Heterologous expression of OsSIZ1, a rice SUMO E3 ligase,enhances broad abiotic stress tolerance in transgenic creeping bentgrass

Sumoylation is a posttranslational regulatory process in higher eukaryotes modifying substrate proteins through conjugation of small ubiquitin‐related modifiers (SUMOs). Sumoylation modulates protein stability, subcellular localization and activity; thus, it regulates most cellular functions including response to environmental stress in plants. To study the feasibility of manipulating SUMO E3 ligase, one of the important components in the sumoylation pathway in transgenic (TG) crop plants for improving overall plant performance under adverse environmental conditions, we have analysed TG creeping bentgrass (Agrostis stolonifera L.) plants constitutively expressing OsSIZ1, a rice SUMO E3 ligase. Overexpression of OsSIZ1 led to increased photosynthesis and overall plant growth. When subjected to water deficiency and heat stress, OsSIZ1 plants exhibited drastically enhanced performance associated with more robust root growth, higher water retention and cell membrane integrity than wild‐type (WT) controls. OsSIZ1 plants also displayed significantly better growth than WT controls under phosphate‐starvation conditions, which was associated with a higher uptake of phosphate (Pi) and other minerals, such as potassium and zinc. Further analysis revealed that overexpression of OsSIZ1 enhanced stress‐induced SUMO conjugation to substrate in TG plants, which was associated with modified expression of stress‐related genes. This strongly supports a role sumoylation plays in regulating multiple molecular pathways involved in plant stress response, establishing a direct link between sumoylation and plant response to environmental adversities. Our results demonstrate the great potential of genetic manipulation of sumoylation process in TG crop species for improved resistance to broad abiotic stresses.     

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.     

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.     

Overexpression of CpSIZ1, a SIZ/PIAS-Type SUMO E3 Ligase from Wintersweet (Chimonanthus praecox), Delays Flowering,Accelerates Leaf Senescence and Enhances Cold Tolerance in Arabidopsis

Wintersweet (Chimonanthus praecox L.) is a traditional winter-flowering plant in China and a popular cut flower in winter. Its unique flowering characteristics under cold stress may involve the regulation of a large number of proteins. Protein post-translational modification is an important regulating type for the gene function. However, little is known about the post-translational modification in wintersweet. SUMOylation is an important post-translational modification in eukaryotes. Small ubiquitin-like modifier (SUMO) E3 ligases perform multiple functional regulatory activities in plants via SUMOylation. Here, we cloned and identified a SIZ/PIAS-type SUMO E3 ligase, CpSIZ1, from wintersweet. CpSIZ1 shared high similarity with other SIZ1 proteins. Quantitative real-time PCR (qRT-PCR) indicated that CpSIZ1 was expressed in all tissues tested, with the highest expression in flower wither period of stage 6, and followed by mature leaves except for different flower development stages. The ectopic expression of CpSIZ1 in Arabidopsis, including the CpSIZ1 overexpression in siz1-2 mutant (HB line) and CpSIZ1 overexpression in WT (OE line), not only promoted vegetative growth, delayed flowering and accelerated leaf senescence, but also improve the cold tolerance in Arabidopsis. Therefore, our studies have enrich the understanding of function of SIZ1 gene in woody plant, and provide a good foundation for further research on the post-translational modification regulation mechanism in this winter-flowering plant.

     

Overexpression of Tomato SIZ2 in Arabidopsis Improves Plant Salinity Tolerance

Small ubiquitin-like modifier (SUMO) conjugation to target proteins is an important post-translational modification, which regulates plant tolerance to biotic and abiotic stresses. SIZ1, a well-characterized SUMO E3 ligase, facilitates the conjugation of SUMO to target proteins. Here, a SIZ/PAIS-type protein SlSIZ2 was identified in tomato (Solanum lycopersicum) that is a homolog of AtSIZ1 and SlSIZ1. SlSIZ2 was expressed in tomato vegetative and reproductive tissues, and induced by ABA and NaCl. Nucleus-localized SlSIZ2 partially rescued atsiz1-2 dwarfism and also alleviated the sensitivity of atsiz1-2 to ABA and NaCl, suggesting the functional replacement of SlSIZ2 to AtSIZ1. Moreover, SlSIZ2-overexpressing Arabidopsis has higher cotyledon expansion rate, lateral root density and survival rate under salinity stress. These results suggested the contribution of SlSIZ2 to the tolerance of salinity stress.

     

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.     

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.     

低温潜沼性逆境下不同耐受能力水稻根系动态变化

水稻根系生长分布与地上部生长及产量形成密切相关,定量研究低温潜沼性逆境下水稻根系分布特征对改进稻田管理方式及耐潜品种的选育等具有重要意义。以4个耐潜等级的8个水稻品种为实验材料,在水稻栽培环境模拟系统中连续栽植3年(2012—2014年),研究水稻根系在低温潜沼性逆境下的动态变化规律和分布规律,明确低温潜沼性逆境下水稻根系变化与产量构成的关系。结果表明:1)低温潜沼性逆境下水稻根系长度和数量的M值(绝对变化率)均大于0,P值(相对变化率)均小于0。表明,低温潜沼性逆境下水稻根系仍有一定增长,但与非低温潜沼性逆境相比,根系长度和数量均显著降低,降低程度因品种耐潜等级的高低而不同,耐潜指数高的品种(泸优5号和C优130)降低程度显著低于耐潜指数低的品种(川谷优204和协优027)。2)低温潜沼性逆境对0—10 cm土层根系的影响在5月底6月初最大;对10—30 cm土层根系的影响在6月底最大;对30—50 cm土层根系的影响在6月30日前后最大。3)低温潜沼性逆境下,水稻孕穗-抽穗期根系长度和数量与穗粒数、实粒数、结实率、产量之间呈显著正相关;与单株有效穗数和千粒重呈显著负相关,根系长度和数量的M值与P值,尤其是白根长度和数量的M值与P值可作为一个水稻耐潜能力评价指标进行考虑。4)低温潜沼性逆境下,不同土层水稻根系与产量构成因素间的相关性不同。0—10 cm土层根系长度和数量与有效分蘖数呈显著负相关;10—20 cm土层白根长度和数量与结实率呈显著正相关;30—50 cm土层根系长度和数量与着粒数呈显著正相关。     

SUMOylation represses SnRK1 signaling in Arabidopsis

The SnRK1 protein kinase balances cellular energy levels in accordance with extracellular conditions and is thereby key for plant stress tolerance. In addition, SnRK1 has been implicated in numerous growth and developmental processes from seed filling and maturation to flowering and senescence. Despite its importance, the mechanisms that regulate SnRK1 activity are poorly understood. Here, we demonstrate that the SnRK1 complex is SUMOylated on multiple subunits and identify SIZ1 as the E3 Small Ubiquitin‐like Modifier (SUMO) ligase responsible for this modification. We further show that SnRK1 is ubiquitinated in a SIZ1‐dependent manner, causing its degradation through the proteasome. In consequence, SnRK1 degradation is deficient in siz1‐2 mutants, leading to its accumulation and hyperactivation of SnRK1 signaling. Finally, SnRK1 degradation is strictly dependent on its activity, as inactive SnRK1 variants are aberrantly stable but recover normal degradation when expressed as SUMO mimetics. Altogether, our data suggest that active SnRK1 triggers its own SUMOylation and degradation, establishing a negative feedback loop that attenuates SnRK1 signaling and prevents detrimental hyperactivation of stress responses.     

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