Autophagy, a critical process for the vacuolar degradation of proteins and organelles, is governed by multiple conserved autophagy-related (ATG) proteins. The central component of the ATG machinery is the ubiquitin-like protein ATG8, which is essential for multiple steps of the autophagy process, including phagophore expansion, autophagosome closure, trafficking and fusion with the lysosome/vacuole, and selective cargo recruitment. Currently, our understanding of the roles of ATG8 in plant autophagy and the functional specialization of ATG8 family members is limited due to genetic redundancy. To assess the roles of ATG8 genes in plant autophagy, here we used CRISPR/Cas9 technology to systematically knockout the Arabidopsis ATG8 genes. By analyzing the atg8 mutants, we found that in contrast to mammalian ATG8s, in which the LC3s and GABARAP subfamilies play distinct roles in the autophagic process, Arabidopsis ATG8s perform an overlapping function in controlling autophagic flux. Combinatorial mutations of Clade I and Clade II ATG8s resulted in severely impaired autophagy under nutrient-starved conditions. Furthermore, we found that RABG3 proteins, members of the RAB7/RABG GTPase family, interact with ATG8s through AIM-LDS interfaces, and that such interaction is essential for the association of RABG3 proteins with the autophagosomal membrane and probably for the fusion of autophagosome with the vacuole, but is not required for endosomal trafficking. With the collection of multiple high-order atg8 mutants generated in this study, we now provide a venue to study the roles of ATG8 genes in canonical autophagy and non-canonical autophagy in Arabidopsis. ### Competing Interest Statement The authors have declared no competing interest.

Akin to mammalian extracellular fluids, the plant apoplastic fluid (APF) contains a unique collection of proteins, RNAs, and vesicles that drive many physiological processes ranging from cell wall assembly to defense against environmental challenges. Using an improved method to enrich for the Arabidopsis APF, we better define its composition and discover that the APF harbors active proteasomes though microscopic detection, proteasome-specific activity and immunological assays, and mass spectrometry showing selective enrichment of the core protease. Functional analysis of extracellular (ex)-proteasomes reveals that they help promote basal pathogen defense through proteolytic release of microbe-associated molecular patterns (MAMPs) such as flg22 from bacterial flagellin that induce protective reactive-oxygen-species (ROS) bursts. Flagellin-triggered ROS is also strongly suppressed by the enigmatic Pseudomonas syringae virulence effector syringolin-A that blocks ex-proteasome activity. Collectively, we provide a deep catalog of apoplast proteins and evidence that ex-proteasomes participate in the evolving arms race between pathogens and their plant hosts.

The ubiquitin-like protein ATG8 is a central component of the autophagy process and is required at multiple steps during both bulk and selective autophagy. Currently, our understanding of the roles of ATG8 in plants and the possible functional specialization of its family members is limited by genetic redundancy. Here, we employed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 targeting technology to systematically inactivate all nine Arabidopsis thaliana ATG8 loci. Subsequent analyses of the resulting mutants revealed that, unlike mammalian ATG8 family members, which have distinct roles, Arabidopsis isoforms largely overlap in their functions controlling autophagic flux. Notably, combinatorial mutations have similarly impaired autophagy and misregulated proteomes much like other autophagy mutants. We further examined the functional redundancy of Arabidopsis ATG8s in late autophagy stages by investigating their interactions with Rab GTPase (RABG)3/RAB7 proteins. We found that all ATG8 representatives could interact with RABG3 proteins via ATG8-interacting motif-LC3-interacting region-docking site interfaces. Such interactions are crucial for RABG3 binding to the autophagosome membrane and probably for the fusion of autophagosomes with the vacuole. However, they are not necessary for endosomal trafficking. With this collection of multiple high-order atg8 mutants, we provide a venue to selectively study the roles of individual ATG8 isoforms during both canonical and noncanonical autophagy in Arabidopsis.

The apoplastic space surrounding plant cells, encompassing the cell wall matrix, extracellular spaces, and xylem, is one of the least understood compartments within plant tissues due to its lack of limiting membranes and its unavoidable damage upon tissue homogenization. Using a streamlined vacuum-infiltration/centrifugation protocol to enrich for the Arabidopsis apoplastic fluid (APF) combined with in-depth tandem mass spectrometry, we provide an improved view of its proteome that includes over 1500 proteins possibly assigned to this compartment with minimized cytosolic contamination. Included are large and varied collections of polypeptides associated with cell wall metabolism, oxido-reductase reactions, cell-cell signaling, proteolysis, and pathogen protection via basal defense pathways. While numerous apoplast proteins were predicted to house N-terminal signal peptide sequences that direct extracellular secretion, many did not, suggesting widespread use of non-classical export route(s). Among APF constituents are numerous pathogenesis-related proteins, glycosidases, aspartyl and subtilisin-type serine proteases, and the complement of subunits that assemble the core particle of the 26S proteasome. When this APF proteome is compared with those based on two prior isolation methods, a consensus collection of 338 polypeptides emerges that offers a comprehensive view of the core APF proteome that manages the cell wall and interfaces with the environment.

The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.

Nonclimacteric varieties of guava (Psidium guajava L.), such as ‘Jen-Ju Bar’ (‘JJB’), have been cultivated in tropical areas due to their crispy texture and long storability. However, the molecular mechanism resulting in different ripening behaviors among guava varieties is still obscure. To elucidate the underlying mechanism, four cDNA clones of two ACC synthases (ACS) and two ACC oxidases (ACO), the key enzymes of ethylene biosynthesis, were isolated based on the conserved nucleotide sequences. Their enzymatic activities were confirmed by heterologous expression in Escherichia coli. The expression levels of PgACS1, PgACO1, and PgACO2 increased dramatically with the advance of ripening in the climacteric cultivar ‘Li-Tzy Bar’ (‘LTB’), whereas the transcripts of these genes, especially PgACS1 and PgACO2, were barely detected in ‘JJB’ throughout the phase. In addition, PgACS1 and PgACO2 transcription were induced by ethylene and repressed by 1-methylcyclopropene, an ethylene action inhibitor, which exhibited the features of system-2 ethylene production genes. These results suggested that PgACS1 and PgACO2 are two important coordinators for controlling autocatalytic ethylene biosynthesis in guava fruit. The major defect of system-2 ethylene production in ‘JJB’ is caused by the silencing of PgACS1 during ripening. Consequently, ‘JJB’ behaves nonclimacterically and has a long storability.

Increasing nitrogen use efficiency (NUE) is critical to improve crop yield, reduce N fertilizer demand and alleviate environmental pollution. N remobilization is a key component of NUE. The nitrate transporter NRT1.7 is responsible for loading excess nitrate stored in source leaves into phloem and facilitates nitrate allocation to sink leaves. Under N starvation, the nrt1.7 mutant exhibits growth retardation, indicating that NRT1.7-mediated source-to-sink remobilization of stored nitrate is important for sustaining growth in plants. To energize NRT1.7-mediated nitrate recycling, we introduced a hyperactive chimeric nitrate transporter NC4N driven by the NRT1.7 promoter into the nrt1.7 mutant. NRT1.7p::NC4N::3 ′ transgenic plants accumulated more nitrate in younger leaves, and 15 NO 3 − tracing analysis revealed that more 15 N was remobilized into sink tissues. Consistently, transgenic Arabidopsis , tobacco and rice plants showed improved growth or yield. Our study suggests that enhancing source-to-sink nitrate remobilization represents a new strategy for enhancing NUE and crop production.

Nitrogen accounts for approximately 60% of the fertilizer consumed each year; thus, it represents one of the major input costs for most nonlegume crops. Nitrate is one of the two major forms of nitrogen that plants acquire from the soil. Mechanistic insights into nitrate transport and signaling have enabled new strategies for enhancing nitrogen utilization efficiency, for lowering input costs for farming, and, more importantly, for alleviating environmental impacts (e.g., eutrophication and production of the greenhouse gas N2O). Over the past decade, significant progress has been made in understanding how nitrate is acquired from the surroundings, how it is efficiently distributed into different plant tissues in response to environmental changes, how nitrate signaling is perceived and transmitted, and how shoot and root nitrogen status is communicated. Several key components of these processes have proven to be novel tools for enhancing nitrate-and nitrogen-use efficiency. In this review, we focus on the roles of NRT1 and NRT2 in nitrate uptake and nitrate allocation among different tissues; we describe the functions of the transceptor NRT1.1, transcription factors, and small signaling peptides in nitrate signaling and tissue communication; and we compile the new strategies for improving nitrogen-use efficiency.

Plants have evolved sophisticated mechanisms to adapt to fluctuating environmental nitrogen availability. However, more underlying genes regulating the response to nitrate have yet to be characterized. We report here the identification of a nitrate regulatory mutant whose mutation mapped to the Cleavage and Polyadenylation Specificity Factor 30 gene (CPSF30-L). In the mutant, induction of nitrate-responsive genes was inhibited independent of the ammonium conditions and was restored by expression of the wild-type 65 kDa encoded by CPSF30-L. Molecular and genetic evidence suggests that CPSF30-L works upstream of NRT1.1 and independently of NLP7 in response to nitrate. Analysis of the 3'-UTR of NRT1.1 showed that the pattern of polyadenylation sites was altered in the cpsf30 mutant. Transcriptome analysis revealed that four nitrogen-related clusters were enriched in the differentially expressed genes of the cpsf30 mutant. Nitrate uptake was decreased in the mutant along with reduced expression of the nitrate transporter/sensor gene NRT1.1, while nitrate reduction and amino acid content were enhanced in roots along with increased expression of several nitrate assimilatory genes. These findings indicate that the 65 kDa protein encoded by CPSF30-L mediates nitrate signaling in part by regulating NRT1.1 expression, thus adding an important component to the nitrate signaling network.

The non-climacteric ripening characteristics, including delayed peel coloration and pulp softening, of 'Jen-Ju Bar' ('JJB') guava (Psidium guajava L.) is a result of a reduction in the usually massive amount of ethylene produced during the ripening stage. To elucidate the underlying physiological mechanism, four cDNA clones of ACC synthase (PgACS1 and PgACS2) and ACC oxidase (PyACO1 and PgACO2), the key enzymes in the ethylene biosynthetic pathway, from guava pericarp were isolated and were analyzed for their expression patterns in the fruit. The expressions of PgACS1, PgACO1, and PgACO2 increased with the advance of fruit ripening in 'Li-Tzy Bar' ('LTB'), a climacteric cultivar of guava, at 20 degrees C. On the other hand, all three genes, especially PgACS1, displayed a low and steady pattern of gene expression in 'JJB' fruit over 12-day storage. The fact that exogenous ethylene treatment up-regulated the gene expression as well as 1-methylcyclopropene, an ethylene action inhibitor, suppressed the transcript accumulation in mature-green 'LTB' fruit revealed an autocatalysis or System-2 property of PgACS1 expression. Because not enough ACC is produced by PgACS1 to support massive ethylene synthesis at ripening, 'JJB' fruit behaved in a non-climacteric manner.

The ripening characteristics and physiology of two guava cultivars, 'Jen-Ju Bar' ('JJB') and 'Li-Tzy Bar' ('LTB') were investigated and compared because of their divergent storability. During storage at 20 degrees C, mature-green 'LTB' fruit exhibited ripening behavior typical for a climacteric fruit, including increases in respiration and ethylene production (3.75 mmol CO2 kg(-1) h(-1) and 1.37 x 10(3) nmol C2H4 kg(-1) h(-1) at peak levels, respectively), yellow color development, flesh softening, and volatile production. By contrast, 'JJB' is considered a nonclimacteric fruit because it features low and stable rates of respiration (0.61-0.73 mmol CO2 kg(-1) h(-1)) and ethylene production (less than 4 nmol C2H4 kg(-1) h(-1)) while retaining its pale-green color and firmness throughout a 20d observation period. Furthermore, the nonclimacteric nature of 'JJB' fruit was revealed through its responses to various levels of applied ethylene, as its respiration rate increased in a concentration-dependent manner. This respiration enhancement occurred only when exogenous ethylene was present and could be evoked repeatedly in the same 'JJB' fruit. The ripening characteristics of 'JJB' do not result from a defect in ethylene signaling, but instead result from a lack of autocatalytic ethylene synthesis, as propylene ventilation stimulated normal fruit ripening and an upsurge of respiration without a massive endogenous ethylene release. However, a cutting treatment of 'JJB' was able to induce stress-responsive ethylene production expected from system 1 ethylene biosynthesis. The finding that 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC synthase (ACS) activity were significantly lower in 'JJB' than in 'LTB' fruit after harvest, together with the finding that treatment with ACC (rather than S-adenosyl-t-methionine) led to both fully ripened 'JJB' fruit and endogenous ethylene production, strongly suggest that insufficient ACC synthesis, i.e., the reaction catalyzed by ACS, prevents system 2 ethylene biosynthesis in 'JJB' guava fruit. (C) 2012 Elsevier B.V. All rights reserved.