The conjugation of SUMO to the transcription factor MYC2 functions in blue light-mediated seedling development in Arabidopsis.

A key function of photoreceptor signaling is the coordinated regulation of a large number of genes to optimize plant growth and development. The basic helix loop helix (bHLH) transcription factor MYC2 is crucial for regulating gene expression in Arabidopsis thaliana during development in blue light. Here we demonstrate that blue light induces the SUMOylation of MYC2. Non-SUMOylatable MYC2 is less effective in suppressing blue light-mediated photomorphogenesis than wild-type (WT) MYC2. MYC2 interacts physically with the SUMO proteases SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2. Blue light exposure promotes the degradation of SPF1 and SPF2 and enhances the SUMOylation of MYC2. Phenotypic analysis revealed that SPF1/SPF2 function redundantly as positive regulators of blue light-mediated photomorphogenesis. Our data demonstrate that SUMO conjugation does not affect the dimerization of MYC transcription factors but modulates the interaction of MYC2 with its cognate DNA cis-element and with the ubiquitin ligase Plant U-box 10 (PUB10). Finally, we show that non-SUMOylatable MYC2 is less stable and interacts more strongly with PUB10 than the WT. Taken together, we conclude that SUMO functions as a counterpoint to the ubiquitin-mediated degradation of MYC2, thereby enhancing its function in blue light signaling.

The converging path of protein SUMOylation in phytohormone signalling: highlights and new frontiers.

KEY MESSAGE: The intersection of phytohormone signalling pathways with SUMOylation, a key post-translational modification, offers an additional layer of control to the phytohormone signalling for sophisticated regulation of plant development. Plants live in a constantly changing environment that are often challenging for the growth and development of plants. Phytohormones play a critical role in modulating molecular-level changes for enabling plants to resist climatic aberrations. The orchestration of such effective molecular responses entails rapid regulation of phytohormone signalling at transcriptional, translational and post-translational levels. Post-translational modifications have emerged as a key player in modulating hormonal pathways. The current review lays emphasis on the role of SUMOylation, a key post-translational modification, in manipulating individual hormone signalling pathways for better plant adaptability. Here, we discuss the recent advancement in the field and highlights how SUMO targets key signalling intermediates including transcription factors to provide a quick response to different biotic or abiotic stresses, sometimes even prior to changes in hormone levels. The understanding of the convergence of SUMOylation and hormonal pathways will offer an additional layer of control to the phytohormone signalling for an intricate and sophisticated regulation of plant development and can be utilised as a tool to generate climate-resilient crops.

Towards understanding the multifaceted role of SUMOylation in plant growth and development.

Post-translational modifications (PTMs) play a critical role in regulating plant growth and development through the modulation of protein functionality and its interaction with its partners. Analysis of the functional implication of PTMs on plant cellular signalling presents grand challenges in understanding their significance. Proteins decorated or modified with another chemical group or polypeptide play a significant role in regulating physiological processes as compared with non-decorated or non-modified proteins. In the past decade, SUMOylation has been emerging as a potent PTM influencing the adaptability of plants to growth, in response to various environmental cues. Deciphering the SUMO-mediated regulation of plant stress responses and its consequences is required to understand the mechanism underneath. Here, we will discuss the recent advances in the role and significance of SUMOylation in plant growth, development and stress response.

SUMO Suppresses the Activity of the Jasmonic Acid Receptor CORONATINE INSENSITIVE1.

Plants respond rapidly to sudden environmental cues, often responding prior to changes in the hormone levels that coordinate these responses. How this is achieved is not fully understood. The integrative role of the phytohormone jasmonic acid (JA) relies upon the plant's ability to control the levels of JASMONATE ZIM (JAZ) domain-containing repressor proteins. Here, we demonstrate that regardless of intrinsic JA levels, Small Ubiquitin-like Modifier (SUMO)-conjugated JAZ proteins inhibit the JA receptor CORONATINE INSENSITIVE1 (COI1) from mediating non-SUMOylated JAZ degradation. The SUMO-deconjugating proteases OVERLY TOLERANT TO SALT1 (OTS1) and OTS2 regulate JAZ protein SUMOylation and stability. The ots1 ots2 double mutants accumulate SUMOylated and non-SUMOylated JAZ repressor proteins but show no change in endogenous JA levels compared with wild-type plants. SUMO1-conjugated JAZ proteins bind to COI1 independently of the JA mimic coronatine. SUMO inhibits JAZ binding to COI1. We identify the SUMO interacting motif in COI1 and demonstrate that this is vital to SUMO-dependent inhibition of COI1. Necrotroph infection of Arabidopsis thaliana promotes SUMO protease degradation, and this increases JAZ SUMOylation and abundance, which in turn inhibits JA signaling. This study reveals a mechanism for rapidly regulating JA responses, allowing plants to adapt to environmental changes.

Exploiting protein modification systems to boost crop productivity: SUMO proteases in focus.

In recent years, post-translational modification (PTM) of proteins has emerged as a key process that integrates plant growth and response to a changing environment. During the processes of domestication and breeding, plants were selected for various yield and adaptational characteristics. The post-translational modifier small ubiquitin-like modifier (SUMO) protein is known to have a role in the regulation of a number of these characteristics. Using bioinformatics, we mined the genomes of cereal and Brassica crops and their non-crop relatives Arabidopsis thaliana and Brachypodium distachyon for ubiquitin-like protease (ULP) SUMO protease sequences. We discovered that the SUMO system in cereal crops is disproportionately elaborate in comparison with that in B. distachyon. We use these data to propose deSUMOylation as a mechanism for specificity in the SUMO system.

Rice SUMO protease Overly Tolerant to Salt 1 targets the transcription factor, OsbZIP23 to promote drought tolerance in rice.

Conjugation of SUMO (Small Ubiquitin-like Modifier) protein to cellular targets is emerging as a very influential protein modification system. Once covalently bound, SUMO conjugation can change the stability or functionality of its cognate target proteins. SUMO protease can rapidly reverse SUMO conjugation making this modification system highly dynamic. A major factor in the variation of SUMO-target function is the balance between the conjugated/de-conjugated forms. The mechanistic role of these regulatory SUMO proteases in mediating stress responses has not been defined in any crops. In this study, we reveal the role of the SUMO protease, OsOTS1 in mediating tolerance to drought in rice. OsOTS1 depleted transgenic plants accumulate more ABA and exhibit more productive agronomic traits during drought while OsOTS1 overexpressing lines are drought sensitive but ABA insensitive. Drought and ABA treatment stimulates the degradation of OsOTS1 protein indicating that SUMO conjugation is an important response to drought stress in rice achieved through down-regulation of OTS1/2 activity. We reveal that OsOTS1 SUMO protease directly targets the ABA and drought responsive transcription factor OsbZIP23 for de-SUMOylation affecting its stability. OsOTS-RNAi lines show increased abundance of OsbZIP23 and increased drought responsive gene expression while OsOTS1 overexpressing lines show reduced levels of OsbZIP23 leading to suppressed drought responsive gene expression. Our data reveal a mechanism in which rice plants govern ABA-dependant drought responsive gene expression by controlling the stability of OsbZIP23 by SUMO conjugation through manipulating specific SUMO protease levels.

Rice OVERLY TOLERANT TO SALT 1 (OTS1) SUMO protease is a positive regulator of seed germination and root development.

Salinity is one of the major environmental stresses affecting rice production worldwide. Improving rice salt tolerance is a critical step for sustainable food production. Posttranslational modifications of proteins greatly expand proteome diversity, increase functionality and allow quick responses to environmental stresses, all at low cost to the cell. SUMO mediated modification of substrate proteins is a highly dynamic process governed by the balance of activities of SUMO E3 ligases and deconjugating SUMO proteases. In recent years, SUMO (Small Ubiquitin like Modifier) conjugation of proteins has emerged as an influential regulator of stress signaling in the model plant Arabidopsis. However SUMOylation remain largely under studied in crop plants. We recently identified the SUMO protease gene family in rice and demonstrated a role for OsOTS1 SUMO proteases in salt stress. Interestingly, rice plants silencing OsOTS1 also show significantly reduced germination rate. Knockdown of OsOTS1 gene expression affects root growth by primarily reducing cell size rather than cell division.

SUMO proteases: uncovering the roles of deSUMOylation in plants.

Plants have evolved to cope with changing environmental conditions. One way plants achieve this is through post-translational modification of target proteins by ubiquitination and SUMOylation. These post-translational modifiers (PMs) can alter stability, protein-protein interactions, and the overall fate of the protein. Both of these systems have remarkable similarities in terms of the process leading to attachment of the PM to its substrate : having to undertake activation, conjugation, and finally ligation to the target. In the ubiquitin system, there are a vast number of ubiquitin ligase enzymes (E3s) that provide specificity for the attachment of ubiquitin. With the SUMO system, only a small number of SUMO E3 ligases have so far been identified in the fully sequenced plant genomes. In Arabidopsis thaliana, there are only two SUMO E3s, compared to over 1400 ubiquitin E3s, a trend also observed in crop species such as Oryza sativa and Zea mays Recent research indicates that removing SUMO from its substrate by the enzymatically active SUMO proteases is a vital part of this system. A class of SUMO proteases called ubiquitin-like proteases (ULPs) are widespread in all eukaryotes; within plants, both monocot and dicot kingdoms have conserved and divergent ULPs and ULP-like proteases. This paper examines the roles ULPs have in stress responses and highlights the 'fine-tuning' of SUMO attachment/removal in balancing growth versus stress.

SUMO Is a Critical Regulator of Salt Stress Responses in Rice.

SUMO (Small Ubiquitin-like Modifier) conjugation onto target proteins has emerged as a very influential class of protein modification systems. SUMO1/2 double mutant plants are nonviable, underlining the importance of SUMO conjugation to plant survival. Once covalently bound, SUMO can alter a conjugated protein's stability and/or function. SUMO conjugation is a highly dynamic process that can be rapidly reversed by the action of SUMO proteases. The balance between the conjugated/deconjugated forms is a major determinant in the modulation of SUMO-target function. Despite the important mechanistic role of SUMO proteases in model plants, until now the identity or the function of these regulatory enzymes has not been defined in any crop plant. In this report, we reveal the ubiquitin-like protease class of SUMO protease gene family in rice (Oryza sativa) and demonstrate a critical role for OsOTS1 SUMO protease in salt stress. OsOTS-RNAi rice plants accumulate high levels of SUMO-conjugated proteins during salt stress and are highly salt sensitive; however, in non-salt conditions, they are developmentally indistinguishable from wild-type plants. Transgenic rice plants overexpressing OsOTS1 have increased salt tolerance and a concomitant reduction in the levels of SUMOylated proteins. We demonstrate that OsOTS1 confers salt tolerance in rice by increasing root biomass. High salinity triggers OsOTS1 degradation, indicating that increased SUMO conjugation in rice plants during salt stress is in part achieved by down-regulation of OTS1/2 activity. OsOTS1 is nuclear localized indicating a direct requirement of OsOTS1-dependent deSUMOylation activity in rice nuclei for salt tolerance.

Short Hypocotyl in White Light1 Interacts with Elongated Hypocotyl5 (HY5) and Constitutive Photomorphogenic1 (COP1) and Promotes COP1-Mediated Degradation of HY5 during Arabidopsis Seedling Development.

Arabidopsis (Arabidopsis thaliana) Short Hypocotyl in White Light1 (SHW1) encodes a Ser-Arg-Asp-rich protein that acts as a negative regulator of photomorphogenesis. SHW1 and Constitutive Photomorphogenic1 (COP1) genetically interact in an additive manner to suppress photomorphogenesis. Elongated Hypocotyl5 (HY5) is a photomorphogenesis promoting a basic leucine zipper transcription factor that is degraded by COP1 ubiquitin ligase in the darkness. Here, we report the functional interrelation of SHW1 with COP1 and HY5 in Arabidopsis seedling development. The in vitro and in vivo molecular interaction studies show that SHW1 physically interacts with both COP1 and HY5. The genetic studies reveal that SHW1 and HY5 work in an antagonistic manner to regulate photomorphogenic growth. Additional mutation of SHW1 in hy5 mutant background is able to suppress the gravitropic root growth defect of hy5 mutants. This study further reveals that the altered abscisic acid responsiveness of hy5 mutants is modulated by additional loss of SHW1 function. Furthermore, this study shows that SHW1 promotes COP1-mediated degradation of HY5 through enhanced ubiquitylation in the darkness. Collectively, this study highlights a mechanistic view on coordinated regulation of SHW1, COP1, and HY5 in Arabidopsis seedling development.

Destabilization of interaction between cytokinin signaling intermediates AHP1 and ARR4 modulates Arabidopsis development.

Eukaryotic two-component signaling involves the His-Asp-His-Asp multistep phosphorelay (MSP). In Arabidopsis thaliana, cytokinin-mediated MSP signaling intermediates include histidine kinases (HKs), histidine phosphotransfer proteins (Hpts) and response regulators (RRs). The structure-function relationship of interaction between Hpt (e.g. AHP1) and RR (e.g. ARR4) is poorly understood. Using a homology model and yeast two-hybrid analysis, we identified key amino acids of ARR4 at the AHP1-ΔARR4((16-175)) interaction interface. Mutating them in Arabidopsis (arr3,4,5,6,8,9 hextuple mutant background) and performing root length assays provided functional relevance, and coimmunoprecipitation (coIP) assay provided biochemical evidence for the interaction. The homology model mimics crystal structures of Hpt-RR complexes. Mutating selected interface residues of ARR4 either abolished or destabilized the interaction. D45A and Y96A mutations weakened interaction with AHP1, and exhibited weaker rescue of root elongation in the hextuple mutants. CoIP analysis using cytokinin-treated transgenic Arabidopsis seedlings provided biochemical evidence for weakened AHP1-ARR4 interaction. The relevance of the selected residues for the interaction was further validated in two independent pairs of Hpt-RR proteins from Arabidopsis and rice (Oryza sativa). Our data provide evidence of a link between Hpt-RR interaction affinity and regulation of downstream functions of RRs. This establishes a structure-function relationship for the final step of a eukaryotic MSP signal cascade.

Cell cycle stage specific application of municipal landfill leachates to assess the genotoxicity in root meristem cells of barley (Hordeum vulgare).

Municipal solid wastes (MSW) are unavoidable sources of environmental pollution. Improper disposal of municipal waste results in the leaching of toxic metals and organic chemicals, which can contaminate the surface and ground water leading to serious health hazard. In this study, the toxic effects of the leachate prepared from municipal solid waste samples were examined in root meristem cells of barley (Hordeum vulgare L.) at various stages of cell cycle, i.e., G1, S, and G2. Seeds of barley were exposed to 2.5, 5, and 10 % of leachates in soil and aqueous media in 48 h at different cell cycle stages. The physicochemical data of the present study revealed that municipal solid waste leachate contains high amount of heavy metals, which significantly affected growth and physiological activities of barley. Significant inhibition in hypocotyl length, germination, and mitotic index were observed at all concentration of leachate treatment. Induction of chromosomal aberrations (CA's) and micronuclei (MN) formation were also observed with different concentrations of leachate treatment at 7, 17, and 27 h of presoaking durations, which falls in G1, S, and G2 phase of the cell cycle, respectively. Also, exposure of leachate at S phase of the cell cycle had significant effects in barley through chromosomal aberration and micronuclei formation.

Z-box binding transcription factors (ZBFs): a new class of transcription factors in Arabidopsis seedling development.

One set of genes encoding diverse groups of transcription factors that interact with the Z-box (ATACGTGT; a potential Z-DNA forming sequence) is called ZBFs (Z-box Binding Factors). ZBFs include ZBF1, ZBF2, and ZBF3, which encode ZBF1/MYC2 (bHLH), ZBF2/GBF1 (bZIP), and ZBF3/CAM7 (Calmodulin) proteins, respectively. With several recent reports, it is becoming increasingly evident that ZBFs play crucial roles in Arabidopsis seedling photomorphogenesis. ZBFs integrate signals from various wavelengths of light to coordinate the regulation of transcriptional networks that affect multiple facets of plant growth and development. The function of each ZBF is qualitatively and quantitatively distinct. The zbf mutants display pleiotropic effects including altered hypocotyl elongation, cotyledon expansion, lateral root development, and flowering time. In this inaugural review, we discuss the identification, molecular functions, and interacting partners of ZBFs in light-mediated Arabidopsis seedling development.

Exogenous application of salicylic acid to alleviate the toxic effects of insecticides in Vicia faba L.

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting plants from insecticides toxicity. The seeds of Vicia faba var IIVR Selection-1 were treated with different concentrations (1.5, 3.0, and 6.0 ppm) of the insecticides alphamethrin (AM) and endosulfan (ES) for 6 h with and without 12 h conditioning treatment of SA (0.01 mM). Insecticides treatment caused a significant decrease in mitotic index (MI) and induction of different types of chromosomal abnormalities in the meristematic cells of broad bean roots. Pretreatment of seeds with SA resulted in increased MI and significant reduction of chromosomal abnormalities. SA application also regulated proline accumulation and carotenoid content in the leaf tissues. SA resulted in the decrement of insecticides induced increase in proline content and increased the carotenoids content. These results illustrate the ameliorating effect of SA under stress conditions and reveal that SA is more effective in alleviating the toxic effects of insecticides at higher concentrations than that at lower concentrations.

Cell cycle stage specific application of cypermethrin and carbendazim to assess the genotoxicity in somatic cells of Hordeum vulgare L.

In this study, the genotoxicity of commonly used insecticide cypermethrin and fungicide carbendazim were examined on root meristem cells of Hordeum vulgare L. Cypermethrin and carbendazim were applied with concentrations of 0.05%, 0.1% and 0.5% at different presoaking durations of 7 h, 17 h and 27 h which falls into G(1), S and G(2) phases of cell cycle, respectively. The cytogenetic studies of the root meristem cells of Hordeum vulgare L. showed that S phase of the cell cycle was more sensitive in comparison to other phases of cell cycle.