Effect of Cu content on the activity of Cu/ZnSOD1 in the Arabidopsis SUMO E3 ligase siz1 mutant

In a previous study, we found copper (Cu) accumulated to a higher level in the aerial parts of soil-grown plants of the SUMO E3 ligase siz1 mutant than in those of the wild-type. Here, we found that all superoxide dismutase (SOD) isoforms, such as FeSOD, MnSOD and different types of Cu/ZnSOD, were more active in the siz1 mutant than in the wild type under normal growth conditions. We further examined the expression and enzymatic activity of Cu/ZnSOD1 (CSD1) in shoots of the siz1 mutant under excess Cu. Shoot CSD1 protein level and activity were reduced in siz1 with excess Cu but induced in the wild type. SIZ1-dependent SUMOylation may be involved in maintaining CSD1 protein stability or repelling a feedback regulation under Cu stress.Key words: Cu/Zn SOD, CSD1, SUMO E3 ligase, SIZ1, Cu stress     

Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds

Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. Beta-glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues.     

Salicylic acid-mediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase

Reversible modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins are involved in many cellular processes in yeast and animals. Yet little is known about the function of sumoylation in plants. Here, we show that the SIZ1 gene, which encodes an Arabidopsis SUMO E3 ligase, regulates innate immunity. Mutant siz1 plants exhibit constitutive systemic-acquired resistance (SAR) characterized by elevated accumulation of salicylic acid (SA), increased expression of pathogenesis-related (PR) genes, and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Transfer of the NahG gene to siz1 plants results in reversal of these phenotypes back to wild-type. Analyses of the double mutants, npr1 siz1, pad4 siz1 and ndr1 siz1 revealed that SIZ1 controls SA signalling. SIZ1 interacts epistatically with PAD4 to regulate PR expression and disease resistance. Consistent with these observations, siz1 plants exhibited enhanced resistance to Pst DC3000 expressing avrRps4, a bacterial avirulence determinant that responds to the EDS1/PAD4-dependent TIR-NBS-type R gene. In contrast, siz1 plants were not resistant to Pst DC3000 expressing avrRpm1, a bacterial avirulence determinant that responds to the NDR1-dependent CC-NBS-type R gene. Jasmonic acid (JA)-induced PDF1.2 expression and susceptibility to Botrytis cinerea were unaltered in siz1 plants. Taken together, these results demonstrate that SIZ1 is required for SA and PAD4-mediated R gene signalling, which in turn confers innate immunity in Arabidopsis.     

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.     

Alternative Functions of Arabidopsis YELLOW STRIPE-LIKE3: From Metal Translocation to Pathogen Defense

YELLOW STRIPE-LIKE1 (YSL1) and YSL3 are involved in iron (Fe) and copper (Cu) translocation. Previously, we reported that upregulation of YSL1 and YSL3 under excess Cu caused high accumulation of Cu in the siz1 mutant, impaired in small ubiquitin-like modifier (SUMO) E3 ligase. Interestingly, the siz1 mutant contains high levels of salicylic acid (SA), involved in plant defense against biotrophic pathogens. In this study, we found that YSL1 and YSL3 were upregulated by SA. SA-regulated YSL3 but not YSL1 depended on NONEXPRESSOR OF PR1 (NPR1). Susceptibility to the pathogen Pseudomonas syringe pv. tomato (Pst) DC3000 was greater for ysl3 than the wild type. Also, during Pst DC3000 infection, YSL3 was positively regulated by SA signaling through NPR1 and the upregulation was enhanced in the coi1 mutant that defective in the jasmonic acid (JA) receptor, CORONATINE INSENSITIVE1. This line of evidence indicates that the regulation of YSL3 is downstream of SA signaling and interplays with JA signaling for involvement in pathogen-induced defense. We provide new insights into the biological function of the metal transporter YSL3 in plant pathogen defense.     

Rice SIZ1, a SUMO E3 ligase, controls spikelet fertility through regulation of anther dehiscence

? Sumoylation, a post-translational modification, has important functions in both animals and plants. However, the biological function of the SUMO E3 ligase, SIZ1, in rice (Oryza sativa) is still under investigation. ? In this study, we employed two different genetic approaches, the use of siz1 T-DNA mutant and SIZ1-RNAi transgenic plants, to characterize the function of rice SIZ1. ? Genetic results revealed the co-segregation of single T-DNA insertional recessive mutation with the observed phenotypes in siz1. In addition to showing reduced plant height, tiller number and seed set percentage, both the siz1 mutant and SIZ1-RNAi transgenic plants showed obvious defects in anther dehiscence, but not pollen viability. The anther indehiscence in siz1 was probably a result of defects in endothecium development before anthesis. Interestingly, rice orthologs of AtIRX and ZmMADS2, which are essential for endothecium development during anther dehiscence, were significantly down-regulated in siz1. Compared with the wild-type, the sumoylation profile of high-molecular-weight proteins in mature spikelets was reduced significantly in siz1 and the SIZ1-RNAi line with notably reduced SIZ1 expression. The nuclear localization signal located in the SIZ1 C-terminus was sufficient for its nuclear targeting in bombarded onion epidermis. ? The results suggest the functional role of SIZ1, a SUMO E3 ligase, in regulating rice anther dehiscence.     

A loss-of-function mutation in AtYSL1 reveals its role in iron and nicotianamine seed loading

The Arabidopsis Yellow Stripe 1-Like (YSL) proteins have been identified by homology with the maize (Zea mays) Yellow Stripe 1 (YS1) transporter which is responsible for iron-phytosiderophore (PS) uptake by roots in response to iron shortage. Although dicotyledonous plants do not synthesize PS, they do synthesize the PS precursor nicotianamine, a strong metal chelator essential for maintenance of iron homeostasis and copper translocation. Furthermore, ZmYS1 and the rice (Oryza sativa) protein OsYSL2 have metal-nicotianamine transport activities in heterologous expression systems. In this work, we have characterized the function of AtYSL1 in planta. Two insertional loss-of-function ysl1 mutants of Arabidopsis were found to exhibit increased nicotianamine accumulation in shoots. More importantly, seeds of both ysl1 knockouts contained less iron and nicotianamine than wild-type seeds, even when produced by plants grown in the presence of an excess of iron. This phenotype could be reverted by expressing the wild-type AtYSL1 gene in ysl1 plants. ysl1 seeds germinated slowly, but this defect was rescued by an iron supply. AtYSL1 was expressed in the xylem parenchyma of leaves, where it was upregulated in response to iron excess, as well as in pollen and in young silique parts. This pattern is consistent with long-distance circulation of iron and nicotianamine and their delivery to the seed. Taken together, our work provides strong physiological evidence that iron and nicotianamine levels in seeds rely in part on AtYSL1 function.     

A novel tomato SUMO E3 ligase,SlSIZ1, confers drought tolerance in transgenic tobacco

SUMOylation is an important post‐translational modification process that regulates different cellular functions in eukaryotes. SIZ/PIAS‐type SAP and Miz1 (SIZ1) proteins exhibit SUMO E3 ligase activity, which modulates SUMOylation. However, SIZ1 in tomato has been rarely investigated. In this study, a tomato SIZ1 gene (SlSIZ1) was isolated and its molecular characteristics and role in tolerance to drought stress are described. SlSIZ1 was up‐regulated by cold, sodium chloride (NaCl), polyethylene glycol (PEG), hydrogen peroxide (H₂O₂) and abscisic acid (ABA), and the corresponding proteins were localized in the nucleus. The expression of SlSIZ1 in Arabidopsis thaliana (Arabidopsis) siz1‐2 mutants partially complemented the phenotypes of dwarf, cold sensitivity and ABA hypersensitivity. SlSIZ1 also exhibited the activity of SUMO E3 ligase to promote the accumulation of SUMO conjugates. Under drought stress, the ectopic expression of SlSIZ1 in transgenic tobacco lines enhanced seed germination and reduced the accumulation of reactive oxygen species. SlSIZ1 overexpression conferred the plants with improved growth, high free proline content, minimal malondialdehyde accumulation and increased accumulation of SUMO conjugates. SlSIZ1 is a functional homolog of Arabidopsis SIZ1 with SUMO E3 ligase activity. Therefore, overexpression of SlSIZ1 enhanced the tolerance of transgenic tobacco to drought stress.     

Whole-plant mineral partitioning throughout the life cycle in Arabidopsis thaliana ecotypes Columbia, Landsberg erecta, Cape Verde Islands, and the mutant line ysl1ysl3

Minimal information exists on whole-plant dynamics of mineral flow through Arabidopsis thaliana or on the source tissues responsible for mineral export to developing seeds. Understanding these phenomena in a model plant could help in the development of nutritionally enhanced crop cultivars. A whole-plant partitioning study, using sequential harvests, was conducted to characterize growth and mineral concentrations and contents of rosettes, cauline leaves, stems, immature fruit, mature fruit hulls, and seeds of three WT lines (Col-0, Ler, and Cvi) and one mutant line (Col-0::ysl1ysl3). Shoot mineral content increased throughout the life cycle for all minerals, although tissue-specific mineral partitioning differed between genotypes. In particular, iron (Fe), zinc (Zn), and copper (Cu) were aberrantly distributed in ysl1ysl3. Remobilization was observed for several minerals from various tissues, including cauline leaves and silique hulls, but the amounts were generally far below the total mineral accretion observed in seeds. When YSL1 and YSL3 are nonfunctional, Cu, Fe, and Zn are not effectively remobilized from, or do not effectively pass through, leaf and maternal fruit tissues. With respect to seed mineral accretion in Arabidopsis, continued uptake and translocation of minerals to source tissues during seed fill are as important, if not more important, than remobilization of previously stored minerals.     

An Arabidopsis SUMO E3 Ligase,SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.     

SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean

SIZ1 is a small ubiquitin‐related modifier (SUMO) E3 ligase that mediates post‐translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1‐2, including dwarfism, constitutively activated expression of pathogen‐related genes, and ABA‐sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)‐mediated gene silencing decreased heat shock‐induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1‐2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean.     

Mutation in SUMO E3 ligase, SIZ1, disrupts the mature female gametophyte in Arabidopsis

Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance.     

OTS1‐dependent deSUMOylation increases tolerance to high copper levels in Arabidopsis

The conjugation of SUMO(small ubiquitin-like modifier) to protein substrates is a reversible process(SUMOylation/deSUMOylation) that regulates plant development and stress responses. The essential metal copper(Cu) is required for normal plant growth, but excess amounts are toxic. The SUMO E_3 ligase, SIZ_1, and SIZ_1-mediated SUMOylation function in plant tolerance to excess Cu. It is unknown whether deSUMOylation also contributes to Cu tolerance in plants. Here, we report that OTS_1, a protease that cleaves SUMO from its substrate proteins,participates in Cu tolerance in Arabidopsis thaliana(Arabidopsis). OTS_1 loss-of-function mutants(ots_(1-2) and ots_(1-3))displayed increased sensitivity to excess Cu. Redox homeostasis and the balance between SUMOylation and deSUMOylation were disrupted in the ots_(1-3) mutant under excess Cu conditions. The ots_(1-3) mutant accumulated higher levels of Cu in both shoots and roots compared to wild type.Specific Cu-related metal transporter genes were upregulated due to the loss-of-function of OTS_1, which might explain the high Cu levels in ots_(1-3). These results suggest that the SUMOylation/deSUMOylation machinery is activated in response to excess Cu, and modulates Cu homeostasis and tolerance by regulating both Cu uptake and detoxification. Together, our findings provide insight into the biological function and regulatory role of SUMOylation/deSUMOylation in plant tolerance to Cu.     

Misregulation of 2 microm circle copy number in a SUMO pathway mutant

Attachment of the ubiquitin-like protein SUMO to other proteins is an essential process in Saccharomyces cerevisiae. However, yeast mutants lacking the SUMO ligases Siz1 and Siz2/Nfi1 are viable, even though they show dramatically reduced levels of SUMO conjugation. This siz1Delta siz2Delta double mutant is cold sensitive and has an unusual phenotype in that it forms irregularly shaped colonies that contain sectors of wild-type-appearing cells as well as sectors of enlarged cells that are arrested in G(2)/M. We have found that these phenotypes result from misregulation of the copy number of the endogenous yeast plasmid, the 2 microm circle. siz1Delta siz2Delta mutants have up to 40-fold-higher levels of 2 microm than do wild-type strains. Furthermore, 2 microm is responsible for the siz1Delta siz2Delta mutant's obvious growth defects, as siz1Delta siz2Delta [cir(0)] strains, which lack 2 microm, are no longer heterogeneous and show growth characteristics similar to those of the wild type. Possible mechanisms for SUMO's effect on 2 microm are suggested by the finding that both Flp1 recombinase and Rep2, two of the four proteins encoded by 2 microm, are covalently modified by SUMO. Our data suggest that SUMO attachment negatively regulates Flp1 levels, which may partially account for the increased 2 microm copy number in the siz1Delta siz2Delta strain.