The Histone-Modifying Complex PWR/HOS15/HD2C Epigenetically Regulates Cold Tolerance.

Cold stress is a major environmental stress that severely affects plant growth and crop productivity. Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15) is a substrate receptor of the CULLIN4-based CLR4 ubiquitin E3 ligase complex, which epigenetically regulates cold tolerance by degrading HISTONE DEACETYLASE2C (HD2C) to switch from repressive to permissive chromatin structure in response to cold stress. In this study, we characterized a HOS15-binding protein, POWERDRESS (PWR), and analyzed its function in the cold stress response. PWR loss-of-function plants (pwr) showed lower expression of cold-regulated (COR) genes and sensitivity to freezing. PWR interacts with HD2C through HOS15, and cold-induced HD2C degradation by HOS15 is diminished in the pwr mutant. The association of HOS15 and HD2C to promoters of cold-responsive COR genes was dependent on PWR. Consistent with these observations, the high acetylation levels of histone H3 by cold-induced and HOS15-mediated HD2C degradation were significantly reduced in pwr under cold stress. PWR also interacts with C-repeat element-binding factor transcription factors to modulate their cold-induced binding to the promoter of COR genes. Collectively, our data signify that the PWR-HOS15-HD2C histone-modifying complex regulates the expression of COR genes and the freezing tolerance of plants.

High-Temperature Conditions Promote Soybean Flowering through the Transcriptional Reprograming of Flowering Genes in the Photoperiod Pathway.

Global warming has an impact on crop growth and development. Flowering time is particularly sensitive to environmental factors such as day length and temperature. In this study, we investigated the effects of global warming on flowering using an open-top Climatron chamber, which has a higher temperature and CO2 concentration than in the field. Two different soybean cultivars, Williams 82 and IT153414, which exhibited different flowering times, were promoted flowering in the open-top Climatron chamber than in the field. We more specifically examined the expression patterns of soybean flowering genes on the molecular level under high-temperature conditions. The elevated temperature induced the expression of soybean floral activators, GmFT2a and GmFT5a as well as a set of GmCOL genes. In contrast, it suppressed floral repressors, E1 and E2 homologs. Moreover, high-temperature conditions affected the expression of these flowering genes in a day length-independent manner. Taken together, our data suggest that soybean plants properly respond and adapt to changing environments by modulating the expression of a set of flowering genes in the photoperiod pathway for the successful production of seeds and offspring.

Phenylephrine, a small molecule, inhibits pectin methylesterases.

Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1 µM. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.

AvrBsT acetylates Arabidopsis ACIP1, a protein that associates with microtubules and is required for immunity.

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1's defense function, which is linked to the activation of ETI.

N-terminal arm of orchardgrass Hsp17.2 (DgHsp17.2) is essential for both in vitro chaperone activity and in vivo thermotolerance in yeast.

Small heat shock proteins are well-known to function as chaperone in the protection of proteins and subcellular structures against stress-induced denaturation in many cell compartments. Irrespective of such general functional assignment, a proof of function in a living organism is missing. Here, we used heat-induced orchardgrass small Hsp17.2 (DgHsp17.2). Its function in in vitro chaperone properties has shown in protecting the model substrate, malate dehydrogenase (MDH) and citrate synthase (CS). Overexpression of DgHsp17.2 triggering strong chaperone activity enhanced in vivo thermotolerance of yeast cells. To identify the functional domain on DgHsp17.2 and correlationship between in vitro chaperone property and in vivo thermotolerance, we generated truncation mutants of DgHsp17.2 and showed essentiality of the N-terminal arm of DgHsp17.2 for the chaperone function. In addition, beyond for acquisition of thermotolerance irrespective of sequences are diverse among the small Hsps. However, any truncation mutants of DgHsp17.2 did not exhibit strong interaction with orchardgrass heat shock protein 70 (DgHsp70) different from mature DgHsp17.2, indicating that full-length DgHsp17.2 is necessary for cooperating with Hsp70 protein. Our study indicates that the N-terminal arm of DgHsp17.2 is an important region for chaperone activity and thermotolerance.

Different Inhibitory Effects of Erythromycin and Chlortetracycline on Early Growth of Brassica campestris Seedlings.

Veterinary antibiotics, including erythromycin (Ery) and chlortetracycline (CTC), are often detected in agricultural land. Although these contaminants affect plant growth and development, their effects on crops remain elusive. In this study, the effects of Ery and CTC on plant growth were investigated and compared by analyzing transcript abundance in Brassica campestris seedlings. Treatment with Ery and/or CTC reduced chlorophyll content in leaves and photosynthetic efficiency. Examination of the chloroplast ultrastructure revealed the presence of abnormally shaped plastids in response to Ery and CTC treatments. The antibiotics produced similar phenotypes of lower accumulation of photosynthetic genes, including RBCL and LHCB1.1. Analysis of the transcript levels revealed that Ery and CTC differentially down-regulated genes involved in the tetrapyrrole biosynthetic pathway and primary root growth. In the presence of Ery and CTC, chloroplasts were undeveloped and photosynthesis efficiency was reduced. These results suggest that both Ery and CTC individually affect gene expression and influence plant physiological activity, independently of one another.

Auxin-Glucose Conjugation Protects the Rice (Oryza sativa L.) Seedlings Against Hydroxyurea-Induced Phytotoxicity by Activating UDP-Glucosyltransferase Enzyme.

Hydroxyurea (HU) is the replication stress known to carry out cell cycle arrest by inhibiting ribonucleotide reductase (RNR) enzyme upon generating excess hydrogen peroxide (H2O2) in plants. Phytohormones undergo synergistic and antagonistic interactions with reactive oxygen species (ROS) and redox signaling to protect plants against biotic and abiotic stress. Therefore, in this study, we investigated the protective role of Indole-3-acetic acid (IAA) in mitigating HU-induced toxicity in rice seedlings. The results showed that IAA augmentation improved the growth of the seedlings and biomass production by maintaining photosynthesis metabolism under HU stress. This was associated with reduced H2O2 and malondialdehyde (MDA) contents and improved antioxidant enzyme [superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD)] activity that was significantly affected under HU stress. Furthermore, we showed that the HU stress-induced DNA damage leads to the activation of uridine 5'-diphosphate-glucosyltransferase (UGT), which mediates auxin homeostasis by catalyzing IAA-glucose conjugation in rice. This IAA-glucose conjugation upregulates the RNR, transcription factor 2 (E2F2), cyclin-dependent kinase (CDK), and cyclin (CYC) genes that are vital for DNA replication and cell division. As a result, perturbed IAA homeostasis significantly enhanced the key phytohormones, such as abscisic acid (ABA), salicylic acid (SA), cytokinin (CTK), and gibberellic acid (GA), that alter plant architecture by improving growth and development. Collectively, our results contribute to a better understanding of the physiological and molecular mechanisms underpinning improved growth following the HU + IAA combination, activated by phytohormone and ROS crosstalk upon hormone conjugation via UGT.

Arabidopsis CCoAOMT1 Plays a Role in Drought Stress Response via ROS- and ABA-Dependent Manners.

Plants possess adaptive reprogramed modules to prolonged environmental stresses, including adjustment of metabolism and gene expression for physiological and morphological adaptation. CCoAOMT1 encodes a caffeoyl CoA O-methyltransferase and is known to play an important role in adaptation of Arabidopsis plants to prolonged saline stress. In this study, we showed that the CCoAOMT1 gene plays a role in drought stress response. Transcript of CCoAOMT1 was induced by salt, dehydration (drought), and methyl viologen (MV), and loss of function mutants of CCoAOMT1, ccoaomt1-1, and ccoaomt1-2 exhibit hypersensitive phenotypes to drought and MV stresses. The ccoaomt1 mutants accumulated higher level of H2O2 in the leaves and expressed lower levels of drought-responsive genes including RD29B, RD20, RD29A, and ERD1, as well as ABA3 3 and NCED3 encoding ABA biosynthesis enzymes during drought stress compared to wild-type plants. A seed germination assay of ccoaomt1 mutants in the presence of ABA also revealed that CCoAOMT1 functions in ABA response. Our data suggests that CCoAOMT1 plays a positive role in response to drought stress response by regulating H2O2 accumulation and ABA signaling.

Specific domain structures control abscisic acid-, salicylic acid-, and stress-mediated SIZ1 phenotypes.

SIZ1 (for yeast SAP and MIZ1) encodes the sole ortholog of mammalian PIAS (for protein inhibitor of activated STAT) and yeast SIZ SUMO (for small ubiquitin-related modifier) E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (for scaffold attachment factor A/B/acinus/PIAS domain), PINIT (for proline-isoleucine-asparagine-isoleucine-threonine), SP-RING (for SIZ/PIAS-RING), and SXS (for serine-X-serine, where X is any amino acid) motifs. SIZ1 contains, in addition, a PHD (for plant homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. Salicylic acid (SA) accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity, and abscisic acid inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of abscisic acid. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment.

Functional characterization of the SIZ/PIAS-type SUMO E3 ligases, OsSIZ1 and OsSIZ2 in rice.

Sumoylation is a post-translational regulatory process in diverse cellular processes in eukaryotes, involving conjugation/deconjugation of small ubiquitin-like modifier (SUMO) proteins to other proteins thus modifying their function. The PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. Here, we report the isolation and molecular characterization of Oryza sativa SIZ1 (OsSIZ1) and SIZ2 (OsSIZ2), rice homologs of Arabidopsis SIZ1. The rice SIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Our analysis showed increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABA-hypersensitivity of siz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation.

AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana.

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca(2+) signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca(2+)-dependent electrophoretic mobility shift and Ca(2+) binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.

Influence of Sulfonamide Contamination Derived from Veterinary Antibiotics on Plant Growth and Development.

Veterinary antibiotics such as sulfonamides are widely used to increase feed efficiency and to protect against disease in livestock production. The sulfonamide antimicrobial mechanism involves the blocking of folate biosynthesis by inhibiting bacterial dihydropteroate synthase (DHPS) activity competitively. Interestingly, most treatment antibiotics can be released into the environment via manure and result in significant diffuse pollution in the environment. However, the physiological effects of sulfonamide during plant growth and development remain elusive because the plant response is dependent on folate biosynthesis and the concentration of antibiotics. Here, we present a chemical interaction docking model between Napa cabbage (Brassica campestris) DHPS and sulfamethoxazole and sulfamethazine, which are the most abundant sulfonamides detected in the environment. Furthermore, seedling growth inhibition was observed in lentil bean (Lens culinaris), rice (Oryza sativa), and Napa cabbage plants upon sulfonamide exposure. The results revealed that sulfonamide antibiotics target plant DHPS in a module similar to bacterial DHPS and affect early growth and the development of crop seedlings. Taking these results together, we suggest that sulfonamides act as pollutants in crop fields.

Investigating the Induced Systemic Resistance Mechanism of 2,4-Diacetylphloroglucinol (DAPG) using DAPG Hydrolase-Transgenic Arabidopsis.

Plant immune responses can be triggered by chemicals, microbes, pathogens, insects, or abiotic stresses. In particular, induced systemic resistance (ISR) refers to the activation of the immune system due to a plant's interaction with beneficial microorganisms. The phenolic compound, 2,4-diacetylphloroglucinol (DAPG), which is produced by beneficial Pseudomonas spp., acts as an ISR elicitor, yet DAPG's mechanism in ISR remains unclear. In this study, transgenic Arabidopsis thaliana plants overexpressing the DAPG hydrolase gene (phlG) were generated to investigate the functioning of DAPG in ISR. DAPG was applied onto 3-week-old A. thaliana Col-0 and these primed plants showed resistance to the pathogens Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000. However, in the phlG transgenic A. thaliana, the ISR was not triggered against these pathogens. The DAPG-mediated ISR phenotype was impaired in transgenic A. thaliana plants overexpressing phlG, thus showing similar disease severity when compared to untreated control plants. Furthermore, the DAPG-treated A. thaliana Col-0 showed an increase in their gene expression levels of PDF1.2 and WRKY70 but this failed to occur in the phlG transgenic lines. Collectively, these experimental results indicate that jasmonic acid/ethylene signal-based defense system is effectively disabled in phlG transgenic A. thaliana lines.

Erythromycin Treatment of Brassica campestris Seedlings Impacts the Photosynthetic and Protein Synthesis Pathways.

Erythromycin (Ery) is a commonly used veterinary drug that prevents infections and promotes the growth of farm animals. Ery is often detected in agricultural fields due to the effects of manure application in the ecosystem. However, there is a lack of information on Ery toxicity in crops. In this study, we performed a comparative proteomic analysis to identify the molecular mechanisms of Ery toxicity during seedling growth based on our observation of a decrease in chlorophyll (Chl) contents using Brassica campestris. A total of 452 differentially abundant proteins (DAPs) were identified including a ribulose-1,5-bisphosphate carboxylase (RuBisCO). The proteomic analysis according to gene ontology (GO) classification revealed that many of these DAPs responding to Ery treatment functioned in a cellular process and a metabolic process. The molecular function analysis showed that DAPs classified within catalytic activity were predominantly changed by Ery, including metabolite interconversion enzyme and protein modifying enzyme. An analysis of functional pathways using MapMan revealed that many photosynthesis components were downregulated, whereas many protein biosynthesis components were upregulated. A good relationship was observed between protein and transcript abundance in a photosynthetic pathway, as determined by qPCR analysis. These combined results suggest that Ery affects plant physiological activity by downregulating protein abundance in the photosynthetic pathway.

AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs.

Cell surface receptors perceive signals from the environment and transfer them to the interior of the cell. The Arabidopsis thaliana PR5 receptor-like kinase (AtPR5K) subfamily consists of three members with extracellular domains that share sequence similarity with the PR5 proteins. In this study, we characterized the role of AtPR5K2 in plant drought-stress signaling. AtPR5K2 is predominantly expressed in leaves and localized to the plasma membrane. The atpr5k2-1 mutant showed tolerance to dehydration stress, while AtPR5K2-overexpressing plants was hypersensitive to drought. Bimolecular fluorescence complementation assays showed that AtPR5K2 physically interacted with the type 2C protein phosphatases ABA-insensitive 1 (ABI1) and ABI2 and the SNF1-related protein kinase 2 (SnRK2.6) proteins, all of which are involved in the initiation of abscisic acid (ABA) signaling; however, these interactions were inhibited by treatments of exogenous ABA. Moreover, AtPR5K2 was found to phosphorylate ABI1 and ABI2, but not SnRK2.6. Taken together, these results suggest that AtPR5K2 participates in ABA-dependent drought-stress signaling through the phosphorylation of ABI1 and ABI2.

Overexpression of the alfalfa DnaJ-like protein (MsDJLP) gene enhances tolerance to chilling and heat stresses in transgenic tobacco plants.

Heat shock proteins (HSPs) are generally considered as important molecular chaperones; they are known to perform critical functions in plant development and abiotic stress response processes. In this study, we examined the role of a HSP, the Medicago sativa DnaJ-like protein (MsDJLP), in alfalfa and its potential application for the development of abiotic stress tolerance in plants. We found that expression of the MsDJLP gene was induced by chilling (4 °C) and heat (42 °C), but not by cadmium (500 µM) or arsenic (500 µM) stresses. We then cloned the MsDJLP gene downstream of the strong constitutive CaMV 35S promoter and transformed it into tobacco plants. Ectopic expression of MsDJLP conferred enhanced tolerance to both chilling and heat stresses in transgenic tobacco plants. Under chilling stress, the transgenic tobacco plants showed lower H2O2 accumulation and electrolyte leakage (EL) activity, and better photosystem II efficiency than wild-type (WT) plants, indicating that photoinhibition was less severe in transgenic compared to WT plants. Following heat treatment, the transgenic plants showed better relative chlorophyll and water contents, and lower malondialdehyde accumulation than WT plants. Our study provides evidence for a pivotal role of MsDJLP for chilling and heat stress tolerance in transgenic tobacco plants.

Antifungal Effect of Arabidopsis SGT1 Proteins via Mitochondrial Reactive Oxygen Species.

The highly conserved SGT1 (suppressor of the G2 alleles of skp1) proteins from Arabidopsis are known to contribute to plant resistance to pathogens. While SGT1 proteins respond to fungal pathogens, their antifungal activity is not reported and the mechanism for this inhibition is not well understood. Therefore, recombinant Arabidopsis SGT1 proteins were cloned, expressed, and purified to evaluate their antifungal activity, resulting in their potent inhibition of pathogen growth. Dye-labeled proteins are localized to the cytosol of Candida albicans cells without the disruption of the cell membrane. Moreover, we showed that entry of the proteins into C. albicans cells resulted in the accumulation of reactive oxygen species (ROS) and cell death via altered mitochondrial potential. Morphological changes of C. albicans cells in the presence of proteins were visualized by scanning electron microscopy. Our data suggest that AtSGT1 proteins play a critical role in plant resistance to pathogenic fungal infection and they can be classified to a new plant antifungal protein.

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin.

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.

Pathogenesis-related gene expression by specific calmodulin isoforms is dependent on NIM1, a key regulator of systemic acquired resistance.

Plants produce numerous calmodulin isoforms that exhibit differential gene expression patterns and sense different Ca2+ signals. This diversity results in different physiological responses to particular stimuli. Gm-CaM-4 and -5 are two divergent calmodulin isoforms from the soybean (Glycine max) that have been reported to be involved in plant disease resistance. However, little is known about the pathway by which these specific isoforms transduce the defense signal and up-regulate pathogenesis-related (PR) genes. Here we report that overexpression of GmCaM-4/-5 induces constitutive PR gene expression and enhances disease resistance in wild-type Arabidopsis, but not in the nim1 mutant of Arabidopsis. GmCaM-4/-5 also appear to activate trans-acting elements that bind to cis-acting elements in the Arabidopsis PR-1 promoter. Thus up-regulation of PR genes by these GmCaM isoforms is dependent on NIM1 (Non immunity 1) and unknown transcription factors.

Identification and molecular properties of SUMO-binding proteins in Arabidopsis.

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.