JcDREB2, a Physic Nut AP2/ERF Gene, Alters Plant Growth and Salinity Stress Responses in Transgenic Rice.
ABSTRACT: Transcription factors of the AP2/ERF family play important roles in plant growth, development, and responses to biotic and abiotic stresses. In this study, a physic nut AP2/ERF gene, JcDREB2, was functionally characterized. Real-time PCR analysis revealed that JcDREB2 was expressed mainly in the leaf and could be induced by abscisic acid but suppressed by gibberellin (GA) and salt. Transient expression of a JcDREB2-YFP fusion protein in Arabidopsis protoplasts cells suggested that JcDREB2 is localized in the nucleus. Rice plants overexpressing JcDREB2 exhibited dwarf and GA-deficient phenotypes with shorter shoots and roots than those of wild-type plants. The dwarfism phenotype could be rescued by the application of exogenous GA3. The expression levels of GA biosynthetic genes including OsGA20ox1, OsGA20ox2, OsGA20ox4, OsGA3ox2, OsCPS1, OsKO2, and OsKAO were significantly reduced in plants overexpressing JcDREB2. Overexpression of JcDREB2 in rice increased sensitivity to salt stress. Increases in the expression levels of several salt-tolerance-related genes in response to salt stress were impaired in JcDREB2-overexpressing plants. These results demonstrated not only that JcDREB2 influences GA metabolism, but also that it can participate in the regulation of the salt stress response in rice.
Project description:The APETALA 2/ethylene response factors (AP2/ERF) are widespread in the plant kingdom and play essential roles in regulating plant growth and development as well as defense responses. In this study, a novel rice AP2/ERF transcription factor gene, OsRPH1, was isolated and functionally characterized. OsRPH1 falls into group-IVa of the AP2/ERF family. OsRPH1 protein was found to be localized in the nucleus and possessed transcriptional activity. Overexpression of OsRPH1 resulted in a decrease in plant height and length of internode and leaf sheath as well as other abnormal characters in rice. The length of the second leaf sheath of OsRPH1-overexpressing (OE) plants recovered to that of Kitaake (non-transgenic recipient) in response to exogenous gibberellin A3 (GA3) application. The expression of GA biosynthesis genes (OsGA20ox1-OsGA20ox4, OsGA3ox1, and OsGA3ox2) was significantly downregulated, whereas that of GA inactivation genes (OsGA2ox7, OsGA2ox9, and OsGA2ox10) was significantly upregulated in OsRPH1-OE plants. Endogenous bioactive GA contents significantly decreased in OsRPH1-OE plants. OsRPH1 interacted with a blue light receptor, OsCRY1b, in a blue light-dependent manner. Taken together, our results demonstrate that OsRPH1 negatively regulates plant height and bioactive GA content by controlling the expression of GA metabolism genes in rice. OsRPH1 is involved in blue light inhibition of leaf sheath elongation by interacting with OsCRY1b.
Project description:It has been shown that seed development is regulated by a network of transcription factors in Arabidopsis including LEC1 (LEAFY COTYLEDON1), L1L (LEC1-like) and the B3 domain factors LEC2, FUS3 (FUSCA3) and ABI3 (ABA-INSENSITIVE3); however, molecular and genetic regulation of seed development in cereals is poorly understood. To understand seed development and seed germination in cereals, a large-scale screen was performed using our T-DNA mutant population, and a mutant germination-defective1 (gd1) was identified. In addition to the severe germination defect, the gd1 mutant also shows a dwarf phenotype and abnormal flower development. Molecular and biochemical analyses revealed that GD1 encodes a B3 domain-containing transcription factor with repression activity. Consistent with the dwarf phenotype of gd1, expression of the gibberelic acid (GA) inactivation gene OsGA2ox3 is increased dramatically, accompanied by reduced expression of GA biosynthetic genes including OsGA20ox1, OsGA20ox2 and OsGA3ox2 in gd1, resulting in a decreased endogenous GA? level. Exogenous application of GA not only induced GD1 expression, but also partially rescued the dwarf phenotype of gd1. Furthermore, GD1 binds to the promoter of OsLFL1, a LEC2/FUS3-like gene of rice, via an RY element, leading to significant up-regulation of OsLFL1 and a large subset of seed maturation genes in the gd1 mutant. Plants over-expressing OsLFL1 partly mimic the gd1 mutant. In addition, expression of GD1 was induced under sugar treatment, and the contents of starch and soluble sugar are altered in the gd1 mutant. These data indicate that GD1 participates directly or indirectly in regulating GA and carbohydrate homeostasis, and further regulates rice seed germination and seedling development.
Project description:Plant height (PH) is one of the most important agronomic traits of rice, as it directly affects the yield potential and lodging resistance. Here, semi-dwarf mutant lines were developed through CRISPR/Cas9-based editing of OsGA20ox2 in an indica rice cultivar. Total 24 independent lines were obtained in T0 generation with the mean mutation rate of 73.5% including biallelic (29.16%), homozygous (47.91%) and heterozygous (16.66%) mutations, and 16 T-DNA-free lines (50%) were obtained in T1 generation without off-target effect in four most likely sites. Mutations resulted in a changed amino acid sequence of mutant plants and reduced gibberellins (GA) level and PH (22.2%), flag leaf length (FLL) and increased yield per plant (YPP) (6.0%), while there was no effect on other agronomic traits. Mutants restored their PH to normal by exogenous GA3 treatment. The expression of the OsGA20ox2 gene was significantly suppressed in mutant plants, while the expression level was not affected for other GA biosynthesis (OsGA2ox3 and OsGA3ox2) and signaling (D1, GIDI and SLR1) genes. The mutant lines showed decreased cell length and width, abnormal cell elongation, while increased cell numbers in the second internode sections at mature stage. Total 30 protein spots were exercised, and 24 proteins were identified, and results showed that OsGA20ox2 editing altered protein expression. Five proteins including, glyceraldehyde-3-phosphate dehydrogenase, putative ATP synthase, fructose-bisphosphate aldolase 1, S-adenosyl methionine synthetase 1 and gibberellin 20 oxidase 2, were downregulated in dwarf mutant lines which may affect the plant growth. Collectively, our results provide the insights into the role of OsGA20ox2 in PH and confirmed that CRISPR-Cas9 is a powerful tool to understand the gene functions.
Project description:The AP2/ERF transcription factors play crucial roles in plant growth, development and responses to biotic and abiotic stresses. A total of 119 AP2/ERF genes (JcAP2/ERFs) have been identified in the physic nut genome; they include 16 AP2, 4 RAV, 1 Soloist, and 98 ERF genes. Phylogenetic analysis indicated that physic nut AP2 genes could be divided into 3 subgroups, while ERF genes could be classed into 11 groups or 43 subgroups. The AP2/ERF genes are non-randomly distributed across the 11 linkage groups of the physic nut genome and retain many duplicates which arose from ancient duplication events. The expression patterns of several JcAP2/ERF duplicates in the physic nut showed differences among four tissues (root, stem, leaf, and seed), and 38 JcAP2/ERF genes responded to at least one abiotic stressor (drought, salinity, phosphate starvation, and nitrogen starvation) in leaves and/or roots according to analysis of digital gene expression tag data. The expression of JcERF011 was downregulated by salinity stress in physic nut roots. Overexpression of the JcERF011 gene in rice plants increased its sensitivity to salinity stress. The increased expression levels of several salt tolerance-related genes were impaired in the JcERF011-overexpressing plants under salinity stress.
Project description:Osmotin promoter binding protein 1 (OPBP1), an AP2/ERF transcription factor of tobacco, has been demonstrated to function in disease resistance and salt tolerance in tobacco. To increase stress tolerant capability of rice, we generated rice plants with an OPBP1 overexpressing construct. Salinity shock treatment with 250 mM NaCl indicated that most of the OPBP1 transgenic plants can survive, whereas the control seedlings cannot. Similar recovery was found by using the seedlings grown in 200 mM NaCl for two weeks. The OPBP1 transgenic and control plants were also studied for oxidative stress tolerance by treatment with paraquat, showing the transgenic lines were damaged less in comparison with the control plants. Further, the OPBP1 overexpression lines exhibited enhanced resistance to infections of Magnaporthe oryzae and Rhizoctonia solani pathogens. Gene expressing analysis showed increase in mRNA accumulation of several stress related genes. These results suggest that expression of OPBP1 gene increase the detoxification capability of rice.
Project description:Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na(+)/K(+) ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels.
Project description:Members of the ERF transcription factor family play important roles in regulating gene expression in response to biotic and abiotic stresses. In soybean (Glycine max L.), however, only a few ERF genes have been studied so far. In this study, 98 unigenes that contained a complete AP2/ERF domain were identified from 63,676 unique sequences in the DFCI Soybean Gene Index database. The phylogeny, gene structures, and putative conserved motifs in soybean ERF proteins were analysed, and compared with those of Arabidopsis and rice. The members of the soybean ERF family were divided into 12 subgroups, similar to the case for Arabidopsis. AP2/ERF domains were conserved among soybean, Arabidopsis, and rice. Outside the AP2/ERF domain, many soybean-specific conserved motifs were detected. Expression analysis showed that nine unigenes belonging to six ERF family subgroups were induced by both biotic/abiotic stresses and hormone treatment, suggesting that they were involved in cross-talk between biotic and abiotic stress-responsive signalling pathways. Overexpression of two full-length genes from two different subgroups enhanced the tolerances to drought, salt stresses, and/or pathogen infection of the tobacco plants. These results will be useful for elucidating ERF gene-associated stress response signalling pathways in soybean.
Project description:AP2/ERF-type transcription factors regulate important functions of plant growth and development as well as responses to environmental stimuli. A rice AP2/ERF transcription factor, OsEREBP1 is a downstream component of a signal transduction pathway in a specific interaction between rice (Oryza sativa) and its bacterial pathogen, Xoo (Xanthomonas oryzae pv. oryzae). Constitutive expression of OsEREBP1 in rice driven by maize ubiquitin promoter did not affect normal plant growth. Microarray analysis revealed that over expression of OsEREBP1 caused increased expression of lipid metabolism related genes such as lipase and chloroplastic lipoxygenase as well as several genes related to jasmonate and abscisic acid biosynthesis. PR genes, transcription regulators and Aldhs (alcohol dehydrogenases) implicated in abiotic stress and submergence tolerance were also upregulated in transgenic plants. Transgenic plants showed increase in endogenous levels of ?-linolenate, several jasmonate derivatives and abscisic acid but not salicylic acid. Soluble modified GFP (SmGFP)-tagged OsEREBP1 was localized to plastid nucleoids. Comparative analysis of non-transgenic and OsEREBP1 overexpressing genotypes revealed that OsEREBP1 attenuates disease caused by Xoo and confers drought and submergence tolerance in transgenic rice. Our results suggest that constitutive expression of OsEREBP1 activates the jasmonate and abscisic acid signalling pathways thereby priming the rice plants for enhanced survival under abiotic or biotic stress conditions. OsEREBP1 is thus, a good candidate gene for engineering plants for multiple stress tolerance.
Project description:Background APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors play essential roles in plant growth, development, metabolism, and responses to biotic and abiotic stresses. However, few studies concerning AP2/ERF genes in sugarcane which are the most critical sugar and energy crops worldwide. Results A total of 218 AP2/ERF genes were identified in the Saccharum spontaneum genome. Phylogenetic analysis showed that these genes could be divided into four groups, including 43 AP2s, 160 ERFs and Dehydration-responsive element-binding (DREB) factors, 11 ABI3/VPs (RAV), and four Soloist genes. These genes were unevenly distributed on 32 chromosomes. The structural analysis of SsAP2/ERF genes showed that 91 SsAP2/ERFs lacked introns. Sugarcane and sorghum had a collinear relationship between 168 SsAP2/ERF genes and sorghum AP2/ERF genes that reflected their similarity. Multiple cis-regulatory elements (CREs) present in the SsAP2/ERF promoter were related to abiotic stresses, suggesting that SsAP2/ERF activity could contribute to sugarcane adaptation to environmental changes. The tissue-specific analysis showed spatiotemporal expression of SsAP2/ERF in the stems and leaves of sugarcane at different development stages. In ten sugarcane samples, 39 SsAP2/ERFs were not expressed, whereas 58 SsAP2/ERFs were expressed in all samples. Quantitative PCR experiments showed that SsERF52 expression was up-regulated under salt stress, but suppressed under dehydration stress. SsSoloist4 had the most considerable upregulation in response to treatment with the exogenous hormones ABA and GA. Within 3?h of ABA or PEG6000 treatment, SsSoloist4 expression was up-regulated, indicating that this gene could play a role in the responses to ABA and GA-associated dehydration stress. Analysis of AP2/ERF gene expression patterns under different treatments indicated that SsAP2/ERF genes played an essential role in dehydration and salt stress responses of S. spontaneum. Conclusions In this study, a total of 218 members of the AP2 / ERF superfamily were identified in sugarcane, and their genetic structure, evolution characteristics, and expression patterns were studied and analyzed. The results of this study provide a foundation for future analyses to elucidate the importance of AP2/ERF transcription factors in the function and molecular breeding of sugarcane.
Project description:The AP2/ERF transcription factors (TFs) comprise one of the largest gene superfamilies in plants. These TFs perform vital roles in plant growth, development, and responses to biotic and abiotic stresses. In this study, 171 AP2/ERF TFs were identified in cauliflower (Brassica oleracea L. var. botrytis), one of the most important horticultural crops in Brassica. Among these TFs, 15, 9, and 1 TFs were classified into the AP2, RAV, and Soloist family, respectively. The other 146 TFs belong to ERF family, which were further divided into the ERF and DREB subfamilies. The ERF subfamily contained 91 TFs, while the DREB subfamily contained 55 TFs. Phylogenetic analysis results indicated that the AP2/ERF TFs can be classified into 13 groups, in which 25 conserved motifs were confirmed. Some motifs were group- or subgroup- specific, implying that they are significant to the functions of the AP2/ERF TFs of these clades. In addition, 35 AP2/ERF TFs from the 13 groups were selected randomly and then used for expression pattern analysis under salt and drought stresses. The majority of these AP2/ERF TFs exhibited positive responses to these stress conditions. In specific, Bra-botrytis-ERF054a, Bra-botrytis-ERF056, and Bra-botrytis-CRF2a demonstrated rapid responses. By contrast, six AP2/ERF TFs were showed to delay responses to both stresses. The AP2/ERF TFs exhibiting specific expression patterns under salt or drought stresses were also confirmed. Further functional analysis indicated that ectopic overexpression of Bra-botrytis-ERF056 could increase tolerance to both salt and drought treatments. These findings provide new insights into the AP2/ERF TFs present in cauliflower, and offer candidate AP2/ERF TFs for further studies on their roles in salt and drought stress tolerance.