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.

AbstractPhytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8–3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases.

Phytochromes (Phys) are a diverse collection of photoreceptors that regulate numerous physiological and developmental processes in microorganisms and plants through photointerconversion between red-light-absorbing Pr and far-red light-absorbing Pfr states. Light is detected by an N-terminal photo-sensing module (PSM) sequentially comprised of Period/ARNT/Sim (PAS), cGMP-phosphodiesterase/adenylyl cyclase/FhlA (GAF), and Phy-specific (PHY) domains, with the bilin chromophore covalently-bound within the GAF domain. Phys sense light via the Pr/Pfr ratio measured by the light-induced rotation of the bilin D-pyrrole ring that triggers conformational changes within the PSM, which for microbial Phys reaches into an output region. A key step is a β-stranded to α-helical reconfiguration of a hairpin loop extending from the PHY domain to contact the GAF domain. Besides canonical Phys, cyanobacteria express several variants, including a PAS-less subfamily that harbors just the GAF and PHY domains for light detection. Prior 2D-NMR studies of a model PAS-less Phy from Synechococcus_sp._JA-2-3B'a(2-13) (SyB-Cph1) proposed a unique photoconversion mechanism involving an A-pyrrole ring rotation while magic-angle-spinning NMR probing the chromophore proposed the prototypic D-ring flip. To help solve this conundrum, we determined the crystallographic structure of the GAF-PHY region from SyB-Cph1 as Pr. Surprisingly, this structure differs from canonical Phys by having a Pr ZZZsyn,syn,anti bilin configuration but shifted to the activated position in the binding pocket with consequent folding of the hairpin loop to α-helical, an architecture common for Pfr. Collectively, the PSM of SyB-Cph1 as Pr displayed a mix of dark-adapted and photoactivated features whose co-planar A-C pyrrole rings support a D-ring flip mechanism.

Peroxisomes house diverse metabolic pathways that are essential for plant and animal survival, including enzymes that produce or inactivate toxic byproducts. Despite the importance of peroxisomes and their collaborations with other organelles, the mechanisms that trigger or prevent peroxisome turnover and the cellular impacts of impaired peroxisomes are incompletely understood. When Arabidopsis thaliana LON2, a peroxisomal protein with chaperone and protease capacity, is disrupted, metabolic dysfunction and protein instability in peroxisomes ensue. Paradoxically, preventing autophagy in lon2 mutants appears to normalize peroxisomal metabolism and stabilize peroxisomal proteins-hinting at a role for autophagy in causing the peroxisomal defects observed in lon2 seedlings. Using a combination of transcriptomics, proteomics, and in silico investigations, we compared wild type to lon2 and autophagy null mutants and double mutants. Through this analysis, we found that impeding autophagy via an atg2 null mutation alleviated several of the global defects observed when LON2 is absent. Moreover, we revealed processes influenced by LON2 that are independent of autophagy, including impacts on lipid droplet and chloroplast protein levels. Finally, we identified and classified potential LON2 substrates, which include proteins that might provide signal(s) for pexophagy.

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.

The selective turnover of macromolecules by autophagy provides a critical homeostatic mechanism for recycling cellular constituents and for removing superfluous and damaged organelles, membranes, and proteins. To better understand how autophagy impacts seed maturation and nutrient storage, we studied maize (Zea mays) endosperm in its early and middle developmental stages via an integrated multiomic approach using mutants impacting the core macroautophagy factor AUTOPHAGY (ATG)-12 required for autophagosome assembly. Surprisingly, the mutant endosperm in these developmental windows accumulated normal amounts of starch and Zein storage proteins. However, the tissue acquired a substantially altered metabolome, especially for compounds related to oxidative stress and sulfur metabolism, including increases in cystine, dehydroascorbate, cys-glutathione disulfide, glucarate, and galactarate, and decreases in peroxide and the antioxidant glutathione. While changes in the associated transcriptome were mild, the proteome was strongly altered in the atg12 endosperm, especially for increased levels of mitochondrial proteins without a concomitant increase in mRNA abundances. Although fewer mitochondria were seen cytologically, a heightened number appeared dysfunctional based on the accumulation of dilated cristae, consistent with attenuated mitophagy. Collectively, our results confirm that macroautophagy plays a minor role in the accumulation of starch and storage proteins during maize endosperm development but likely helps protect against oxidative stress and clears unneeded/dysfunctional mitochondria during tissue maturation.

The reversible conjugation of small ubiquitin-like modifier (SUMO) to other proteins has pervasive roles in various aspects of plant development and stress defense through its selective attachment to numerous intracellular substrates. An intriguing aspect of SUMO is that it can be further modified by SUMOylation and ubiquitylation, which isopeptide-link either or both polypeptides to internal lysines within previously bound SUMOs. Although detectable by mass spectrometry, the functions of these secondary modifications remain obscure. Here, we generated transgenic Arabidopsis that replaced the two related and essential SUMO isoforms (SUMO1 and SUMO2) with a lysine-null SUMO1 variant (K0) immune to further SUMOylation/ubiquitylation at these residues. Remarkably, homozygous SUMO1(K0) sumo1 sumo2 plants developed normally, were not hypersensitive to heat stress, and have nearly unaltered SUMOylation profiles during heat shock. However, subtle changes in tolerance to salt, paraquat, and the DNA-damaging agents bleomycin and methane methylsulfonate were evident, as were increased sensitivities to ABA and the gibberellic acid biosynthesis inhibitor paclobutrazol, suggesting roles for these secondary modifications in stress defense, DNA repair, and hormone signaling. We also generated viable sumo1 sumo2 lines expressing a SUMO1(K0) variant specifically designed to help isolate SUMO conjugates and map SUMOylation sites, thus offering a new tool for investigating SUMO in planta.

Plants employ a divergent cohort of phytochrome (Phy) photoreceptors to govern many aspects of morphogenesis through reversible photointerconversion between inactive Pr and active Pfr conformers. The two most influential are PhyA whose retention of Pfr enables sensation of dim light, while the relative instability of Pfr for PhyB makes it better suited for detecting full sun and temperature. To better understand these contrasts, we solved, by cryo-electron microscopy, the three-dimensional structure of full-length PhyA as Pr. Like PhyB, PhyA dimerizes through head-to-head assembly of its C-terminal histidine kinase-related domains (HKRDs), while the remainder assembles as a head-to-tail light-responsive platform. Whereas the platform and HKRDs associate asymmetrically in PhyB dimers, these lopsided connections are absent in PhyA. Analysis of truncation and site-directed mutants revealed that this decoupling and altered platform assembly have functional consequences for Pfr stability of PhyA and highlights how plant Phy structural diversification has extended light and temperature perception.

The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.

The 5’ untranslated region (UTR) sequence of eukaryotic mRNAs may contain upstream open reading frames (uORFs), which can regulate translation of the main open reading frame (mORF). The current model of translational regulation by uORFs posits that when a ribosome scans an mRNA and encounters a uORF, translation of that uORF can prevent ribosomes from reaching the mORF and cause decreased mORF translation. In this study, we first observed that rare variants in the 5’ UTR dysregulate protein abundance. Upon further investigation, we found that rare variants near the start codon of uORFs can repress or derepress mORF translation, causing allelic changes in protein abundance. This finding holds for common variants as well, and common variants that modify uORF start codons also contribute disproportionately to metabolic and whole-plant phenotypes, suggesting that translational regulation by uORFs serves an adaptive function. These results provide evidence for the mechanisms by which natural sequence variation modulates gene expression, and ultimately, phenotype.

Synthetic mRNA technology is a promising avenue for treating and preventing disease. Key to the technology is the incorporation of modified nucleotides such as N1-methylpseudouridine (m1Ψ) to decrease immunogenicity of the RNA. However, relatively few studies have addressed the effects of modified nucleotides on the decoding process. Here, we investigate the effect of m1Ψ and the related modification pseudouridine (Ψ) on translation. In a reconstituted system, we find that m1Ψ does not significantly alter decoding accuracy. More importantly, we do not detect an increase in miscoded peptides when mRNA containing m1Ψ is translated in cell culture, compared with unmodified mRNA. We also find that m1Ψ does not stabilize mismatched RNA-duplex formation and only marginally promotes errors during reverse transcription. Overall, our results suggest that m1Ψ does not significantly impact translational fidelity, a welcome sign for future RNA therapeutics.

Many aspects of plant photoperception are mediated by the phytochrome (Phy) family of bilin-containing photoreceptors that reversibly interconvert between inactive Pr and active Pfr conformers1,2. Despite extensive biochemical studies, full understanding of plant Phy signalling has remained unclear due to the absence of relevant 3D models. Here we report a cryo-electron microscopy structure of Arabidopsis PhyB in the Pr state that reveals a topologically complex dimeric organization that is substantially distinct from its prokaryotic relatives. Instead of an anticipated parallel architecture, the C-terminal histidine-kinase-related domains (HKRDs) associate head-to-head, whereas the N-terminal photosensory regions associate head-to-tail to form a parallelogram-shaped platform with near two-fold symmetry. The platform is internally linked by the second of two internal Per/Arnt/Sim domains that binds to the photosensory module of the opposing protomer and a preceding 'modulator' loop that assembles tightly with the photosensory module of its own protomer. Both connections accelerate the thermal reversion of Pfr back to Pr, consistent with an inverse relationship between dimer assembly and Pfr stability. Lopsided contacts between the HKRDs and the platform create profound asymmetry to PhyB that might imbue distinct signalling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate-sensitive surface for conformation-dependent interactions between plant Phy photoreceptors and their signalling partners.

Mechanosensitive (MS) ion channels are an evolutionarily conserved way for cells to sense mechanical forces and transduce them into ionic signals. The channel properties of Arabidopsis thaliana MscS-Like (MSL)10 have been well studied, but how MSL10 signals remains largely unknown. To uncover signaling partners of MSL10, we employed a proteomic screen and a forward genetic screen; both unexpectedly implicated endoplasmic reticulum–plasma membrane contact sites (EPCSs) in MSL10 function. The proteomic screen revealed that MSL10 associates with multiple proteins associated with EPCSs. Of these, only VAMP-associated proteins (VAP)27-1 and VAP27-3 interacted directly with MSL10. The forward genetic screen, for suppressors of a gain-of-function MSL10 allele ( msl10-3G, MSL10 S640L ), identified mutations in the synaptotagmin (SYT)5 and SYT7 genes. We also found that EPCSs were expanded in leaves of msl10-3G plants compared to the wild type. Taken together, these results indicate that MSL10 associates and functions with EPCS proteins, providing a new cell-level framework for understanding MSL10 signaling. In addition, placing a mechanosensory protein at EPCSs provides new insight into the function and regulation of this type of subcellular compartment.

As central effectors of ubiquitin (Ub)-mediated proteolysis, proteasomes are regulated at multiple levels, including degradation of unwanted or dysfunctional particles via autophagy (termed proteaphagy). In yeast, inactive proteasomes are exported from the nucleus, sequestered into cytoplasmic aggresomes via the Hsp42 chaperone, extensively ubiquitylated, and then tethered to the expanding phagophore by the autophagy receptor Cue5. Here, we demonstrate the need for ubiquitylation driven by the trio of Ub ligases (E3s), San1, Rsp5, and Hul5, which together with their corresponding E2s work sequentially to promote nuclear export and Cue5 recognition. Whereas San1 functions prior to nuclear export, Rsp5 and Hul5 likely decorate aggresome-localized proteasomes in concert. Ultimately, topologically complex Ub chain(s) containing both K48 and K63 Ub-Ub linkages are assembled, mainly on the regulatory particle, to generate autophagy-competent substrates. Because San1, Rsp5, Hul5, Hsp42, and Cue5 also participate in general proteostasis, proteaphagy likely engages a fundamental mechanism for eliminating inactive/misfolded proteins.