The molecular basis of heat stress responses in plants.

Global warming impacts crop production and threatens food security. Elevated temperatures are sensed by different cell components. Temperature increases are classified as either mild warm temperatures or excessively hot temperatures, which are perceived by distinct signaling pathways in plants. Warm temperatures induce thermomorphogenesis, while high-temperature stress triggers heat acclimation and has destructive effects on plant growth and development. In this review, we systematically summarize the heat-responsive genetic networks in Arabidopsis and crop plants based on recent studies. In addition, we highlight the strategies used to improve grain yield under heat stress from a source-sink perspective. We also discuss the remaining issues regarding the characteristics of thermosensors and the urgency required to explore the basis of acclimation under multifactorial stress combination.

VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana.

Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.

Arabidopsis ORP2A mediates ER-autophagosomal membrane contact sites and regulates PI3P in plant autophagy.

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) in vivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.

A genetic module at one locus in rice protects chloroplasts to enhance thermotolerance.

How the plasma membrane senses external heat-stress signals to communicate with chloroplasts to orchestrate thermotolerance remains elusive. We identified a quantitative trait locus, Thermo-tolerance 3 (TT3), consisting of two genes, TT3.1 and TT3.2, that interact together to enhance rice thermotolerance and reduce grain-yield losses caused by heat stress. Upon heat stress, plasma membrane-localized E3 ligase TT3.1 translocates to the endosomes, on which TT3.1 ubiquitinates chloroplast precursor protein TT3.2 for vacuolar degradation, implying that TT3.1 might serve as a potential thermosensor. Lesser accumulated, mature TT3.2 proteins in chloroplasts are essential for protecting thylakoids from heat stress. Our findings not only reveal a TT3.1-TT3.2 genetic module at one locus that transduces heat signals from plasma membrane to chloroplasts but also provide the strategy for breeding highly thermotolerant crops.

A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana.

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Plant Rho GTPase signaling promotes autophagy.

The roles of Rho family guanosine triphosphatases (GTPases) of plants (ROPs) in modulating plant growth and development have been well characterized. However, little is known about the roles of ROP signaling pathways in regulating plant autophagy and autophagosome formation. In this study, we identify a unique ROP signaling mechanism, which mediates developmental to autophagic transition under stress conditions in the model plant Arabidopsis. Loss-of-function mutants of ROP8 showed stress-induced hypersensitive phenotypes and compromised autophagic flux. Similar to other ROPs in the ROP/RAC family, ROP8 exhibits both plasma membrane and cytosolic punctate localization patterns. Upon autophagic induction, active ROP8 puncta colocalize with autophagosomal markers and are degraded inside the vacuole. In human cells, RalB, an RAS subfamily GTPase, engages its effector Exo84 for autophagosome assembly. However, a RalB counterpart is missing in the plant lineage. Intriguingly, we discovered that plant ROP8 promotes autophagy via its downstream effector Sec5. Live-cell super-resolution imaging showed that ROP8 and Sec5 reside on phagophores for autophagosome formation. Taken together, our findings highlight a previously unappreciated role of an ROP8-Sec5 signaling axis in autophagy promotion, providing new insights into how plants utilize versatile ROP signaling networks to coordinate developmental and autophagic responses depending on environmental changes.

MYB117 is a negative regulator of flowering time in Arabidopsis.

Plant flowering is crucial for the onset and progression of reproduction processes. The control of flowering time is a sophisticated system with multiple known regulatory mechanisms in plants. Here, we show that MYB117 participates in the flowering time regulation in Arabidopsis as myb117 mutants exhibited early flowering phenotypes under long-day condition. Transcriptome analysis of myb117 mutants revealed 410 differentially expressed genes between wild type and myb117-1 mutants, where selective genes including the Flowering Locus T (FT) were further confirmed by qRT-PCR analysis. Further, in vivo dual-luciferase and chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) assays showed that MYB117 directly binds to the promoter of FT to suppress its expression. Taken together, we have revealed the transcriptome profile of myb117 mutants and identified MYB117 as a negative regulator in controlling flowering time through regulating the expression of FT in Arabidopsis.

MYB106 is a negative regulator and a substrate for CRL3BPM E3 ligase in regulating flowering time in Arabidopsis thaliana.

Flowering time is crucial for successful reproduction in plants, the onset and progression of which are strictly controlled. However, flowering time is a complex and environmentally responsive history trait and the underlying mechanisms still need to be fully characterized. Post-translational regulation of the activities of transcription factors (TFs) is a dynamic and essential mechanism for plant growth and development. CRL3BPM E3 ligase is a CULLIN3-based E3 ligase involved in orchestrating protein stability via the ubiquitin proteasome pathway. Our study shows that the mutation of MYB106 induced early flowering phenotype while over-expression of MYB106 delayed Arabidopsis flowering. Transcriptome analysis of myb106 mutants reveals 257 differentially expressed genes between wild type and myb106-1 mutants, including Flowering Locus T (FT) which is related to flowering time. Moreover, in vitro electrophoretic mobility shift assays (EMSA), in vivo chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) assays and dual luciferase assays demonstrate that MYB106 directly binds to the promoter of FT to suppress its expression. Furthermore, we confirm that MYB106 interacts with BPM proteins which are further identified by CRL3BPM E3 ligases as the substrate. Taken together, we have identified MYB106 as a negative regulator in the control of flowering time and a new substrate for CRL3BPM E3 ligases in Arabidopsis.

Transient Expression of Fluorescent Fusion Proteins in Arabidopsis Protoplasts.

Transient expression using protoplasts isolated from Arabidopsis suspension culture cells is a fast and useful tool for analyzing protein subcellular localization and dynamics in plant cells. Recently, super-resolution imaging techniques such as N-SIM (Nikon, Structured Illumination Microscopy) are widely used in cell biology study, allowing cell biologists to obtain unattainable details and relationships of cell structures and functions by conventional confocal imaging. To facilitate the usage of protoplasts transient expression and super-resolution imaging for protein localization and dynamic analysis in plant cell biology research, here we describe updated protocols of protoplasts isolation from Arabidopsis suspension culture cells and transient expression assay for protein trafficking and localization study. Further, using GFP-tagged ERES (Endoplasmic Reticulum Exit Site) marker proteins and RFP-tagged Golgi marker as examples, we illustrate the major tools and methods for protein localization analysis using super-resolution imaging.

New insights into AtNBR1 as a selective autophagy cargo receptor in Arabidopsis.

Selective autophagy, mediated by cargo receptors and recruiting specific targets to autophagosomes for degradation and recycling, plays an important role in quality control and cellular homeostasis in eukaryotes. The Arabidopsis AtNBR1 shares a similar domain organization with the mammalian autophagic receptors p62 and NBR1. We recently demonstrated that AtNBR1 functions as a selective autophagy receptor for the exocyst component AtExo70E2, a marker for the Exocyst-positive organelle (EXPO), which was achieved via a specific ATG8-AtNBR1-AtExo70E2 interaction in Arabidopsis. Here we further showed that nbr1 CRISPR mutants exhibit an early senescence phenotype under short-day growth conditions, which can be restored by complementation with expression of AtNBR1pro::AtNBR1-GFP in the mutant. Interestingly, in addition to the typical cytosolic and punctate patterns, YFP-AtNBR1 also exhibited a microtubule pattern particularly in the cortical layer. Treatments with the microtubule depolymerizer oryzalin but not the microfilament depolymerizer latrunculin B abolished the microtubule pattern and affected the vacuolar delivery of YFP-AtNBR1 upon autophagy induction. These results indicated that microtubules may be required for AtNBR1 to shuttle its cargos to the vacuole during plant autophagy. The present study thus sheds new light on the recognition and movement pattern of AtNBR1 in selective autophagy in Arabidopsis.

AtNBR1 Is a Selective Autophagic Receptor for AtExo70E2 in Arabidopsis.

Selective autophagy is a subcellular process whereby cytoplasmic materials are selectively sequestered into autophagosomes for subsequent delivery to the vacuole for degradation and recycling. Arabidopsis (Arabidopsis thaliana) NBR1 (next to BRCA1 gene 1 protein; AtNBR1) has been proposed to function as a selective autophagy receptor in plants, whereby AtNBR1 anchors the ubiquitinated targets to autophagosomes for degradation. However, the specific cargos of AtNBR1 remain elusive. We previously showed that Arabidopsis exocyst subunit EXO70 family protein E2 (AtExo70E2), a marker for exocyst-positive organelle (EXPO), colocalized with the autophagosome marker Arabidopsis autophagy-related protein8 (AtATG8) and was delivered to the vacuole for degradation upon autophagic induction. Here, through multiple analyses, we demonstrate that AtNBR1 is a selective receptor for AtExo70E2 during autophagy in Arabidopsis. First, two novel loss-of-function nbr1 CRISPR mutants (nbr1-c1 and nbr1-c2) showed an early-senescence phenotype under short-day growth conditions. Second, during autophagic induction, the vacuolar delivery of AtExo70E2 or EXPO was significantly reduced in nbr1 mutants compared to wild-type plants. Third, biochemical and recruitment assays demonstrated that AtNBR1 specifically interacted and recruited AtExo70E2 or its EXPO to AtATG8-positive autophagosomes in a ubiquitin-associated (UBA)-independent manner during autophagy. Taken together, our data indicate that AtNBR1 functions as a selective receptor in mediating vacuolar delivery of AtExo70E2 or EXPO in a UBA-independent manner in plant autophagy.

The interplay between endomembranes and autophagy in plants.

Autophagosomes are unique double-membrane organelles that enclose a portion of intracellular components for lysosome/vacuole delivery to maintain cellular homeostasis in eukaryotic cells. Genetic screening has revealed the requirement of autophagy-related proteins for autophagosome formation, although the origin of the autophagosome membrane remains elusive. The endomembrane system is a series of membranous organelles maintained by dynamic membrane flow between various compartments. In plants, there is accumulating evidence pointing to a link between autophagy and the endomembrane system, in particular between the endoplasmic reticulum and autophagosome. Here, we highlight and discuss about recent findings on plant autophagosome formation. We also look into the functional roles of endomembrane machineries in regard to the autophagy pathway in plants.

Exocyst-Positive Organelles and Autophagosomes Are Distinct Organelles in Plants.

Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner.

A two-locus interaction causes interspecific hybrid weakness in rice.

Reproductive barriers perform a vital role during speciation. Hybrid weakness, the poorer development of hybrids compared with their parents, hinders gene exchange between different species at the postzygotic stage. Here we show that two incompatible dominant loci (Hwi1 and Hwi2) involving three genes are likely to determine the high temperature-dependent expression of hybrid weakness in interspecific hybrids of rice. Hwi1 comprises two leucine-rich repeat receptor-like kinase (LRR-RLK) genes, 25L1 and 25L2, which are specific to wild rice (Oryza rufipogon) and induce hybrid weakness. Hwi2, a rare allele that is predominantly distributed in indica rice (Oryza sativa), encodes a secreted putative subtilisin-like protease. Functional analysis indicated that pyramiding of Hwi1 and Hwi2 activates the autoimmune response in the basal nodes of hybrids, interrupting root formation and then impairing shoot growth. These findings bring new insights into our understanding of reproductive isolation and may benefit rice breeding.

An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells.

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the pre-vacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here we developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture medium along with their corresponding cargo proteins. Indeed, transgenic Arabidopsis culture cell lines expressing VSR N-terminal binding domains (VSRNTs) were shown to secrete truncated VSRs (BP80NT, AtVSR1NT and AtVSR4NT) with attached cargo molecules into the culture medium. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by localization studies and interaction analysis with VSRs. The screening strategy described here should be applicable to all VSRs and will help identify and study cargo proteins for individual VSR proteins. This method should be useful for both cargo identification and protein-protein interaction in vivo.

The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3.

Increased crop yields are required to support rapid population growth worldwide. Grain weight is a key component of rice yield, but the underlying molecular mechanisms that control it remain elusive. Here, we report the cloning and characterization of a new quantitative trait locus (QTL) for the control of rice grain length, weight and yield. This locus, GL3.1, encodes a protein phosphatase kelch (PPKL) family - Ser/Thr phosphatase. GL3.1 is a member of the large grain WY3 variety, which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety. GL3.1-WY3 influences protein phosphorylation in the spikelet to accelerate cell division, thereby resulting in longer grains and higher yields. Further studies have shown that GL3.1 directly dephosphorylates its substrate, Cyclin-T1;3, which has only been rarely studied in plants. The downregulation of Cyclin-T1;3 in rice resulted in a shorter grain, which indicates a novel function for Cyclin-T in cell cycle regulation. Our findings suggest a new mechanism for the regulation of grain size and yield that is driven through a novel phosphatase-mediated process that affects the phosphorylation of Cyclin-T1;3 during cell cycle progression, and thus provide new insight into the mechanisms underlying crop seed development. We bred a new variety containing the natural GL3.1 allele that demonstrated increased grain yield, which indicates that GL3.1 is a powerful tool for breeding high-yield crops.