network (TGN) is a dynamic organelle that functions as a relay station for receiving endocytosed cargo, directing secretory cargo, and trafficking to the vacuole. TGN-localized SYP41-interacting protein (TNO1) is a large, TGN-localized, coiled-coil protein that associates with the membrane fusion protein SYP41, a target SNARE, and is required for efficient protein trafficking to the vacuole. Here, we show that a tno1 mutant has auxin transport-related defects. Mutant roots have delayed lateral root emergence, decreased gravitropic bending of plant organs and increased sensitivity to the auxin analog 2,4-dichlorophenoxyacetic acid and the natural auxin 3-indoleacetic acid. Auxin asymmetry at the tips of elongating stage II lateral roots was reduced in the tno1 mutant, suggesting a role for TNO1 in cellular auxin transport during lateral root emergence. During gravistimulation, tno1 roots exhibited delayed auxin transport from the columella to the basal epidermal cells. Endocytosis to the TGN was unaffected in the mutant, indicating that bulk endocytic defects are not responsible for the observed phenotypes. Together these studies demonstrate a role for TNO1 in mediating auxin responses during root development and gravistimulation, potentially through trafficking of auxin transport proteins

Autophagy in plants and algae

Autophagy is a major cellular degradation pathway in which materials are delivered to the vacuole in double-membrane vesicles known as autophagosomes, broken down, and recycled (Li and Vierstra, 2012; Liu and Bassham, 2012). In photosynthetic organisms, the pathway is strongly activated by biotic and abiotic stresses, including nutrient limitation, oxidative, salt and drought stress and pathogen infection, and during senescence (Perez-Perez et al., 2012; Lv et al., 2014). Mutation of genes required for autophagy causes hypersensitivity to stress, indicating that autophagy is important for tolerance of multiple stresses. While autophagy is often non-selective, a growing number of examples of selectivity are now evident, in which specific cargos are recruited into autophagosomes via cargo receptors (Floyd et al., 2012; Li and Vierstra, 2012). In this Research Topic, a series of original research articles and reviews highlight areas of current focus in plant and algal autophagy research, including mechanisms and cargos of selective autophagy, lipid degradation, and metabolic and physiological consequences of the autophagy pathway

Functional redundancy between trans-Golgi network SNARE family members in Arabidopsis thaliana

Background Vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system. Fusion is mediated by t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) fusion proteins on the target membrane and v-SNAREs on the vesicle membrane; v-and t-SNAREs interact with each other, driving vesicle fusion with the target membrane. The Arabidopsis thaliana trans-Golgi network resident SNAREs SYP41 and VTI12, along with YKT61/62, have been shown to function in vesicle fusion in vitro, consistent with immunoprecipitation results showing their interaction in Arabidopsis cell extracts. Conflicting published results have indicated that SYP4 family members are either functionally redundant or have distinct and essential functions; the reason for this discrepancy is unclear. Results Here we used a proteoliposome fusion assay to demonstrate that SYP42 and SYP43 can substitute for SYP41 in driving lipid mixing, providing support for functional overlap between family members. Previous reports have also suggested that VTI11 and VTI12 SNAREs show partial overlap in function, despite having mostly distinct localizations and binding partners. We show that VTI11 can substitute for VTI12 in in vitro lipid mixing reactions, providing molecular support for the genetic evidence for partial functional redundancy in vivo. Conclusions Our data provide biochemical evidence for functional overlap in membrane fusion between members of the SYP4 or VTI1 SNARE groups, supporting previous genetic data suggesting redundancy

A Functional Unfolded Protein Response Is Required for Normal Vegetative Development

The unfolded protein response (UPR) is activated in plants in response to endoplasmic reticulum stress and plays an important role in mitigating stress damage. Multiple factors act in the UPR, including the membrane-associated transcription factor, BASIC LEUCINE ZIPPER 17 (bZIP17), and the membrane-associated RNA splicing factor, INOSITOL REQUIRING ENZYME1 (IRE1). We have analyzed an Arabidopsis (Arabidopsis thaliana) ire1a ire1b bzip17 triple mutant, with defects in stress signaling, and found that the mutant is also impaired in vegetative plant growth under conditions without externally applied stress. This raised the possibility that the UPR functions in plant development in the same manner as it does in responding to stress. bZIP17 is mobilized to the nucleus in response to stress, and through the analysis of a mobilization-defective bZIP17 mutant, we found that to support normal plant development bZIP17 must be capable of mobilization. Likewise, through the analysis of ire1 mutants defective in either protein kinase or RNase activities, we found that both must be operative to promote normal development. These findings demonstrate that the UPR, which is associated with stress responses in plants, also functions under unstressed conditions to support normal development

Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots

Super-resolution fluorescence microscopy has generated tremendous success in revealing detailed subcellular structures in animal cells. However, its application to plant cell biology remains extremely limited due to numerous technical challenges, including the generally high fluorescence background of plant cells and the presence of the cell wall. In the current study, stochastic optical reconstruction microscopy (STORM) imaging of intact Arabidopsis thaliana seedling roots with a spatial resolution of 20–40 nm was demonstrated. Using the super-resolution images, the spatial organization of cortical microtubules in different parts of a whole Arabidopsis root tip was analyzed quantitatively, and the results show the dramatic differences in the density and spatial organization of cortical microtubules in cells of different differentiation stages or types. The method developed can be applied to plant cell biological processes, including imaging of additional elements of the cytoskeleton, organelle substructure, and membrane domains

Dynamics of Autophagosome Formation

Autophagy, literally defined as “self-eating,” functions as a degradation process by recycling cytoplasmic contents under stress conditions or during development. Upon activation of autophagy, a membrane structure known as a phagophore forms and expands, finally closing to form a double-membrane vesicle called an autophagosome (Fig. 1; Lamb et al., 2013; Yin et al., 2016). The completed autophagosome, which contains the autophagic cargo, is delivered to the vacuole (plants and yeast) or lysosome (animals). The outer membrane fuses with the vacuolar/lysosomal membrane, and the inner membrane and contents are released into the vacuole/lysosome as an autophagic body and are degraded by hydrolases. The breakdown products are transported back into the cytoplasm for reuse by the cell (Yang and Bassham, 2015)

5AtRabD2b and AtRabD2c have overlapping functions in pollen development and pollen tube growth

<p>Abstract</p> <p>Background</p> <p>Rab GTPases are important regulators of endomembrane trafficking, regulating exocytosis, endocytosis and membrane recycling. Many Rab-like proteins exist in plants, but only a subset have been functionally characterized.</p> <p>Results</p> <p>Here we report that AtRabD2b and AtRabD2c play important roles in pollen development, germination and tube elongation. <it>AtrabD2b </it>and <it>AtrabD2c </it>single mutants have no obvious morphological changes compared with wild-type plants across a variety of growth conditions. An <it>AtrabD2b/2c </it>double mutant is also indistinguishable from wild-type plants during vegetative growth; however its siliques are shorter than those in wild-type plants. Compared with wild-type plants, <it>AtrabD2b/2c </it>mutants produce deformed pollen with swollen and branched pollen tube tips. The shorter siliques in the <it>AtrabD2b/2c </it>double mutant were found to be primarily due to the pollen defects. <it>AtRabD2b </it>and <it>AtRabD2c </it>have different but overlapping expression patterns, and they are both highly expressed in pollen. Both AtRabD2b and AtRabD2c protein localize to Golgi bodies.</p> <p>Conclusions</p> <p>These findings support a partially redundant role for AtRabD2b and AtRabD2c in vesicle trafficking during pollen tube growth that cannot be fulfilled by the remaining AtRabD family members.</p

The Ins and Outs of Autophagic Ribosome Turnover

Ribosomes are essential for protein synthesis in all organisms and their biogenesis and number are tightly controlled to maintain homeostasis in changing environmental conditions. While ribosome assembly and quality control mechanisms have been extensively studied, our understanding of ribosome degradation is limited. In yeast or animal cells, ribosomes are degraded after transfer into the vacuole or lysosome by ribophagy or nonselective autophagy, and ribosomal RNA can also be transferred directly across the lysosomal membrane by RNautophagy. In plants, ribosomal RNA is degraded by the vacuolar T2 ribonuclease RNS2 after transport by autophagy-related mechanisms, although it is unknown if a selective ribophagy pathway exists in plants. In this review, we describe mechanisms of turnover of ribosomal components in animals and yeast, and, then, discuss potential pathways for degradation of ribosomal RNA and protein within the vacuole in plants