Functional analysis reveals pleiotropic effects of rice RING-H2 finger protein gene OsBIRF1 on regulation of growth and defense responses against abiotic and biotic stresses

RING finger proteins comprise a large family and play key roles in regulating growth/developmental processes, hormone signaling and responses to biotic and abiotic stresses in plants. A rice gene, OsBIRF1, encoding a putative RING-H2 finger protein, was cloned and identified. OsBIRF1 encodes a 396 amino acid protein belonging to the ATL family characterized by a conserved RING-H2 finger domain (C-X2-C-X15-C-X1-H-X2-H-X2-C-X10-C-X2-C), a transmembrane domain at the N-terminal, a basic amino acid rich region and a characteristic GLD region. Expression of OsBIRF1 was up-regulated in rice seedlings after treatment with benzothaidiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid and jasmonic acid, and was induced differentially in incompatible but not compatible interactions between rice and Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants that constitutively express OsBIRF1 exhibit enhanced disease resistance against tobacco mosaic virus and Pseudomonas syringae pv. tabaci and elevated expression levels of defense-related genes, e.g. PR-1, PR-2, PR-3 and PR-5. The OsBIRF1-overexpressing transgenic tobacco plants show increased oxidative stress tolerance to exogenous treatment with methyl viologen and H2O2, and up-regulate expression of oxidative stress-related genes. Reduced ABA sensitivity in root elongation and increased drought tolerance in seed germination were also observed in OsBIRF1 transgenic tobacco plants. Furthermore, the transgenic tobacco plants show longer roots and higher plant heights as compared with the wild-type plants, suggesting that overexpression of OsBIRF1 promote plant growth. These results demonstrate that OsBIRF1 has pleiotropic effects on growth and defense response against multiple abiotic and biotic stresses.     

Expression analysis of RING zinc finger genes from Triticum aestivum and identification of TaRZF70 that contains four RING-H2 domains and differentially responds to water deficit between leaf and root

RING zinc finger proteins are known for their role predominantly in targeted protein degradation and participate in gene regulation through interaction with other regulatory proteins. In this study seven RING zinc finger genes from Triticum aestivum (bread wheat) were analysed for expression profiles in various organs (leaf, root, stem, spike, endosperm and embryo) and during leaf development and aging as well as in their responses to water deficit. Expression levels of six of these seven genes varied markedly among the six organs examined. All seven genes changed their expression levels in the leaf from the growing to senescing stage. Four genes were responsive to water deficit. A RING-H2 zinc finger gene, TaRZF70 showed differential response to water deprivation, namely up-regulation in the leaf and down-regulation in the root. This differential response was also observed in abscisic acid (ABA)-treated plants. Sequence analysis revealed that TaRZF70 contained four RING-H2 domains, the largest number of RING-H2 domains in any RING-H2 zinc finger proteins reported to date. These results indicate that these RING zinc finger genes are involved in diverse physiological processes in wheat, including response to drought.     

Active site residues and amino acid specificity of the ubiquitin carrier protein-binding RING-H2 finger domain

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.     

Isolation and characterization of a novel RING-H2 finger gene induced in response to cold and drought in the interfertile Citrus relative Poncirus trifoliata

Many genes in different organisms encode proteins with really interesting gene (RING) finger domain(s). The RING zinc finger domain is involved in a wide variety of functions in diverse organisms. A cDNA clone showing homology with RING zinc finger genes and nine-fold induction in response to cold was previously identified during a gene expression study in the interfertile Citrus relative Poncirus trifoliata (L.) Raf. In this study, the full-length cDNA of this clone was isolated from 2-day cold-acclimated P. trifoliata by a rapid amplification of cDNA ends method using gene-specific primers. The full-length cDNA was 956 bp containing a complete open reading frame of 474 bp encoding a polypeptide of 158 amino acids. The full-length cDNA showed a high level of homology with genes encoding putative RING zinc finger proteins in plants. The deduced amino acid sequence of this gene contained a signature sequence motif for a RING zinc finger close to the C terminus of the protein. The RING zinc finger domain was significantly similar to previously characterized RING zinc finger proteins from different organisms. Additionally, it had a histidine residue at the fifth co-ordination site, indicating that this gene encodes a RING-H2 finger protein. Northern blot hybridization showed that the expression of the RING finger gene was induced in response to cold in cold-hardy P. trifoliata but not to the same extent in cold-sensitive Citrus grandis L. Osb. (pummelo). However, the gene was induced by drought stress similarly in both the species. To our knowledge, this study presents the first isolation of the full-length sequence of a RING zinc finger gene induced in response to abiotic stress in plants and the initial characterization of this gene in Citrus .     

High precision NMR structure and function of the RING-H2 finger domain of EL5, a rice protein whose expression is increased upon exposure to pathogen-derived oligosaccharides

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of two-stranded beta-sheets (beta1, Ala(147)-Phe(149); beta2, Gly(156)-His(158)), one alpha-helix (Cys(161)-Leu(166)), and two large N- and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N- and C-terminal loops and the alpha-helix. We demonstrated that the fusion protein with EL5-(96-181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.     

Genome-wide analysis of the RING finger gene family in apple

The RING finger protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, no detailed information concerning this family is available for apple (Malus × domestica L. Borkh) due to the limited information on whole genome sequences. In this study, 688 RING domains in 663 predicted proteins were identified in apple. Based on the spacing between metal ligands or substitutions at one or more of the metal ligand positions, nine RING types were identified: RING-H2, RING-HC, RING-C2, RING-v, RING-D, RING-S/T, RING-G, RING-mH2, and RING-mHC, in which the first seven types were described previously in Arabidopsis, while the latter two were newly identified in apple. Proteins containing RING finger motifs were further classified into 57 groups according to the different known or unknown domains outside the RING domains. A total of 643 retrieved proteins appear to be distributed over all 17 linkage groups with different densities. Microarray and expressed sequence tag data revealed that only a few of these RING finger proteins may be involved in fruit development. As a first step towards genome-wide analyses of the RING-containing genes in apple, our results provide valuable information for understanding the classification and putative functions of the RING finger gene family in higher plants.     

OsSIDP366, a DUF1644 gene,positively regulates responses to drought and salt stresses in rice

Domain of unknown function 1644(DUF1644) is a Rhighly conserved amino acid sequence motif present only in plants. Analysis of expression data of the family of DUF1644-containing genes indicated that they may regulate responses to abiotic stress in rice. Here we present our discovery of the role of Os SIDP366, a member of the DUF1644 gene family, in response to drought and salinity stresses in rice. Transgenic rice plants overexpressing Os SIDP366 showed enhanced drought and salinity tolerance and reduced water loss as compared to that in the control, whereas plants with downregulated Os SIDP366 expression levels using RNA interference(RNAi) were more sensitive to salinity and drought treatments. The sensitivity to abscisic acid(ABA)treatment was not changed in Os SIDP366-overexpressing plants, and Os SIDP366 expression was not affected in ABAdeficient mutants. Subcellular localization analysis revealed that Os SIDP366 is presented in the cytoplasmic foci that colocalized with protein markers for both processing bodies(PBs) and stress granules(SGs) in rice protoplasts. Digital gene expression(DGE) profile analysis indicated that stress-related genes such as SNAC1, Os HAK5 and PRs were upregulated in Os SIDP366-overexpressing plants. These results suggest that Os SIDP366 may function as a regulator of the PBs/SGs and positively regulate salt and drought resistance in rice.     

水稻OsWRKY转录因子对非生物胁迫响应的重叠表达特性分析

WRKY转录因子是植物一类比较大的基因家族,在水稻中已鉴定出102个成员。研究表明WRKY转录因子在植物生长发育、抗病耐逆等方面都具有重要的作用。本研究利用基因芯片数据结合实时定量分析,对水稻Os WRKY转录因子基因在不同的非生物逆境下的表达进行了分析,发现至少有33个Os WRKY基因同时对任何两种非生物胁迫因子做出响应,且所选20个基因中,13个基因可被ABA所诱导。OsWRKY基因这种重叠表达的特性,预示着这些基因在非生物逆境中具有功能多效性,对于培育抗逆境水稻品种具有重要的理论与实践意义。     

The RING finger motif of photomorphogenic repressor COP1 specifically interacts with the RING-H2 motif of a novel Arabidopsis protein.

The constitutive photomorphogenic 1 (COP1) protein of Arabidopsis functions as a molecular switch for the seedling developmental fates: photomorphogenesis under light conditions and skotomorphogenesis in darkness. The COP1 protein contains a cysteine-rich zinc-binding RING finger motif found in diverse groups of regulatory proteins. To understand the role of the COP1 RING finger in mediating protein-protein interaction, we have performed a yeast two-hybrid screen and isolated a novel protein with a RING-H2 motif, a variant type of the RING finger. This protein, designated COP1 Interacting Protein 8 (CIP8), is encoded by a single copy gene and localized to cytosol in a transient assay. In addition to the RING-H2 motif, the predicted protein has a C4 zinc finger, an acidic region, a glycine-rich cluster, and a serine-rich cluster. The COP1 RING finger and the CIP8 RING-H2 domains are sufficient for their interaction with each other both in vitro and in yeast, whereas neither motif displayed significant self-association. Moreover, site-directed mutagenesis studies demonstrated that the expected zinc-binding ligands of the RING finger and RING-H2 fingers are essential for their interaction. Our findings indicate that the RING finger motif, in this case, serves as autonomous protein-protein interaction domain. The allele specific effect of cop1 mutations on the CIP8 protein accumulation in seedlings indicates that its stability in vivo is dependent on the COP1 protein.     

Expression diversity and evolutionary dynamics of rice duplicate genes

Duplicate genes are believed to be a major source of new gene functions over evolutionary time. In order to evaluate the evolutionary dynamics of rice duplicate genes, formed principally by paleoployploidization prior to the speciation of the Poaceae family, we have employed a public microarray dataset including 155 gene expression omnibus sample plates and bioinformatics tools. At least 57.4% of old ~70 million years ago (MYA) duplicate gene pairs exhibit divergences in expression over the given experimental set, whereas at least 50.9% of young ~7.7-MYA duplicate gene pairs were shown to be divergent. When grouping the rice duplicate genes according to functional categories, we noted a striking and significant enrichment of divergent duplicate metabolism-associated genes, as compared to that observed in non-divergent duplicate genes. While both non-synonymous substitution (Ka) and synonymous substitution (Ks) values between non- and divergent duplicate gene pairs evidenced significant differences, the Ka/Ks values between them exhibited no significant differences. Interestingly, the average numbers of conserved motifs of the duplicate gene pairs revealed a pattern of decline along with an increase in expression diversity, partially supporting the subfunctionalization model with degenerative complementation in regulatory motifs. Duplicate gene pairs with high local similarity (HLS) segments, which might be formed via conversion between rice paleologs, evidenced higher expression correlations than were observed in the gene pairs without the HLS segments; this probably resulted in an increased likelihood of gene conversion in promoters of the gene pairs harboring HLS segments. More than 60% of the rice gene families exhibited similar high expression diversity between members as compared to that of randomly selected gene pairs. These findings are likely reflective of the evolutionary dynamics of rice duplicate genes for gene retention. An erratum to this article can be found at     

In search of function for hypothetical proteins encoded by genes of SA-JA pathways in Oryza sativa by in silico comparison and structural modeling

Knowledge of rice genome brings new dimensions to the management of abiotic stresses; however, gene sequences in the rice genome are yet to be assigned structure and function. Hydrogen peroxide, salicylates and jasmonates act as signal molecules in plants employing common machinery to manage abiotic stress. The present work is primarily focused to assign a structurefunction relationship by modeling of the hypothetical proteins of SA-JA signaling pathway known in Arabidopsis thaliana and compare them with corresponding proteins in rice in silico. Thirteen known gene sequences with their encoded proteins for SA/JA pathway in model plant A. thaliana were obtained and similar gene sequences from rice were retrieved at NCBI. Five rice gene sequences Os09g0392100, Os03g0233200, OsJ_33269, OsJ_23610 and Os01g0194300 resulted in hypothetical protein products with unknown structure and function. Modeling and comparison of 5 proteins from rice and Arabidopsis showed 73 - 98% identity with acceptable RMSD values of 0.6 - 1.7 upon superimposition. Results suggest conserved nature of these proteins during evolution. The hypothetical protein from rice contains similar functional protein domain as that in A. thaliana and therefore are likely to perform similar functions in rice. There is a cross talk between the genes in SA/JA pathway wherein Os09g0392100 or EDS1, Os03g0233200 or PR5, OsJ_33269 or PAD4 and OsJ_23610 or SFD-1 activates the pathway and Os01g0194300 or NPR1 inhibit the pathway. Further investigation through wet-lab experiments are in progress to look into suppression/activation of the genes of SAJA signaling in rice plants exposed to abiotic stress.     

Molecular characterization and concerted evolution of two genes encoding RING-C2 type proteins in rice

RING (Really Interesting New Gene) finger proteins are believed to play a critical role in mediating the transfer of ubiquitin to heterogeneous substrate(s). While the two canonical types, RING-H2 and RING-HC, have been well-characterized, the molecular functions of the modified types, particularly the RING-C2 types, remain elusive. We isolated two rice genes harboring the RING-C2 domain on the distal parts of rice chromosomes 11 and 12, termed OsRINGC2-1 and OsRINGC2-2, respectively. A comparison of sequence divergences between 10 duplicate pairs on the distal parts of rice chromosomes 11 and 12 and randomly selected duplicate pairs suggested that OsRINGC2-1 and OsRINGC2-2 have evolved in concert via gene conversion. An in vitro ubiquitination assay revealed that both proteins possess E3 ligase activity, suggesting that the innate functions of these RING domains have not been affected by their modifications during evolution. Subcellular localizations were strikingly different; OsRINGC2-1 was found only in the cytoplasm with many punctate complexes, whereas OsRINGC2-2 was observed in both the nucleus and cytoplasm. The expression patterns of both genes showed striking differences in response to salt stress, whereas plants heterogeneous for both genes mediated salt tolerance in Arabidopsis, supporting the notion of concerted evolution. These results shed light on the molecular functions of OsRINGC2-1 and OsRINGC2-2 and provide insight into their molecular evolution.     

Molecular cloning and characterization of a novel RING zinc-finger protein gene up-regulated under in vitro salt stress in cassava

Cassava (Manihot esculenta Crantz) is one of the world’s most important food crops. It is cultivated mainly in developing countries of tropics, since its root is a major source of calories for low-income people due to its high productivity and resistance to many abiotic and biotic factors. A previous study has identified a partial cDNA sequence coding for a putative RING zinc finger in cassava storage root. The RING zinc finger protein is a specialized type of zinc finger protein found in many organisms. Here, we isolated the full-length cDNA sequence coding for M. esculenta RZF (MeRZF) protein by a combination of 5′ and 3′ RACE assays. BLAST analysis showed that its deduced amino acid sequence has a high level of similarity to plant proteins of RZF family. MeRZF protein contains a signature sequence motif for a RING zinc finger at its C-terminal region. In addition, this protein showed a histidine residue at the fifth coordination site, likely belonging to the RING-H2 subgroup, as confirmed by our phylogenetic analysis. There is also a transmembrane domain in its N-terminal region. Finally, semi-quantitative RT-PCR assays showed that MeRZF expression is increased in detached leaves treated with sodium chloride. Here, we report the first evidence of a RING zinc finger gene of cassava showing potential role in response to salt stress.