E3 SUMO ligase SIZ1 splicing variants localize and function according to external conditions.

SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms. The SIZ1 gene encodes an 873-amino acid (aa) protein. Among the four SIZ1 splicing variants (SSVs), SSV1 and SSV4 encode identical 885 aa proteins; SSV2 encodes an 832 aa protein; and SSV3 encodes an 884 aa protein. SSV2 mainly localized to the plasma membrane, whereas SIZ1, SSV1/SSV4, and SSV3 localized to the nucleus. Interestingly, SIZ1 and all SSVs exhibited similar E3 SUMO ligase activities and preferred SUMO1 and SUMO2 for their E3 ligase activity. Transcript levels of SSV2 were substantially increased by heat treatment, while those of SSV1, SSV3, and SSV4 transcripts were unaffected by various abiotic stresses. SSV2 directly interacted with and sumoylated cyclic nucleotide-gated ion channel 6 (CNGC6), a positive thermotolerance regulator, enhancing the stability of CNGC6. Notably, transgenic siz1-2 mutants expressing SSV2 exhibited greater heat stress tolerance than wild-type plants, whereas those expressing SIZ1 were sensitive to heat stress. Furthermore, transgenic cngc6 plants overaccumulating a mutated mCNGC6 protein (K347R, a mutation at the sumoylation site) were sensitive to heat stress, similar to the cngc6 mutants, while transgenic cngc6 plants overaccumulating CNGC6 exhibited restored heat tolerance. Together, we propose that alternative splicing is an important mechanism that regulates the function of SSVs during development or under adverse conditions, including heat stress.

Arabidopsis retromer subunit AtVPS29 is involved in SLY1-mediated gibberellin signaling.

KEY MESSAGE: Retromer protein AtVPS29 upregulates the SLY1 protein and downregulates the RGA protein, positively stimulating the development of the root meristematic zone, which indicates an important role of AtVPS29 in gibberellin signaling. In plants, the large retromer complex is known to play roles in multiple development processes, including cell polarity, programmed cell death, and root hair growth in Arabidopsis. However, many of its roles in plant development remain unknown. Here, we show that Arabidopsis trimeric retromer protein AtVPS29 (vacuolar protein sorting 29) modulates gibberellin signaling. The SLEEPY1 (SLY1) protein, known as a positive regulator of gibberellic acid (GA) signaling, exhibited lower abundance in vps29-3 mutants compared to wild-type (WT) plants. Conversely, the DELLA repressor protein, targeted by the E3 ubiquitin ligase SCF (Skp, Cullin, F-box) complex and acting as a negative regulator of GA signaling, showed increased abundance in vps29-3 mutants compared to WT. The vps29-3 mutants exhibited decreased sensitivity to exogenous GA supply in contrast to WT, despite an upregulation in the expression of GA receptor genes within the vps29-3 mutants. In addition, the expression of the GA synthesis genes was downregulated in vps29-3 mutants, implying that the loss of AtVPS29 causes the downregulation of GA synthesis and signaling. Furthermore, vps29-3 mutants exhibited a reduced meristematic zone accompanied by a decreased cell number. Together, these data indicate that AtVPS29 positively regulates SLY1-mediated GA signaling and plant growth.

Arabidopsis COP1 guides stomatal response in guard cells through pH regulation.

Plants rely on precise regulation of their stomatal pores to effectively carry out photosynthesis while managing water status. The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a critical light signaling repressor, is known to repress stomatal opening, but the exact cellular mechanisms remain unknown. Here, we show that COP1 regulates stomatal movement by controlling the pH levels in guard cells. cop1-4 mutants have larger stomatal apertures and disrupted pH dynamics within guard cells, characterized by increased vacuolar and cytosolic pH and reduced apoplastic pH, leading to abnormal stomatal responses. The altered pH profiles are attributed to the increased plasma membrane (PM) H+-ATPase activity of cop1-4 mutants. Moreover, cop1-4 mutants resist to growth defect caused by alkali stress posed on roots. Overall, our study highlights the crucial role of COP1 in maintaining pH homeostasis of guard cells by regulating PM H+-ATPase activity, and demonstrates how proton movement affects stomatal movement and plant growth.

COP1 mutation causes low leaf temperature under various abiotic stresses in Arabidopsis thaliana.

Stomata are microscopic pores on epidermal cells of leaves and stems that regulate water loss and gas exchange between the plant and its environment. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase that is involved in plant growth and development and multiple abiotic stress responses by regulating the stability of various target proteins. However, little is known about how COP1 controls stomatal aperture and leaf temperature under various environmental conditions. Here, we show that COP1 participates in leaf temperature and stomatal closure regulation under normal and stress conditions in Arabidopsis. Leaf temperature of cop1 mutants was relatively lower than that of wild type (WT) under drought, salt, and heat stress and after abscisic acid (ABA), CaCl2, and H2O2 treatments. However, leaf temperature was generally higher in both WT and cop1 mutants after abiotic stress and chemical treatment than that of untreated WT and cop1 mutants. Stomatal aperture was wider in cop1 mutants than that in WT under all conditions tested, although the extent of stomatal closure varied between WT and cop1 mutants. Under dark conditions, leaf temperature was also lower in cop1 mutants than that in WT. Expression of the genes encoding ABA receptors, ABA biosynthesis proteins, positive regulators of stomatal closure and heat tolerance, and ABA-responsive proteins was lower in cop1 mutants that that in WT. In addition, expression of respiration-related genes was lower in cop1 mutants that that in WT. Taken together, the data provide evidence that mutations in COP1 lead to wider stomatal aperture and higher leaf temperature under normal and stress conditions, indicating that leaf temperature is highly correlated with stomatal aperture.

COP1 controls salt stress tolerance by modulating sucrose content.

The E3 ubiquitin ligase Constitutive Photomorphogenic 1 (COP1) plays evolutionarily conserved and divergent roles. In plants, COP1 regulates a large number of developmental processes including photomorphogenesis, seedling emergence, and gravitropism. Nevertheless, its function in abiotic stress tolerance remains largely unknown. Here, we demonstrate the role of COP1 in salt stress tolerance in Arabidopsis thaliana. In soil, cop1-4 and cop1-6 mutants were more tolerant to high salinity than wild-type (WT) plants during vegetative growth. However, in high salt-containing Murashige and Skoog (MS) medium, cop1-4 and cop1-6 seedlings exhibited significantly impaired growth compared with WT plants. Notably, cop1-4 and cop1-6 seedlings recovered their growth to the WT level upon exogenous sucrose treatment even under high salinity conditions. Compared with WT plants, the sucrose content of cop1-4 mutants was much higher at the vegetative growth stage but similar at the seedling stage. Upon exogenous sucrose supply, root elongation was significantly stimulated in cop1-4 seedlings but only slightly stimulated in WT plants. Thus, no significant difference was observed in root length between the two genotypes. Altogether, our data indicate that cop1 mutants are more tolerant to salt stress than WT plants, and the salt tolerance of cop1 mutants is correlated with their sucrose content.

Intronic long noncoding RNA, RICE FLOWERING ASSOCIATED (RIFLA), regulates OsMADS56-mediated flowering in rice.

Long noncoding RNAs (lncRNAs) are known to play important roles in several plant processes such as flowering, organ development and stress response. However, studies exploring the diversity and complexity of lncRNAs and their mechanism of action in plants are far fewer that those in animals. Here, we show that an intronic lncRNA in rice (Oryza sativa L.), RICE FLOWERING ASSOCIATED (RIFLA), is required for the inhibition of OsMADS56 expression. RIFLA is produced from the first intron of the OsMADS56 gene. Overexpression of RIFLA in rice repressed OsMADS56 expression but activated the expression of flowering inducers Hd3a and RFT1. Additionally, RIFLA-overexpressing transgenic rice plants flowered earlier than the wild type. Under normal conditions, the transcript level of the rice enhancer of zeste gene OsiEZ1, a homolog of Arabidopsis histone H3K27-specific methyltransferase genes SWINGER (SWN) and CURLY LEAF (CLF), was as low as that of RIFLA, whereas the transcript level of OsMADS56 was relatively high. In the osiez1 mutant, OsMADS56 expression was upregulated, whereas RIFLA expression was downregulated. Additionally, RIFLA formed a complex with OsiEZ1. Together, these results suggest that the floral repressor activity of OsMADS56 is epigenetically regulated by RIFLA and OsiEZ1.

A mutation in the pPLA-IIα gene encoding PATATIN-RELATED PHOSPHOLIPASE a causes late flowering in Arabidopsis.

Arabidopsis PATATIN-RELATED PHOSPHOLIPASE 2A (pPLA-IIα) participates in the responses to various growth conditions. The factors affecting pPLA-IIα gene expression and pPLA-IIα protein activity for gycerolipids have been studied thoroughly, but the role of pPLA-IIα during the reproductive phase remains unclear. The effect of pPLA-IIα on flowering time was therefore investigated. ppla-iiα mutants flowered later than wild-type plants under long day conditions. Expression of the floral stimulators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) was downregulated in ppla-iiα mutants compared with their expression in wild-type plants, but expression of the floral repressor FLOWERING LOCUS C (FLC) was upregulated. In addition, expression levels of COLDAIR, a long intronic noncoding RNA, decreased in ppla-iiα mutants. Taken together, these data indicate that pPLA-IIα acts as a positive regulator of flowering time through repression of FLC expression.

Overexpression of Rice OsS1Fa1 Gene Confers Drought Tolerance in Arabidopsis.

Small peptides and proteins play critical regulatory roles in plant development and environmental stress responses; however, only a few of these molecules have been identified and characterized to date because of their poor annotation and other experimental challenges. Here, we present that rice (Oryza sativa L.) OsS1Fa1, a small 76-amino acid protein, confers drought stress tolerance in Arabidopsis thaliana. OsS1Fa1 was highly expressed in leaf, culm, and root tissues of rice seedlings during vegetative growth and was significantly induced under drought stress. OsS1Fa1 overexpression in Arabidopsis induced the expression of selected drought-responsive genes and enhanced the survival rate of transgenic lines under drought. The proteasome inhibitor MG132 protected the OsS1Fa1 protein from degradation. Together, our data indicate that the small protein OsS1Fa1 is induced by drought and is post-translationally regulated, and the ectopic expression of OsS1Fa1 protects plants from drought stress.

Ammonium-mediated reduction in salicylic acid content and recovery of plant growth in Arabidopsis siz1 mutants is modulated by NDR1 and NPR1.

The siz1 mutants exhibit high SA accumulation and consequently severe dwarfism. Although siz1 mutants exhibit growth recovery upon exogenous ammonium supply, the underlying mechanism remains unknown. Here, we investigated the effect of ammonium on SA level and plant growth in SA-accumulating mutants. The growth of siz1-2 and siz1-3 mutants was recovered to wild-type (WT) levels upon exogenous ammonium supply, but that of siz1-3 ndr1 (non-race-specific disease resistance 1) and siz1-3 npr1 (non-expressor of pathogenesis related gene 1) double mutants was unaffected. The SA level was decreased by exogenous ammonium application in siz1-3 ndr1, siz1-3 npr1, and siz1-3 mutants. The level of nitrate reductase (NR) was almost the same in all genotypes (WT, siz1-3, ndr1, npr1, siz1-3 ndr1, and siz1-3 npr1), regardless of the ammonium treatment, suggesting that exogenous ammonium supply to ndr1 siz1-3 and npr1 siz1-3 double mutants does not have any effect on their growth and NR levels, but decreases the SA level. Taken together, these results indicate that ammonium acts as a signaling molecule to regulate the SA amount, and NDR1 and NPR1 play a positive role in the ammonium-mediated growth recovery of siz1 mutants.

Mutation of the OsGlyRS3 gene affects heading date in rice.

Aminoacyl-tRNA synthetases play a critical role in protein synthesis by catalyzing the covalent attachment of amino acids to their cognate tRNAs. However, the role of aminoacyl-tRNA synthetases in the transition from vegetative to reproductive growth in plants remains poorly understood. In this study, a rice (Oryza sativa) glycyl-tRNA synthetase 3, OsGlyRS3, was found to impact heading date in rice. Flowering in osglyrs3, a mutant line containing a T-DNA insertion in OsGlyRS3, was advanced by approximately 2 weeks compared to wild type. Expression analysis of flowering regulator genes showed that transcript levels of Heading date 1 (Hd1), Heading date 3a (Hd3a), and OsMADS51 were elevated in osglyrs3. These data indicate that the loss of OsGlyRS3 activity induces the expression of flowering-activating genes, resulting in early flowering.

A P3A-Type ATPase and an R2R3-MYB Transcription Factor Are Involved in Vacuolar Acidification and Flower Coloration in Soybean.

The determination of flower color mainly depends on the anthocyanin biosynthesis pathway and vacuolar pH; however, unlike the former, the mechanism of vacuolar acidification in soybean remains uncharacterized at the molecular level. To investigate this mechanism, we isolated four recessive purple-blue EMS-induced flower mutants from the purple flower soybean cultivar, Pungsannamul. The petals of all the mutants had increased pH compared with those of wild Pungsannamul. One of the mutants had a single nucleotide substitution in GmPH4, a regulator gene encoding an MYB transcription factor, and the substitution resulted in a premature stop codon in its first exon. The other three mutants had nucleotide substitutions in GmPH5, a single new gene that we identified by physical mapping. It corresponds to Glyma.03G262600 in chromosome 3 and encodes a proton pump that belongs to the P3A-ATPase family. The substitutions resulted in a premature stop codon, which may be a defect in the ATP-binding capacity of GmPH5 and possibly a catalytic inefficiency of GmPH5. The result is consistent with their genetic recessiveness as well as the high pH of mutant petals, suggesting that GmPH5 is directly involved in vacuolar acidification. We also found that the expression of GmPH5 and several putative "acidifying" genes in the gmph4 mutant was remarkably reduced, indicating that GmPH4 may regulate the genes involved in determining the vacuolar pH of soybean petals.

E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds.

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity.

The rice SUMO conjugating enzymes OsSCE1 and OsSCE3 have opposing effects on drought stress.

Posttranslational modification of proteins by the small ubiquitin-related modifier (SUMO) protein is involved in diverse cellular processes. In sumoylation, SUMO-conjugating enzyme (SCE) conjugates SUMO to substrate proteins. Similarly to yeast and animals, Arabidopsis encodes a single SCE gene, but other plants encode at least two SCE genes. In this study, we report the molecular characterization of three Oryza sativa SCE genes. Their levels of expression are commonly upregulated by drought stress but are differentially regulated by hormones and sugars. Only the OsSCE1 gene showed photoperiod- and light-dependent diurnal oscillations in the leaves. Yeast two-hybrid assays showed that OsSCEs do not show SUMO isoform specificity. Three rice OsSCE proteins localize primarily to the nucleus. Interestingly, OsSCE1 is distributed in specific parts of the nucleus and shows sumoylation activities in the absence of a SUMO ligase in E. coli. In addition, overexpression of the OsSCE1 gene alters the biomass and grain yield parameters in transgenic rice plants. Overexpression of the OsSCE3 gene in transgenic rice plants enhances drought stress tolerance. In contrast, OsSCE1-OX transgenic rice plants are hypersensitive to drought stress. Our results suggest that these genes may be involved in different cellular processes.

Biosynthesis of DDMP saponins in soybean is regulated by a distinct UDP-glycosyltransferase.

2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins are one of the major saponin groups that are widely distributed in legumes such as pea, barrel medic, chickpea, and soybean. The steps involved in DDMP saponin biosynthesis remain uncharacterized at the molecular level. We isolated two recessive mutants that lack DDMP saponins from an ethyl methanesulfonate-induced mutant population of soybean cultivar Pungsannamul. Segregation analysis showed that the production of DDMP saponins is controlled by a single locus, named Sg-9. The locus was physically mapped to a 130-kb region on chromosome 16. Nucleotide sequence analysis of candidate genes in the region revealed that each mutant has a single-nucleotide polymorphism in the Glyma.16G033700 encoding a UDP-glycosyltransferase UGT73B4. Enzyme assays and mass spectrum-coupled chromatographic analysis reveal that the Sg-9 protein has glycosyltransferase activity, converting sapogenins and group B saponins to glycosylated products, and that mutant proteins had only partial activities. The tissue-specific expression profile of Sg-9 matches the accumulation pattern of DDMP saponins. This is the first report on a new gene and its function in the biosynthesis of DDMP saponins. Our findings indicate that Sg-9 encodes a putative DDMP transferase that plays a critical role in the biosynthesis of DDMP saponins.

Grain width 2 (GW2) and its interacting proteins regulate seed development in rice (Oryza sativa L.).

BACKGROUND: Seed size has been extensively studied in crop plants, as it determines crop yield. However, the mechanism of seed development remains elusive. In this study, we explored the mechanism of seed development in rice (Oryza sativa L.), and identified proteins affecting seed size. RESULTS: Proteomic analysis showed that glyceraldehyde 3-phosphate dehydrogenase, chitinase 14 (CHT14), and phosphoglycerate kinase (PGK) accumulated to high levels in the seeds of the natural japonica rice mutant Oochikara, which carries a loss-of-function mutation in the grain width 2 (GW2) gene; GW2 encodes a RING-type E3 ubiquitin ligase. In vitro pull-down and ubiquitination assays showed that CHT14 and PGK directly interacted with GW2 but were not ubiquitinated by GW2. Immunoblot analysis revealed that protein disulfide isomerase-like 1-1 accumulated to high levels in young developing seeds of the gw2 mutant compared with the wild type. Histochemical ß-glucuronidase staining showed strong expression of GW2 in leaf and root tissues but weak expression in leaf sheaths and internodes. In addition, transformation of the green fluorescent protein (GFP) gene under the control of the GW2 promoter in rice revealed GFP expression in the aleurone layer of seeds. CONCLUSIONS: Collectively, these results suggest that GW2 regulates seed size through direct interactions with proteins involved in carbohydrate metabolism by modulating their activity or stability and controlling disulfide bond formation in various proteins during seed development. Additionally, GW2 participates in vegetative as well as reproductive growth, and protects the seed from pathogen attack.

GW2 Functions as an E3 Ubiquitin Ligase for Rice Expansin-Like 1.

Seed size is one of the most important traits determining the yield of cereal crops. Many studies have been performed to uncover the mechanism of seed development. However, much remains to be understood, especially at the molecular level, although several genes involved in seed size have been identified. Here, we show that rice Grain Width 2 (GW2), a RING-type E3 ubiquitin ligase, can control seed development by catalyzing the ubiquitination of expansin-like 1 (EXPLA1), a cell wall-loosening protein that increases cell growth. Microscopic examination revealed that a GW2 mutant had a chalky endosperm due to the presence of loosely packed, spherical starch granules, although the grain shape was normal. Yeast two-hybrid and in vitro pull-down assays showed a strong interaction between GW2 and EXPLA1. In vitro ubiquitination analysis demonstrated that EXPLA1 was ubiquitinated by GW2 at lysine 279 (K279). GW2 and EXPLA1 colocalized to the nucleus when expressed simultaneously. These results suggest that GW2 negatively regulates seed size by targeting EXPLA1 for degradation through its E3 ubiquitin ligase activity.

Nitrate Reductases Are Relocalized to the Nucleus by AtSIZ1 and Their Levels Are Negatively Regulated by COP1 and Ammonium.

Nitrate reductases (NRs) catalyze the first step in the reduction of nitrate to ammonium. NR activity is regulated by sumoylation through the E3 ligase activity of AtSIZ1. However, it is not clear how NRs interact with AtSIZ1 in the cell, or how nitrogen sources affect NR levels and their cellular localization. Here, we show that the subcellular localization of NRs is modulated by the E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 and that NR protein levels are regulated by nitrogen sources. Transient expression analysis of GFP fusion proteins in onion epidermal cells showed that the NRs NIA1 and NIA2 localize to the cytoplasmic membrane, and that AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately, whereas NRs and AtSIZ1 localize to the nucleus when co-expressed. Nitrate did not affect the subcellular localization of the NRs, but it caused AtSIZ1 to move from the nucleus to the cytoplasm. NRs were not detected in ammonium-treated cells, whereas the localization of AtSIZ1 was not altered by ammonium treatment. NR protein levels increased in response to nitrate but decreased in response to ammonium. In addition, NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants. NR protein degradation occurred later in cop1-4 than in the wild-type, although the NR proteins did not interact with COP1. Therefore, AtSIZ1 controls nuclear localization of NR proteins, and ammonium negatively regulates their levels. The function and stability of NR proteins might be post-translationally modulated by ubiquitination.

Molecular elucidation of a new allelic variation at the Sg-5 gene associated with the absence of group A saponins in wild soybean.

In soybean, triterpenoid saponin is one of the major secondary metabolites and is further classified into group A and DDMP saponins. Although they have known health benefits for humans and animals, acetylation of group A saponins causes bitterness and gives an astringent taste to soy products. Therefore, several studies are being conducted to eliminate acetylated group A saponins. Previous studies have isolated and characterized the Sg-5 (Glyma.15g243300) gene, which encodes the cytochrome P450 72A69 enzyme and is responsible for soyasapogenol A biosynthesis. In this study, we elucidated the molecular identity of a novel mutant of Glycine soja, 'CWS5095'. Phenotypic analysis using TLC and LC-PDA/MS/MS showed that the mutant 'CWS5095' did not produce any group A saponins. Segregation analysis showed that the absence of group A saponins is controlled by a single recessive allele. The locus was mapped on chromosome 15 (4.3 Mb) between Affx-89193969 and Affx-89134397 where the previously identified Glyma.15g243300 gene is positioned. Sequence analysis of the coding region for the Glyma.15g243300 gene revealed the presence of four SNPs in 'CWS5095' compared to the control lines. One of these four SNPs (G1127A) leads to the amino acid change Arg376Lys in the EXXR motif, which is invariably conserved among the CYP450 superfamily proteins. Co-segregation analysis showed that the missense mutation (Arg376Lys) was tightly linked with the absence of group A saponins in 'CWS5095'. Even though Arg and Lys have similar chemical features, the 3D modelled protein structure indicates that the replacement of Arg with Lys may cause a loss-of-function of the Sg-5 protein by inhibiting the stable binding of a heme cofactor to the CYP72A69 apoenzyme.

In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts.

Nitrate reductase (NR) reduces the major plant nitrogen source, NO3-, into NO2-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude extracts. This protocol offers simple procedure and data analysis to compare NR activity of multiple samples.

Post-translational modifications of Arabidopsis E3 SUMO ligase AtSIZ1 are controlled by environmental conditions.

Sumoylation regulates numerous cellular functions in plants as well as in other eukaryotic systems. However, the regulatory mechanisms controlling E3 small ubiquitin-related modifier (SUMO) ligase are not well understood. Here, post-translational modification of the Arabidopsis E3 SUMO ligase AtSIZ1 was shown to be specifically controlled by abiotic stresses. AtSIZ1 ubiquitination was induced by exposure to heat stress in transgenic plants overexpressing the E3 ubiquitin ligase COP1. In addition, AtSIZ1 ubiquitination was strongly enhanced in transgenic plants overexpressing SUMO isopeptidase ESD4 under heat stress. By contrast, drought stress induced sumoylation rather than ubiquitination of AtSIZ1 and sumoylated forms of AtSIZ1 accumulated in esd4 and cop1-4 mutants. Moreover, siz1 mutants were found to be tolerant to heat and drought stresses. Taken together, these results indicate that ubiquitination and sumoylation of AtSIZ1 in response to abiotic stresses depend on the activities of COP1 and ESD4 and that the activity and stability of AtSIZ1 can be specifically controlled by different abiotic stresses.