Autophagy and multivesicular body pathways cooperate to protect sulfur assimilation and chloroplast functions.

Autophagy and multivesicular bodies (MVBs) represent 2 closely related lysosomal/vacuolar degradation pathways. In Arabidopsis (Arabidopsis thaliana), autophagy is stress-induced, with deficiency in autophagy causing strong defects in stress responses but limited effects on growth. LYST-INTERACTING PROTEIN 5 (LIP5) is a key regulator of stress-induced MVB biogenesis, and mutation of LIP5 also strongly compromises stress responses with little effect on growth in Arabidopsis. To determine the functional interactions of these 2 pathways in Arabidopsis, we generated mutations in both the LIP5 and AUTOPHAGY-RELATED PROTEIN (ATG) genes. atg5/lip5 and atg7/lip5 double mutants displayed strong synergistic phenotypes in fitness characterized by stunted growth, early senescence, reduced survival, and greatly diminished seed production under normal growth conditions. Transcriptome and metabolite analysis revealed that chloroplast sulfate assimilation was specifically downregulated at early seedling stages in the atg7/lip5 double mutant prior to the onset of visible phenotypes. Overexpression of adenosine 5'-phosphosulfate reductase 1, a key enzyme in sulfate assimilation, substantially improved the growth and fitness of the atg7/lip5 double mutant. Comparative multi-omic analysis further revealed that the atg7/lip5 double mutant was strongly compromised in other chloroplast functions including photosynthesis and primary carbon metabolism. Premature senescence and reduced survival of atg/lip5 double mutants were associated with increased accumulation of reactive oxygen species and overactivation of stress-associated programs. Blocking PHYTOALEXIN DEFICIENT 4 and salicylic acid signaling prevented early senescence and death of the atg7/lip5 double mutant. Thus, stress-responsive autophagy and MVB pathways play an important cooperative role in protecting essential chloroplast functions including sulfur assimilation under normal growth conditions to suppress salicylic-acid-dependent premature cell-death and promote plant growth and fitness.

LIP5, a MVB biogenesis regulator, is required for rice growth.

LYST-INTERACTING PROTEIN5 (LIP5) is a conserved regulator of multivesicular body (MVB) biogenesis in eukaryotes. In Arabidopsis, AtLIP5 is a target of stress-responsive MITOGEN-ACTIVATED PROTEIN KINASE3 and 6 and mediates stress-induced MVB biogenesis to promote stress responses. However, Arabidopsis atlip5 knockout mutants are normal in growth and development. Here we report that rice OsLIP5 gene could fully restore both the disease resistance and salt tolerance of the Arabidopsis oslip5 mutant plants to the wild-type levels. Unlike Arabidopsis atlip5 mutants, rice oslip5 mutants were severely stunted, developed necrotic lesions and all died before flowering. Unlike in Arabidopsis, LIP5 regulated endocytosis under both stress and normal conditions in rice. These findings indicate that there is strong evolutionary divergence among different plants in the role of the conserved LIP5-regulated MVB pathway in normal plant growth.

Shared and Related Molecular Targets and Actions of Salicylic Acid in Plants and Humans.

Salicylic acid (SA) is a phenolic compound produced by all plants that has an important role in diverse processes of plant growth and stress responses. SA is also the principal metabolite of aspirin and is responsible for many of the anti-inflammatory, cardioprotective and antitumor activities of aspirin. As a result, the number of identified SA targets in both plants and humans is large and continues to increase. These SA targets include catalases/peroxidases, metabolic enzymes, protein kinases and phosphatases, nucleosomal and ribosomal proteins and regulatory and signaling proteins, which mediate the diverse actions of SA in plants and humans. While some of these SA targets and actions are unique to plants or humans, many others are conserved or share striking similarities in the two types of organisms, which underlie a host of common biological processes that are regulated or impacted by SA. In this review, we compare shared and related SA targets and activities to highlight the common nature of actions by SA as a hormone in plants versus a therapeutic agent in humans. The cross examination of SA targets and activities can help identify new actions of SA and better explain their underlying mechanisms in plants and humans.

Maximizing the Production of Recombinant Proteins in Plants: From Transcription to Protein Stability.

The production of therapeutic and industrial recombinant proteins in plants has advantages over established bacterial and mammalian systems in terms of cost, scalability, growth conditions, and product safety. In order to compete with these conventional expression systems, however, plant expression platforms must have additional economic advantages by demonstrating a high protein production yield with consistent quality. Over the past decades, important progress has been made in developing strategies to increase the yield of recombinant proteins in plants by enhancing their expression and reducing their degradation. Unlike bacterial and animal systems, plant expression systems can utilize not only cell cultures but also whole plants for the production of recombinant proteins. The development of viral vectors and chloroplast transformation has opened new strategies to drastically increase the yield of recombinant proteins from plants. The identification of promoters for strong, constitutive, and inducible promoters or the tissue-specific expression of transgenes allows for the production of recombinant proteins at high levels and for special purposes. Advances in the understanding of RNAi have led to effective strategies for reducing gene silencing and increasing recombinant protein production. An increased understanding of protein translation, quality control, trafficking, and degradation has also helped with the development of approaches to enhance the synthesis and stability of recombinant proteins in plants. In this review, we discuss the progress in understanding the processes that control the synthesis and degradation of gene transcripts and proteins, which underlie a variety of developed strategies aimed at maximizing recombinant protein production in plants.

Chloroplasts Protein Quality Control and Turnover: A Multitude of Mechanisms.

As the organelle of photosynthesis and other important metabolic pathways, chloroplasts contain up to 70% of leaf proteins with uniquely complex processes in synthesis, import, assembly, and turnover. Maintaining functional protein homeostasis in chloroplasts is vitally important for the fitness and survival of plants. Research over the past several decades has revealed a multitude of mechanisms that play important roles in chloroplast protein quality control and turnover under normal and stress conditions. These mechanisms include: (i) endosymbiotically-derived proteases and associated proteins that play a vital role in maintaining protein homeostasis inside the chloroplasts, (ii) the ubiquitin-dependent turnover of unimported chloroplast precursor proteins to prevent their accumulation in the cytosol, (iii) chloroplast-associated degradation of the chloroplast outer-membrane translocon proteins for the regulation of chloroplast protein import, (iv) chloroplast unfolded protein response triggered by accumulated unfolded and misfolded proteins inside the chloroplasts, and (v) vesicle-mediated degradation of chloroplast components in the vacuole. Here, we provide a comprehensive review of these diverse mechanisms of chloroplast protein quality control and turnover and discuss important questions that remain to be addressed in order to better understand and improve important chloroplast functions.

The Mediator Complex: A Central Coordinator of Plant Adaptive Responses to Environmental Stresses.

As sessile organisms, plants are constantly exposed to a variety of environmental stresses and have evolved adaptive mechanisms, including transcriptional reprogramming, in order to survive or acclimate under adverse conditions. Over the past several decades, a large number of gene-specific transcription factors have been identified in the transcriptional regulation of plant adaptive responses. The Mediator complex plays a key role in transducing signals from gene-specific transcription factors to the transcription machinery to activate or repress target gene expression. Since its first purification about 15 years ago, plant Mediator complex has been extensively analyzed for its composition and biological functions. Mutants of many plant Mediator subunits are not lethal but are compromised in growth, development and response to biotic and abiotic stress, underscoring a particularly important role in plant adaptive responses. Plant Mediator subunits also interact with partners other than transcription factors and components of the transcription machinery, indicating the complexity of the regulation of gene expression by plant Mediator complex. Here, we present a comprehensive discussion of recent analyses of the structure and function of plant Mediator complex, with a particular focus on its roles in plant adaptive responses to a wide spectrum of environmental stresses and associated biological processes.

Specialized endoplasmic reticulum-derived vesicles in plants: Functional diversity, evolution, and biotechnological exploitation.

A central role of the endoplasmic reticulum (ER) is the synthesis, folding and quality control of secretory proteins. Secretory proteins usually exit the ER to enter the Golgi apparatus in coat protein complex II (COPII)-coated vesicles before transport to different subcellular destinations. However, in plants there are specialized ER-derived vesicles (ERDVs) that carry specific proteins but, unlike COPII vesicles, can exist as independent organelles or travel to the vacuole in a Golgi-independent manner. These specialized ERDVs include protein bodies and precursor-accumulating vesicles that accumulate storage proteins in the endosperm during seed development. Specialized ERDVs also include precursor protease vesicles that accumulate amino acid sequence KDEL-tailed cysteine proteases and ER bodies in Brassicales plants that accumulate myrosinases that hydrolyzes glucosinolates. These functionally specialized ERDVs act not only as storage organelles but also as platforms for signal-triggered processing, activation and deployment of specific proteins with important roles in plant growth, development and adaptive responses. Some specialized ERDVs have also been exploited to increase production of recombinant proteins and metabolites. Here we discuss our current understanding of the functional diversity, evolutionary mechanisms and biotechnological application of specialized ERDVs, which are associated with some of the highly remarkable characteristics important to plants.

The Cellular and Subcellular Organization of the Glucosinolate-Myrosinase System against Herbivores and Pathogens.

Glucosinolates are an important class of secondary metabolites in Brassicales plants with a critical role in chemical defense. Glucosinolates are chemically inactive but can be hydrolyzed by myrosinases to produce a range of chemically active compounds toxic to herbivores and pathogens, thereby constituting the glucosinolate-myrosinase defense system or the mustard oil bomb. During the evolution, Brassicales plants have developed not only complex biosynthetic pathways for production of a large number of glucosinolate structures but also different classes of myrosinases that differ in catalytic mechanisms and substrate specificity. Studies over the past several decades have made important progress in the understanding of the cellular and subcellular organization of the glucosinolate-myrosinase system for rapid and timely detonation of the mustard oil bomb upon tissue damage after herbivore feeding and pathogen infection. Progress has also been made in understanding the mechanisms that herbivores and pathogens have evolved to counter the mustard oil bomb. In this review, we summarize our current understanding of the function and organization of the glucosinolate-myrosinase system in Brassicales plants and discuss both the progresses and future challenges in addressing this complex defense system as an excellent model for analyzing plant chemical defense.

Biosynthesis and Roles of Salicylic Acid in Balancing Stress Response and Growth in Plants.

Salicylic acid (SA) is an important plant hormone with a critical role in plant defense against pathogen infection. Despite extensive research over the past 30 year or so, SA biosynthesis and its complex roles in plant defense are still not fully understood. Even though earlier biochemical studies suggested that plants synthesize SA from cinnamate produced by phenylalanine ammonia lyase (PAL), genetic analysis has indicated that in Arabidopsis, the bulk of SA is synthesized from isochorismate (IC) produced by IC synthase (ICS). Recent studies have further established the enzymes responsible for the conversion of IC to SA in Arabidopsis. However, it remains unclear whether other plants also rely on the ICS pathway for SA biosynthesis. SA induces defense genes against biotrophic pathogens, but represses genes involved in growth for balancing defense and growth to a great extent through crosstalk with the growth-promoting plant hormone auxin. Important progress has been made recently in understanding how SA attenuates plant growth by regulating the biosynthesis, transport, and signaling of auxin. In this review, we summarize recent progress in the biosynthesis and the broad roles of SA in regulating plant growth during defense responses. Further understanding of SA production and its regulation of both defense and growth will be critical for developing better knowledge to improve the disease resistance and fitness of crops.

Two ubiquitin-associated ER proteins interact with COPT copper transporters and modulate their accumulation.

The endoplasmic reticulum (ER) contains an elaborate protein quality control network that promotes protein folding and prevents accumulation of misfolded proteins. Evolutionarily conserved UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (UBAC2) is involved in ER-associated protein degradation in metazoans. We have previously reported that two close UBAC2 homologs from Arabidopsis (Arabidopsis thaliana) not only participate in selective autophagy of ER components but also interact with plant-specific PATHOGEN-ASSOCIATED MOLECULAR PATTERN (PAMP)-INDUCED COILED COIL (PICC) protein to increase the accumulation of POWDERY MILDEW-RESISTANT 4 callose synthase. Here, we report that UBAC2s also interacted with COPPER (Cu) TRANSPORTER 1 (COPT1) and plasma membrane-targeted members of the Cu transporter family. The ubac2 mutants were significantly reduced in both the accumulation of COPT proteins and Cu content, and also displayed increased sensitivity to a Cu chelator. Therefore, UBAC2s positively regulate the accumulation of COPT transporters, thereby increasing Cu uptake by plant cells. Unlike with POWDERY MILDEW RESISTANCE 4, however, the positive role of UBAC2s in the accumulation of COPT1 is not dependent on PICC or the UBA domain of UBAC2s. When COPT1 was overexpressed under the CaMV 35S promoter, the increased accumulation of COPT1 was strongly UBAC2-dependent, particularly when a signal peptide was added to the N-terminus of COPT1. Further analysis using inhibitors of protein synthesis and degradation strongly suggested that UBAC2s stabilize newly synthesized COPT proteins against degradation by the proteasome system. These results indicate that plant UBAC2s are multifunctional proteins that regulate the degradation and accumulation of specific ER-synthesized proteins.

Regulation and Function of Defense-Related Callose Deposition in Plants.

Plants are constantly exposed to a wide range of potential pathogens and to protect themselves, have developed a variety of chemical and physical defense mechanisms. Callose is a ß-(1,3)-D-glucan that is widely distributed in higher plants. In addition to its role in normal growth and development, callose plays an important role in plant defense. Callose is deposited between the plasma membrane and the cell wall at the site of pathogen attack, at the plasmodesmata, and on other plant tissues to slow pathogen invasion and spread. Since it was first reported more than a century ago, defense-related callose deposition has been extensively studied in a wide-spectrum of plant-pathogen systems. Over the past 20 years or so, a large number of studies have been published that address the dynamic nature of pathogen-induced callose deposition, the complex regulation of synthesis and transport of defense-related callose and associated callose synthases, and its important roles in plant defense responses. In this review, we summarize our current understanding of the regulation and function of defense-related callose deposition in plants and discuss both the progresses and future challenges in addressing this complex defense mechanism as a critical component of a plant immune system.

Cargo Recognition and Function of Selective Autophagy Receptors in Plants.

Autophagy is a major quality control system for degradation of unwanted or damaged cytoplasmic components to promote cellular homeostasis. Although non-selective bulk degradation of cytoplasm by autophagy plays a role during cellular response to nutrient deprivation, the broad roles of autophagy are primarily mediated by selective clearance of specifically targeted components. Selective autophagy relies on cargo receptors that recognize targeted components and recruit them to autophagosomes through interaction with lapidated autophagy-related protein 8 (ATG8) family proteins anchored in the membrane of the forming autophagosomes. In mammals and yeast, a large collection of selective autophagy receptors have been identified that mediate the selective autophagic degradation of organelles, aggregation-prone misfolded proteins and other unwanted or nonnative proteins. A substantial number of selective autophagy receptors have also been identified and functionally characterized in plants. Some of the autophagy receptors in plants are evolutionarily conserved with homologs in other types of organisms, while a majority of them are plant-specific or plant species-specific. Plant selective autophagy receptors mediate autophagic degradation of not only misfolded, nonactive and otherwise unwanted cellular components but also regulatory and signaling factors and play critical roles in plant responses to a broad spectrum of biotic and abiotic stresses. In this review, we summarize the research on selective autophagy in plants, with an emphasis on the cargo recognition and the biological functions of plant selective autophagy receptors.

Expansion and Functional Diversification of TFIIB-Like Factors in Plants.

As sessile organisms, plants have evolved unique patterns of growth and development, elaborate metabolism and special perception and signaling mechanisms to environmental cues. Likewise, plants have complex and highly special programs for transcriptional control of gene expression. A case study for the special transcription control in plants is the expansion of general transcription factors, particularly the family of Transcription Factor IIB (TFIIB)-like factors with 15 members in Arabidopsis. For more than a decade, molecular and genetic analysis has revealed important functions of these TFIIB-like factors in specific biological processes including gametogenesis, pollen tube growth guidance, embryogenesis, endosperm development, and plant-microbe interactions. The redundant, specialized, and diversified roles of these TFIIB-like factors challenge the traditional definition of general transcription factors established in other eukaryotes. In this review, we discuss general transcription factors in plants with a focus on the expansion and functional analysis of plant TFIIB-like proteins to highlight unique aspects of plant transcription programs that can be highly valuable for understanding the molecular basis of plant growth, development and responses to stress conditions.

Coordination and Crosstalk between Autophagosome and Multivesicular Body Pathways in Plant Stress Responses.

In eukaryotic cells, autophagosomes and multivesicular bodies (MVBs) are two closely related partners in the lysosomal/vacuolar protein degradation system. Autophagosomes are double membrane-bound organelles that transport cytoplasmic components, including proteins and organelles for autophagic degradation in the lysosomes/vacuoles. MVBs are single-membrane organelles in the endocytic pathway that contain intraluminal vesicles whose content is either degraded in the lysosomes/vacuoles or recycled to the cell surface. In plants, both autophagosome and MVB pathways play important roles in plant responses to biotic and abiotic stresses. More recent studies have revealed that autophagosomes and MVBs also act together in plant stress responses in a variety of processes, including deployment of defense-related molecules, regulation of cell death, trafficking and degradation of membrane and soluble constituents, and modulation of plant hormone metabolism and signaling. In this review, we discuss these recent findings on the coordination and crosstalk between autophagosome and MVB pathways that contribute to the complex network of plant stress responses.

A Family of NAI2-Interacting Proteins in the Biogenesis of the ER Body and Related Structures.

Plants produce different types of endoplasmic reticulum (ER)-derived vesicles that accumulate and transport proteins, lipids, and metabolites. In the Brassicales, a distinct ER-derived structure called the ER body is found throughout the epidermis of cotyledons, hypocotyls, and roots. NAI2 is a key factor for ER body formation in Arabidopsis (Arabidopsis thaliana). Homologs of NAI2 are found only in the Brassicales and therefore may have evolved specifically to enable ER body formation. Here, we report that three related Arabidopsis NAI2-interacting proteins (NAIP1, NAIP2, and NAIP3) play a critical role in the biogenesis of ER bodies and related structures. Analysis using GFP fusions revealed that all three NAIPs are components of the ER bodies found in the cotyledons, hypocotyls, and roots. Genetic analysis with naip mutants indicates that they have a critical and redundant role in ER body formation. NAIP2 and NAIP3 are also components of other vesicular structures likely derived from the ER that are formed independent of NAI2 and are present not only in the cotyledons, hypocotyls, and roots, but also in the rosettes. Thus, while NAIP1 is a specialized ER body component, NAIP2 and NAIP3 are components of different types of ER-derived structures. Analysis of chimeric NAIP proteins revealed that their N-terminal domains play a major role in the functional specialization between NAIP1 and NAIP3. Unlike NAI2, NAIPs have homologs in all plants; therefore, NAIP-containing ER structures, from which the ER bodies in the Brassicales may have evolved, are likely to be present widely in plants.

Arabidopsis Endoplasmic Reticulum-Localized UBAC2 Proteins Interact with PAMP-INDUCED COILED-COIL to Regulate Pathogen-Induced Callose Deposition and Plant Immunity.

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is initiated upon PAMP recognition by pattern recognition receptors (PRR). PTI signals are transmitted through activation of mitogen-activated protein kinases (MAPKs), inducing signaling and defense processes such as reactive oxygen species (ROS) production and callose deposition. Here, we examine mutants for two Arabidopsis thaliana genes encoding homologs of UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (UBAC2), a conserved endoplasmic reticulum (ER) protein implicated in ER protein quality control. The ubac2 mutants were hypersusceptible to a type III secretion-deficient strain of the bacterial pathogen Pseudomonas syringae, indicating a PTI defect. The ubac2 mutants showed normal PRR biogenesis, MAPK activation, ROS burst, and PTI-associated gene expression. Pathogen- and PAMP-induced callose deposition, however, was compromised in ubac2 mutants. UBAC2 proteins interact with the plant-specific long coiled-coil protein PAMP-INDUCED COILED COIL (PICC), and picc mutants were compromised in callose deposition and PTI. Compromised callose deposition in the ubac2 and picc mutants was associated with reduced accumulation of the POWDERY MILDEW RESISTANT 4 (PMR4) callose synthase, which is responsible for pathogen-induced callose synthesis. Constitutive overexpression of PMR4 restored pathogen-induced callose synthesis and PTI in the ubac2 and picc mutants. These results uncover an ER pathway involving the conserved UBAC2 and plant-specific PICC proteins that specifically regulate pathogen-induced callose deposition in plant innate immunity.

Biogenesis and Function of Multivesicular Bodies in Plant Immunity.

Multivesicular bodies (MVBs) are specialized endosomes that contain intraluminal vesicles generated from invagination and budding of the limiting membrane. In the endocytic pathway, MVBs are late endosomes whose content can be degraded through fusion with lysosomes/vacuoles or released into the extracellular space after fusion with the plasma membrane (PM). The proteins retained on the limiting membrane of MVBs are translocated to the membrane of lysosomes/vacuoles or delivered back to the PM. It has been long suspected that MVBs might fuse with the PM to form paramural bodies in plant cells, possibly leading to release of building blocks for deposition of papillae and antimicrobial molecules against invading pathogens. Over the past decade or so, major progress has been made in establishing the critical roles of MVBs and associated membrane trafficking in pathogen recognition, defense signaling, and deployment of defense-related molecules during plant immune responses. Regulatory proteins and signaling pathways associated with induced biogenesis and trafficking of MVBs during plant immune responses have also been identified and characterized. Recent successful isolation of plant extracellular vesicles and proteomic profiling of their content have provided additional support for the roles of MVBs in plant-pathogen interactions. In this review, we summarize the important progress and discuss how MVBs, particularly through routing of cellular components to different destinations, contribute to the complex network of plant immune system.

Plastid Translation Elongation Factor Tu Is Prone to Heat-Induced Aggregation Despite Its Critical Role in Plant Heat Tolerance.

Translation elongation factor Tu (EF-Tu) is a conserved GTP-binding protein essential for protein translation in prokaryotes and in eukaryotic mitochondria and plastids. EF-Tu also has a GTP/GDP-independent chaperone activity that may function in acclimation to heat. Here, we report that the Arabidopsis (Arabidopsis thaliana) plastid EF-Tu, Rabe1b, rapidly becomes insoluble at temperatures as low as 35°C in vitro and 41°C in vivo, with more than 90% aggregation after 9 h at 45°C in vivo. Based on its established function in protein translation, heat-induced aggregation likely inactivates Rabe1b. To determine the effect of heat-induced aggregation, we isolated an Arabidopsis rabe1b knockdown mutant and discovered it to be highly compromised in heat tolerance. Overexpression of constitutive GTP- or GDP-bound mutant forms of Rabe1b in Arabidopsis and virus-induced silencing of Rabe1b in tomato (Solanum lycopersicum) also reduced heat tolerance. Compromised heat tolerance in the Arabidopsis rabe1b mutant and in the lines overexpressing constitutive GTP- or GDP-bound mutant Rabe1b proteins was associated with reduced plastid translation under heat stress. The Arabidopsis rabe1b mutant also showed compromised heat-induced expression of HsfA2 and its target genes. Constitutive overexpression of HsfA2 activated its target genes but only partially restored the heat tolerance of the rabe1b mutant. These results strongly suggest that the plastid protein EF-Tu is heat sensitive and acts as a critical limiting factor in plant heat stress responses, primarily functioning in plastid protein translation but also in protein folding and retrograde signaling of nuclear heat-responsive gene expression.

Dicot-specific ATG8-interacting ATI3 proteins interact with conserved UBAC2 proteins and play critical roles in plant stress responses.

Selective macroautophagy/autophagy targets specific cargo by autophagy receptors through interaction with ATG8 (autophagy-related protein 8)/MAP1LC3 (microtubule associated protein 1 light chain 3) for degradation in the vacuole. Here, we report the identification and characterization of 3 related ATG8-interacting proteins (AT1G17780/ATI3A, AT2G16575/ATI3B and AT1G73130/ATI3C) from Arabidopsis. ATI3 proteins contain a WxxL LC3-interacting region (LIR) motif at the C terminus required for interaction with ATG8. ATI3 homologs are found only in dicots but not in other organisms including monocots. Disruption of ATI3A does not alter plant growth or development but compromises both plant heat tolerance and resistance to the necrotrophic fungal pathogen Botrytis cinerea. The critical role of ATI3A in plant stress tolerance and disease resistance is dependent on its interaction with ATG8. Disruption of ATI3B and ATI3C also significantly compromises plant heat tolerance. ATI3A interacts with AT3G56740/UBAC2A and AT2G41160/UBAC2B (Ubiquitin-associated [UBA] protein 2a/b), 2 conserved proteins implicated in endoplasmic reticulum (ER)-associated degradation. Disruption of UBAC2A and UBAC2B also compromised heat tolerance and resistance to B. cinerea. Overexpression of UBAC2 induces formation of ATG8- and ATI3-labeled punctate structures under normal conditions, likely reflecting increased formation of phagophores or autophagosomes. The ati3 and ubac2 mutants are significantly compromised in sensitivity to tunicamycin, an ER stress-inducing agent, but are fully competent in autophagy-dependent ER degradation under conditions of ER stress when using an ER lumenal marker for detection. We propose that ATI3 and UBAC2 play an important role in plant stress responses by mediating selective autophagy of specific unknown ER components.