ATG8-Binding UIM Proteins Define a New Class of Autophagy Adaptors and Receptors.

During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.

Plant phytochrome B is an asymmetric dimer with unique signalling potential.

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.

Crystal structure of the photosensory module from a PAS-less cyanobacterial phytochrome as Pr shows a mix of dark-adapted and photoactivated features.

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 ß-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.

Variation in upstream open reading frames contributes to allelic diversity in maize protein abundance.

SignificanceProteins are the machinery which execute essential cellular functions. However, measuring their abundance within an organism can be difficult and resource-intensive. Cells use a variety of mechanisms to control protein synthesis from mRNA, including short open reading frames (uORFs) that lie upstream of the main coding sequence. Ribosomes can preferentially translate uORFs instead of the main coding sequence, leading to reduced translation of the main protein. In this study, we show that uORF sequence variation between individuals can lead to different rates of protein translation and thus variable protein abundances. We also demonstrate that natural variation in uORFs occurs frequently and can be linked to whole-plant phenotypes, indicating that uORF sequence variation likely contributes to plant adaptation.

Autophagy during maize endosperm development dampens oxidative stress and promotes mitochondrial clearance.

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.

Differing biophysical properties underpin the unique signaling potentials within the plant phytochrome photoreceptor families.

Many aspects of photoperception by plants and microorganisms are initiated by the phytochrome (Phy) family of photoreceptors that detect light through interconversion between red light- (Pr) and far-red light-absorbing (Pfr) states. Plants synthesize a small family of Phy isoforms (PhyA to PhyE) that collectively regulate photomorphogenesis and temperature perception through redundant and unique actions. While the selective roles of these isoforms have been partially attributed to their differing abundances, expression patterns, affinities for downstream partners, and turnover rates, we show here from analysis of recombinant Arabidopsis chromoproteins that the Phy isoforms also display distinct biophysical properties. Included are a hypsochromic shift in the Pr absorption for PhyC and varying rates of Pfr to Pr thermal reversion, part of which can be attributed to the core photosensory module in each. Most strikingly, PhyB combines strong temperature dependence of thermal reversion with an order-of-magnitude faster rate to likely serve as the main physiological thermosensor, whereby thermal reversion competes with photoconversion. In addition, comparisons of Pfr occupancies for PhyA and PhyB under a range of red- and white-light fluence rates imply that low-light environments are effectively sensed by PhyA, while high-light environments, such as full sun, are effectively sensed by PhyB. Parallel analyses of the Phy isoforms from potato and maize showed that the unique features within the Arabidopsis family are conserved, thus indicating that the distinct biophysical properties among plant Phy isoforms emerged early in Phy evolution, likely to enable full interrogation of their light and temperature environments.

The SUMO ligase MMS21 profoundly influences maize development through its impact on genome activity and stability.

The post-translational addition of SUMO plays essential roles in numerous eukaryotic processes including cell division, transcription, chromatin organization, DNA repair, and stress defense through its selective conjugation to numerous targets. One prominent plant SUMO ligase is METHYL METHANESULFONATE-SENSITIVE (MMS)-21/HIGH-PLOIDY (HPY)-2/NON-SMC-ELEMENT (NSE)-2, which has been connected genetically to development and endoreduplication. Here, we describe the potential functions of MMS21 through a collection of UniformMu and CRISPR/Cas9 mutants in maize (Zea mays) that display either seed lethality or substantially compromised pollen germination and seed/vegetative development. RNA-seq analyses of leaves, embryos, and endosperm from mms21 plants revealed a substantial dysregulation of the maize transcriptome, including the ectopic expression of seed storage protein mRNAs in leaves and altered accumulation of mRNAs associated with DNA repair and chromatin dynamics. Interaction studies demonstrated that MMS21 associates in the nucleus with the NSE4 and STRUCTURAL MAINTENANCE OF CHROMOSOMES (SMC)-5 components of the chromatin organizer SMC5/6 complex, with in vitro assays confirming that MMS21 will SUMOylate SMC5. Comet assays measuring genome integrity, sensitivity to DNA-damaging agents, and protein versus mRNA abundance comparisons implicated MMS21 in chromatin stability and transcriptional controls on proteome balance. Taken together, we propose that MMS21-directed SUMOylation of the SMC5/6 complex and other targets enables proper gene expression by influencing chromatin structure.

AUTOPHAGY-RELATED14 and Its Associated Phosphatidylinositol 3-Kinase Complex Promote Autophagy in Arabidopsis.

Phosphatidylinositol 3-phosphate (PI3P) is an essential membrane signature for both autophagy and endosomal sorting that is synthesized in plants by the class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of the VPS34 kinase, together with ATG6, VPS15, and either VPS38 or ATG14 as the fourth subunit. Although Arabidopsis (Arabidopsis thaliana) plants missing the three core subunits are infertile, vps38 mutants are viable but have aberrant leaf, root, and seed development, Suc sensing, and endosomal trafficking, suggesting that VPS38 and ATG14 are nonredundant. Here, we evaluated the role of ATG14 through a collection of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and T-DNA insertion mutants disrupting the two Arabidopsis paralogs. atg14a atg14b double mutants were relatively normal phenotypically but displayed pronounced autophagy defects, including reduced accumulation of autophagic bodies and cargo delivery during nutrient stress. Unexpectedly, homozygous atg14a atg14b vps38 triple mutants were viable but showed severely compromised rosette development and reduced fecundity, pollen germination, and autophagy, consistent with a need for both ATG14 and VPS38 to fully actuate PI3P biology. However, the triple mutants still accumulated PI3P, but they were hypersensitive to the PI3K inhibitor wortmannin, indicating that the ATG14/VPS38 component is not essential for PI3P synthesis. Collectively, the ATG14/VPS38 mutant collection now permits the study of plants altered in specific aspects of PI3P biology.

Autophagy Plays Prominent Roles in Amino Acid, Nucleotide, and Carbohydrate Metabolism during Fixed-Carbon Starvation in Maize.

Autophagic recycling of proteins, lipids, nucleic acids, carbohydrates, and organelles is essential for cellular homeostasis and optimal health, especially under nutrient-limiting conditions. To better understand how this turnover affects plant growth, development, and survival upon nutrient stress, we applied an integrated multiomics approach to study maize (Zea mays) autophagy mutants subjected to fixed-carbon starvation induced by darkness. Broad metabolic alterations were evident in leaves missing the core autophagy component ATG12 under normal growth conditions (e.g., lipids and secondary metabolism), while changes in amino acid-, carbohydrate-, and nucleotide-related metabolites selectively emerged during fixed-carbon starvation. Through combined proteomic and transcriptomic analyses, we identified numerous autophagy-responsive proteins, which revealed processes underpinning the various metabolic changes seen during carbon stress as well as potential autophagic cargo. Strikingly, a strong upregulation of various catabolic processes was observed in the absence of autophagy, including increases in simple carbohydrate levels with a commensurate drop in starch levels, elevated free amino acid levels with a corresponding reduction in intact protein levels, and a strong increase in the abundance of several nitrogen-rich nucleotide catabolites. Altogether, this analysis showed that fixed-carbon starvation in the absence of autophagy adjusts the choice of respiratory substrates, promotes the transition of peroxisomes to glyoxysomes, and enhances the retention of assimilated nitrogen.

Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis.

Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. Here we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuing proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.

An evolutionarily distinct chaperone promotes 20S proteasome α-ring assembly in plants.

The core protease (CP) subcomplex of the 26S proteasome houses the proteolytic active sites and assumes a barrel shape comprised of four co-axially stacked heptameric rings formed by structurally related α- and ß-subunits. CP biogenesis typically begins with the assembly of the α-ring, which then provides a template for ß-subunit integration. In eukaryotes, α-ring assembly is partially mediated by two hetero-dimeric chaperones, termed Pba1-Pba2 (Add66) and Pba3-Pba4 (also known as Irc25-Poc4) in yeast. Pba1-Pba2 initially promotes orderly recruitment of the α-subunits through interactions between their C-terminal HbYX or HbF motifs and pockets at the α5-α6 and α6-α7 interfaces. Here, we identified PBAC5 as a fifth α-ring assembly chaperone in Arabidopsis that directly binds the Pba1 homolog PBAC1 to form a trimeric PBAC5-PBAC1-PBAC2 complex. PBAC5 harbors a HbYX motif that docks with a pocket between the α4 and α5 subunits during α-ring construction. Arabidopsis lacking PBAC5, PBAC1 and/or PBAC2 are hypersensitive to proteotoxic, salt and osmotic stresses, and display proteasome assembly defects. Remarkably, whereas PBAC5 is evolutionarily conserved among plants, sequence relatives are also dispersed within other kingdoms, including a scattered array of fungal, metazoan and oomycete species.

Ubiquitylome analysis reveals a central role for the ubiquitin-proteasome system in plant innate immunity.

Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

Genetic Analyses of the Arabidopsis ATG1 Kinase Complex Reveal Both Kinase-Dependent and Independent Autophagic Routes during Fixed-Carbon Starvation.

Under nutrient and energy-limiting conditions, plants up-regulate sophisticated catabolic pathways such as autophagy to remobilize nutrients and restore energy homeostasis. Autophagic flux is tightly regulated under these circumstances through the AuTophaGy-related1 (ATG1) kinase complex, which relays upstream nutrient and energy signals to the downstream components that drive autophagy. Here, we investigated the role(s) of the Arabidopsis (Arabidopsis thaliana) ATG1 kinase during autophagy through an analysis of a quadruple mutant deficient in all four ATG1 isoforms. These isoforms appear to act redundantly, including the plant-specific, truncated ATG1t variant, and like other well-characterized atg mutants, homozygous atg1abct quadruple mutants display early leaf senescence and hypersensitivity to nitrogen and fixed-carbon starvations. Although ATG1 kinase is essential for up-regulating autophagy under nitrogen deprivation and short-term carbon starvation, it did not stimulate autophagy under prolonged carbon starvation. Instead, an ATG1-independent response arose requiring phosphatidylinositol-3-phosphate kinase (PI3K) and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), possibly through phosphorylation of the ATG6 subunit within the PI3K complex by the catalytic KIN10 subunit of SnRK1. Together, our data connect ATG1 kinase to autophagy and reveal that plants engage multiple pathways to activate autophagy during nutrient stress, which include the ATG1 route as well as an alternative route requiring SnRK1 and ATG6 signaling.plantcell;31/12/2973/FX1F1fx1.

PCH1 regulates light, temperature, and circadian signaling as a structural component of phytochrome B-photobodies in Arabidopsis.

The members of the phytochrome (phy) family of bilin-containing photoreceptors are major regulators of plant photomorphogenesis through their unique ability to photointerconvert between a biologically inactive red light-absorbing Pr state and an active far-red light-absorbing Pfr state. While the initial steps in Pfr signaling are unclear, an early event for the phyB isoform after photoconversion is its redistribution from the cytoplasm into subnuclear foci known as photobodies (PBs), which dissipate after Pfr reverts back to Pr by far-red irradiation or by temperature-dependent nonphotochemical reversion. Here we present evidence that PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) functions both as an essential structural component of phyB-containing PBs and as a direct regulator of thermal reversion that is sufficient to stabilize phyB as Pfr in vitro. By examining the genetic interaction between a constitutively active phyBY276H-YFP allele (YHB-YFP) and PCH1, we show that the loss of PCH1 prevents YHB from coalescing into PBs without affecting its nuclear localization, whereas overexpression of PCH1 dramatically increases PB levels. Loss of PCH1, presumably by impacting phyB-PB assembly, compromises a number of events elicited in YHB-YFP plants, including their constitutive photomorphogenic phenotype, red light-regulated thermomorphogenesis, and input of phyB into the circadian clock. Conversely, elevated levels of both phyB and PCH1 generate stable, yet far-red light-reversible PBs that persisted for days. Collectively, our data demonstrate that the assembly of PCH1-containing PBs is critical for phyB signaling to multiple outputs and suggest that altering PB dynamics could be exploited to modulate plant responses to light and temperature.

Improved Spirodela polyrhiza genome and proteomic analyses reveal a conserved chromosomal structure with high abundance of chloroplastic proteins favoring energy production.

Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. Here, we studied the genome and proteome of Spirodela polyrhiza, or greater duckweed, which has the largest body plan yet the smallest genome size in the family (1C=150 Mb). Using Oxford Nanopore sequencing combined with Hi-C scaffolding, we generated a highly contiguous, chromosome-scale assembly of S. polyrhiza line Sp7498 (Sp7498_HiC). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations relative to a recent PacBio assembly of Sp7498, highlighting the need for orthogonal long-range scaffolding techniques such as Hi-C and BioNano optical mapping. Shotgun proteomics of Sp7498 verified the expression of ~2250 proteins and revealed a high abundance of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome illustrates the utility of duckweed as a model for genomics- and proteomics-based education.

Bacteria Exploit Autophagy for Proteasome Degradation and Enhanced Virulence in Plants.

Autophagy and the ubiquitin-proteasome system (UPS) are two major protein degradation pathways implicated in the response to microbial infections in eukaryotes. In animals, the contribution of autophagy and the UPS to antibacterial immunity is well documented and several bacteria have evolved measures to target and exploit these systems to the benefit of infection. In plants, the UPS has been established as a hub for immune responses and is targeted by bacteria to enhance virulence. However, the role of autophagy during plant-bacterial interactions is less understood. Here, we have identified both pro- and antibacterial functions of autophagy mechanisms upon infection of Arabidopsis thaliana with virulent Pseudomonas syringae pv tomato DC3000 (Pst). We show that Pst activates autophagy in a type III effector (T3E)-dependent manner and stimulates the autophagic removal of proteasomes (proteaphagy) to support bacterial proliferation. We further identify the T3E Hrp outer protein M1 (HopM1) as a principle mediator of autophagy-inducing activities during infection. In contrast to the probacterial effects of Pst-induced proteaphagy, NEIGHBOR OF BRCA1-dependent selective autophagy counteracts disease progression and limits the formation of HopM1-mediated water-soaked lesions. Together, we demonstrate that distinct autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection and provide evidence for an intimate crosstalk between proteasome and autophagy in plant-bacterial interactions.

SUMOylome Profiling Reveals a Diverse Array of Nuclear Targets Modified by the SUMO Ligase SIZ1 during Heat Stress.

The posttranslational addition of small ubiquitin-like modifier (SUMO) is an essential protein modification in plants that provides protection against numerous environmental challenges. Ligation is accomplished by a small set of SUMO ligases, with the SAP-MIZ domain-containing SIZ1 and METHYL METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in Arabidopsis thaliana, we used siz1 and mms21 mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.