RESUMO
VQ motif-containing proteins play important roles in plant abiotic and biotic stresses. In this study, we cloned the VQ protein gene TaVQ4-D that is induced by drought stress. Arabidopsis and wheat plants overexpressing TaVQ4-D showed increased tolerance to drought stress. In contrast, wheat lines in which TaVQ4-D expression had been silenced showed decreased drought tolerance. Under drought stress conditions, the contents of superoxide dismutase and proline increased and the content of malondialdehyde decreased in transgenic wheat plants overexpressing TaVQ4-D compared with the wild type. At the same time, the expression of reactive oxygen species-scavenging-related genes and stress-related genes was up-regulated. However, plants of TaVQ4-D-silenced wheat lines showed decreased activities of antioxidant enzymes and reduced expression of some stress-related and antioxidant-related genes. In addition, the TaVQ4-D protein physically interacts with two mitogen-activated protein kinases (MPK3 and MPK6) and plays a role in plant drought stress as the phosphorylated substrates of MPK3 and MPK6. In summary, the results of our study suggest that TaVQ4-D can positively regulate drought stress tolerance in wheat.
Assuntos
Arabidopsis , Proteínas de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Resistência à Seca , Triticum/metabolismo , Antioxidantes/metabolismo , Arabidopsis/metabolismo , Secas , Estresse Fisiológico/genética , Regulação da Expressão Gênica de PlantasRESUMO
Raffinose synthase (RS) plays a crucial role in plant growth and development, as well as in responses to biotic stresses and abiotic stresses, yet few studies have been conducted on its role in bread wheat. Therefore, in this study we screened and identified a family of bread wheat raffinose synthase genes based on bread wheat genome information and analyzed their physicochemical properties, phylogenetic evolutionary relationships, conserved structural domains, promoter cis-acting elements, and expression patterns. The BSMV-induced silencing of TaRS15-3B resulted in the bread wheat seedlings being susceptible to drought and salt stress and reduced the expression levels of stress-related and ROS-scavenging genes in bread wheat plants. This further affected the ability of bread wheat to cope with drought and salt stress. In conclusion, this study revealed that the RS gene family in bread wheat plays an important role in plant response to abiotic stresses and that the TaRS15-3B gene can improve the tolerance of transgenic bread wheat to drought and salt stresses, provide directions for the study of other RS gene families in bread wheat, and supply candidate genes for use in molecular breeding of bread wheat for stress resistance.
Assuntos
Resistência à Seca , Tolerância ao Sal , Tolerância ao Sal/genética , Triticum/metabolismo , Filogenia , Pão , Secas , Estresse Fisiológico/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
In the hexaploid wheat genome, there are three Gα genes, three Gß and twelve Gγ genes, but the function of Gß in wheat has not been explored. In this study, we obtained the overexpression of TaGB1 Arabidopsis plants through inflorescence infection, and the overexpression of wheat lines was obtained by gene bombardment. The results showed that under drought and NaCl treatment, the survival rate of Arabidopsis seedlings' overexpression of TaGB1-B was higher than that of the wild type, while the survival rate of the related mutant agb1-2 was lower than that of the wild type. The survival rate of wheat seedlings with TaGB1-B overexpression was higher than that of the control. In addition, under drought and salt stress, the levels of superoxide dismutase (SOD) and proline (Pro) in the wheat overexpression of TaGB1-B were higher than that of the control, and the concentration of malondialdehyde (MDA) was lower than that of the control. This indicates that TaGB1-B could improve the drought resistance and salt tolerance of Arabidopsis and wheat by scavenging active oxygen. Overall, this work provides a theoretical basis for wheat G-protein ß-subunits in a further study, and new genetic resources for the cultivation of drought-tolerant and salt-tolerant wheat varieties.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Subunidades beta da Proteína de Ligação ao GTP , Arabidopsis/genética , Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/genética , Triticum/genética , Triticum/metabolismo , Secas , Estresse Fisiológico/genética , Plântula/genética , Plântula/metabolismo , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Subunidades beta da Proteína de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Three-amino-loop-extension (TALE) superfamily genes are widely present in plants and function directly in plant growth and development and abiotic stress response. Although TALE genes have been studied in many plant species, members of the TALE family have not been identified in wheat. RESULTS: In this study, we identified 70 wheat TALE protein candidate genes divided into two subfamilies, KNOX (KNOTTED-like homeodomain) and BEL1-like (BLH/BELL homeodomain). Genes in the same subfamily or branch in the phylogenetic tree are similar in structure, and their encoded proteins have similar motifs and conserved structures. Wheat TALE genes are unevenly distributed on 21 chromosomes and expanded on the fourth chromosome. Through gene duplication analysis, 53 pairs of wheat TALE genes were determined to result from segmental duplication events, and five pairs were caused by tandem duplication events. The Ka/Ks between TALE gene pairs indicates a strong purification and selection effect. There are multiple cis-elements in the 2000 bp promoter sequence that respond to hormones and abiotic stress, indicating that most wheat TALE genes are involved in the growth, development, and stress response of wheat. We also studied the expression profiles of wheat TALE genes in different developmental stages and tissues and under different stress treatments. We detected the expression levels of four TALE genes by qRT-PCR, and selected TaKNOX11-A for further downstream analysis. TaKNOX11-A enhanced the drought and salt tolerances of Arabidopsis thaliana. TaKNOX11-A overexpressing plants had decreased malondialdehyde content and increased proline content, allowing for more effective adaptation of plants to unfavorable environments. CONCLUSIONS: We identified TALE superfamily members in wheat and conducted a comprehensive bioinformatics analysis. The discovery of the potential role of TaKNOX11-A in drought resistance and salt tolerance provides a basis for follow-up studies of wheat TALE family members, and also provides new genetic resources for improving the stress resistance of wheat.
Assuntos
Proteínas de Plantas , Triticum , Genoma de Planta , Filogenia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Triticum/genéticaRESUMO
BACKGROUND: VQ motif-containing (VQ) proteins are cofactors of transcriptional regulation that are widely involved in plant growth and development and respond to various stresses. The VQ gene family has been identified and characterized for many plants, but there is little research on VQ gene family proteins in wheat (Triticum aestivum L.). RESULTS: In this study, 113 TaVQ genes (40 homoeologous groups) were identified in the wheat genome. TaVQ proteins all contain the conserved motif FxxhVQxhTG, and most of the TaVQ genes do not contain introns. Phylogenetic analysis demonstrated that TaVQ proteins can be divided into 8 subgroups (I-VIII). The chromosomal location mapping analysis indicated that TaVQ genes are disproportionally distributed on 21 wheat chromosomes. Gene duplication analysis revealed that segmental duplication significantly contributes to the expansion of the TaVQ gene family. Gene expression analysis demonstrated that the expression pattern of TaVQ genes varies in different tissues. The results of quantitative real-time PCR (qRT-PCR) found that TaVQ genes displayed different expression levels under different phytohormones and abiotic stresses. The cis-elements analysis of the promoter region demonstrated that stress responses, hormone responses, growth and development, and WRKY binding elements are all widely distributed. Additionally, a potential regulatory network between TaVQ proteins and WRKY transcription factors was visualized. CONCLUSION: This study systematically analyzed the wheat TaVQ gene family, providing a reference for further functional characterization of TaVQ genes in wheat.
Assuntos
Reguladores de Crescimento de Plantas , Triticum , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Triticum/genética , Triticum/metabolismoRESUMO
The peptidase M24 (Metallopeptidase 24, M24) superfamily is essential for plant growth, stress response, and pathogen defense. At present, there are few systematic reports on the identification and classification of members of the peptidase M24 proteins superfamily in wheat. In this work, we identified 53 putative candidate TaM24 genes. According to the protein sequences characteristics, these members can be roughly divided into three subfamilies: I, II, III. Most TaM24 genes are complex with multiple exons, and the motifs are relatively conserved in each sub-group. Through chromosome mapping analysis, we found that the 53 genes were unevenly distributed on 19 wheat chromosomes (except 3A and 3D), of which 68% were in triads. Analysis of gene duplication events showed that 62% of TaM24 genes in wheat came from fragment duplication events, and there were no tandem duplication events to amplify genes. Analysis of the promoter sequences of TaM24 genes revealed that cis-acting elements were rich in response elements to drought, osmotic stress, ABA, and MeJA. We also studied the expression of TaM24 in wheat tissues at developmental stages and abiotic stress. Then we selected TaM24-9 as the target for further analysis. The results showed that TaM24-9 genes strengthened the drought and salt tolerance of plants. Overall, our analysis showed that members of the peptidase M24 genes may participate in the abiotic stress response and provided potential gene resources for improving wheat resistance.
Assuntos
Regulação da Expressão Gênica de Plantas , Triticum , Genoma de Planta , Família Multigênica , Peptídeo Hidrolases/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Triticum/metabolismoRESUMO
BACKGROUND: The serine carboxypeptidase-like protein (SCPL) family plays a vital role in stress response, growth, development and pathogen defense. However, the identification and functional analysis of SCPL gene family members have not yet been performed in wheat. RESULTS: In this study, we identified a total of 210 candidate genes encoding SCPL proteins in wheat. According to their structural characteristics, it is possible to divide these members into three subfamilies: CPI, CPII and CPIII. We uncovered a total of 209 TaSCPL genes unevenly distributed across 21 wheat chromosomes, of which 65.7% are present in triads. Gene duplication analysis showed that ~ 10.5% and ~ 64.8% of the TaSCPL genes are derived from tandem and segmental duplication events, respectively. Moreover, the Ka/Ks ratios between duplicated TaSCPL gene pairs were lower than 0.6, which suggests the action of strong purifying selection. Gene structure analysis showed that most of the TaSCPL genes contain multiple introns and that the motifs present in each subfamily are relatively conserved. Our analysis on cis-acting elements showed that the promoter sequences of TaSCPL genes are enriched in drought-, ABA- and MeJA-responsive elements. In addition, we studied the expression profiles of TaSCPL genes in different tissues at different developmental stages. We then evaluated the expression levels of four TaSCPL genes by qRT-PCR, and selected TaSCPL184-6D for further downstream analysis. The results showed an enhanced drought and salt tolerance among TaSCPL184-6D transgenic Arabidopsis plants, and that the overexpression of the gene increased proline and decreased malondialdehyde levels, which might help plants adapting to adverse environments. Our results provide comprehensive analyses of wheat SCPL genes that might work as a reference for future studies aimed at improving drought and salt tolerance in wheat. CONCLUSIONS: We conducte a comprehensive bioinformatic analysis of the TaSCPL gene family in wheat, which revealing the potential roles of TaSCPL genes in abiotic stress. Our analysis also provides useful resources for improving the resistance of wheat.
Assuntos
Genoma de Planta , Triticum , Carboxipeptidases , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Triticum/genéticaRESUMO
Drought-response-element binding (DREB)-like transcription factors can significantly enhance plant tolerance to water stress. However, most research on DREB-like proteins to date has been conducted in growth chambers or greenhouses, so there is very little evidence available to support their practical use in the field. In this study, we overexpressed GmDREB1 from soybean in two popular wheat varieties and conducted drought-tolerance experiments across a range of years, sites, and drought-stress regimes. We found that the transgenic plants consistently exhibited significant improvements in yield performance and a variety of physiological traits compared with wild-type plants when grown under limited water conditions in the field, for example showing grain yield increases between 4.79-18.43%. Specifically, we found that the transgenic plants had reduced membrane damage and enhanced osmotic adjustment and photosynthetic efficiency compared to the non-transgenic controls. Three enzymes from the biosynthetic pathway of the phytohormone melatonin were up-regulated in the transgenic plants, and external application of melatonin was found to improve drought tolerance. Together, our results demonstrate the utility of transgenic overexpression of GmDREB1 to improve the drought tolerance of wheat in the field.
Assuntos
Secas , Triticum , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Glycine max/genética , Glycine max/metabolismo , Estresse Fisiológico , Triticum/genética , Triticum/metabolismoRESUMO
Vascular plant one-zinc-finger (VOZ) transcription factor, a plant specific one-zinc-finger-type transcriptional activator, is involved in regulating numerous biological processes such as floral induction and development, defense against pathogens, and response to multiple types of abiotic stress. Six VOZ transcription factor-encoding genes (GmVOZs) have been reported to exist in the soybean (Glycine max) genome. In spite of this, little information is currently available regarding GmVOZs. In this study, GmVOZs were cloned and characterized. GmVOZ genes encode proteins possessing transcriptional activation activity in yeast cells. GmVOZ1E, GmVOZ2B, and GmVOZ2D gene products were widely dispersed in the cytosol, while GmVOZ1G was primarily located in the nucleus. GmVOZs displayed a differential expression profile under dehydration, salt, and salicylic acid (SA) stress conditions. Among them, GmVOZ1G showed a significantly induced expression in response to all stress treatments. Overexpression of GmVOZ1G in soybean hairy roots resulted in a greater tolerance to drought and salt stress. In contrast, RNA interference (RNAi) soybean hairy roots suppressing GmVOZ1G were more sensitive to both of these stresses. Under drought treatment, soybean composite plants with an overexpression of hairy roots had higher relative water content (RWC). In response to drought and salt stress, lower malondialdehyde (MDA) accumulation and higher peroxidase (POD) and superoxide dismutase (SOD) activities were observed in soybean composite seedlings with an overexpression of hairy roots. The opposite results for each physiological parameter were obtained in RNAi lines. In conclusion, GmVOZ1G positively regulates drought and salt stress tolerance in soybean hairy roots. Our results will be valuable for the functional characterization of soybean VOZ transcription factors under abiotic stress.
Assuntos
Desidratação/metabolismo , Glycine max/metabolismo , Proteínas de Plantas/metabolismo , Tolerância ao Sal/genética , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citosol/metabolismo , Desidratação/genética , Secas , Regulação da Expressão Gênica de Plantas/genética , Malondialdeído/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Filogenia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Elementos Reguladores de Transcrição/genética , Estresse Salino/genética , Plântula/genética , Plântula/metabolismo , Glycine max/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fatores de Transcrição/genéticaRESUMO
Pentatricopeptide-repeat (PPR) proteins were identified as a type of nucleus coding protein that is composed of multiple tandem repeats. It has been reported that PPR genes play an important role in RNA editing, plant growth and development, and abiotic stresses in plants. However, the functions of PPR proteins remain largely unknown in soybean. In this study, 179 DYW subgroup PPR genes were identified in soybean genome (Glycine max Wm82.a2.v1). Chromosomal location analysis indicated that DYW subgroup PPR genes were mapped to all 20 chromosomes. Phylogenetic relationship analysis revealed that DYW subgroup PPR genes were categorized into three distinct Clusters (I to III). Gene structure analysis showed that most PPR genes were featured by a lack of intron. Gene duplication analysis demonstrated 30 PPR genes (15 pairs; ~35.7%) were segmentally duplicated among Cluster I PPR genes. Furthermore, we validated the mRNA expression of three genes that were highly up-regulated in soybean drought- and salt-induced transcriptome database and found that the expression levels of GmPPR4 were induced under salt and drought stresses. Under drought stress condition, GmPPR4-overexpressing (GmPPR4-OE) plants showed delayed leaf rolling; higher content of proline (Pro); and lower contents of H2O2, O2- and malondialdehyde (MDA) compared with the empty vector (EV)-control plants. GmPPR4-OE plants exhibited increased transcripts of several drought-inducible genes compared with EV-control plants. Our results provided a comprehensive analysis of the DYW subgroup PPR genes and an insight for improving the drought tolerance in soybean.
Assuntos
Proteínas de Transporte , Regulação da Expressão Gênica de Plantas , Glycine max , Família Multigênica , Pressão Osmótica , Proteínas de Soja , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Desidratação/genética , Desidratação/metabolismo , Estudo de Associação Genômica Ampla , Proteínas de Soja/biossíntese , Proteínas de Soja/genética , Glycine max/genética , Glycine max/metabolismoRESUMO
Melatonin (N-acetyl-5-methoxytryptamine) is involved in many developmental processes and responses to various abiotic stresses in plants. Most of the studies on melatonin focus on its functions and physiological responses in plants, while its regulation mechanism remains unknown. Caffeic acid 3-O-methyltransferase (COMT) functions at a key step of the biosynthesis process of melatonin. In this study, a COMT-like gene, TaCOMT (Traes_1AL_D9035D5E0.1) was identified in common wheat (Triticum aestivum L.). Transient transformation in wheat protoplasts determined that TaCOMT is localized in cytoplasm. TaCOMT in wheat was induced by drought stress, gibberellin (GA)3 and 3-Indoleacetic acid (IAA), but not by ABA. In TaCOMT transgenic Arabidopsis, melatonin contents were higher than that in wild type (WT) plants. Under D-Mannitol treatment, the fresh weight of the transgenic Arabidopsis was significantly higher than WT, and transgenic lines had a stronger root system compared to WT. Drought tolerance assays in pots showed that the survival rate of TaCOMT-overexpression lines was significantly higher than that of WT lines. this phenotype was similar to that the WT lines treated with melatonin under drought condition. In addition, the TaCOMT transgenic lines had higher proline content and lower malondialdehyde (MDA) content compared to WT lines after drought treatment. These results indicated that overexpression of the wheat TaCOMT gene enhances drought tolerance and increases the content of melatonin in transgenic Arabidopsis. It could be one of the potential genes for agricultural applications.
Assuntos
Adaptação Biológica , Arabidopsis/genética , Arabidopsis/metabolismo , Secas , Expressão Gênica , Melatonina/biossíntese , Proteínas Sensíveis a N-Etilmaleimida/genética , Sequência de Aminoácidos , Proteínas Sensíveis a N-Etilmaleimida/química , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais , Estresse Fisiológico/genética , Triticum/genética , Triticum/metabolismoRESUMO
LIM proteins have been found to play important roles in many life activities, including the regulation of gene expression, construction of the cytoskeleton, signal transduction and metabolic regulation. Because of their important roles in many aspects of plant development, LIM genes have been studied in many plant species. However, the LIM gene family has not yet been characterized in foxtail millet. In this study, we analyzed the whole genome of foxtail millet and identified 10 LIM genes. All LIM gene promoters contain MYB and MYC cis-acting elements that are related to drought stress. Based on the presence of multiple abiotic stress-related cis-elements in the promoter of SiWLIM2b, we chose this gene for further study. We analyzed SiWLIM2b expression under abiotic stress and hormone treatments using qRT-PCR. We found that SiWLIM2b was induced by various abiotic stresses and hormones. Under drought conditions, transgenic rice of SiWLIM2b-overexpression had a higher survival rate, higher relative water content and less cell damage than wild type (WT) rice. These results indicate that overexpression of the foxtail millet SiWLIM2b gene enhances drought tolerance in transgenic rice, and the SiWLIM2b gene can potentially be used for molecular breeding of crops with increased resistance to abiotic stress.
Assuntos
Secas , Proteínas com Domínio LIM/genética , Setaria (Planta)/crescimento & desenvolvimento , Sequenciamento Completo do Genoma/métodos , Adaptação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Família Multigênica , Oryza/genética , Oryza/crescimento & desenvolvimento , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Setaria (Planta)/genéticaRESUMO
WRKYs are important regulators in plant development and stress responses. However, knowledge of this superfamily in soybean is limited. In this study, we characterized the drought- and salt-induced gene GmWRKY12 based on RNA-Seq and qRT-PCR. GmWRKY12, which is 714 bp in length, encoded 237 amino acids and grouped into WRKY II. The promoter region of GmWRKY12 included ABER4, MYB, MYC, GT-1, W-box and DPBF cis-elements, which possibly participate in abscisic acid (ABA), drought and salt stress responses. GmWRKY12 was minimally expressed in different tissues under normal conditions but highly expressed under drought and salt treatments. As a nucleus protein, GmWRKY12 was responsive to drought, salt, ABA and salicylic acid (SA) stresses. Using a transgenic hairy root assay, we further characterized the roles of GmWRKY12 in abiotic stress tolerance. Compared with control (Williams 82), overexpression of GmWRKY12 enhanced drought and salt tolerance, increased proline (Pro) content and decreased malondialdehyde (MDA) content under drought and salt treatment in transgenic soybean seedlings. These results may provide a basis to understand the functions of GmWRKY12 in abiotic stress responses in soybean.
Assuntos
Resistência à Doença/fisiologia , Glycine max/metabolismo , Tolerância ao Sal/fisiologia , Plântula/metabolismo , Proteínas de Soja/metabolismo , Fatores de Transcrição/metabolismo , Desidratação , Plântula/genética , Proteínas de Soja/genética , Glycine max/genética , Fatores de Transcrição/genéticaRESUMO
To cope with environmental stress caused by global climate change and excessive nitrogen application, it is important to improve water and nitrogen use efficiencies in crop plants. It has been reported that higher nitrogen uptake could alleviate the damaging impact of drought stress. However, there is scant evidence to explain how nitrogen uptake affects drought resistance. In this study we observed that bZIP transcription factor AtTGA4 (TGACG motif-binding factor 4) was induced by both drought and low nitrogen stresses, and that overexpression of AtTGA4 simultaneously improved drought resistance and reduced nitrogen starvation in Arabidopsis. Following drought stress there were higher nitrogen and proline contents in transgenic AtTGA4 plants than in wild type controls, and activity of the key enzyme nitrite reductase (NIR) involved in nitrate assimilation processes was also higher. Expressions of the high-affinity nitrate transporter genes NRT2.1 and NRT2.2 and nitrate reductase genes NIA1 and NIA2 in transgenic plants were all higher than in wild type indicating that higher levels of nitrate transport and assimilation activity contributed to enhanced drought resistance of AtTGA4 transgenic plants. Thus genetic transformation with AtTGA4 may provide a new approach to simultaneously improve crop tolerance to drought and low nitrogen stresses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Nitratos/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Mudança Climática , Secas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Transportadores de Nitrato , Nitrito Redutases/genética , Nitrito Redutases/metabolismo , Plantas Geneticamente Modificadas , Estresse FisiológicoRESUMO
The vacuolar type H+-ATPase (V-type H+-ATPase) plays important roles in establishing an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this paper, a putative E subunit of the V-type H+-ATPase gene, W36 was isolated from stress-induced wheat de novo transcriptome sequencing combining with 5'-RACE and RT-PCR methods. The full-length of W36 gene was 1097 bp, which contained a 681 bp open reading frame (ORF) and encoded 227 amino acids. Southern blot analysis indicated that W36 was a single-copy gene. The quantitative real-time PCR (qRT-PCR) analysis revealed that the expression level of W36 could be upregulated by drought, cold, salt, and exogenous ABA treatment. A subcellular localization assay showed that the W36 protein accumulated in the cytoplasm. Isolation of the W36 promoter revealed some cis-acting elements responding to abiotic stresses. Transgenic Arabidopsis plants overexpressing W36 were enhanced salt and mannitol tolerance. These results indicate that W36 is involved in the plant response to osmotic stress.
Assuntos
Proteínas de Plantas/metabolismo , Triticum/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Aminobutiratos/farmacologia , Arabidopsis/enzimologia , Secas , Manitol/farmacologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Pressão Osmótica , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Cloreto de Sódio/farmacologia , Regulação para Cima/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Improving wheat drought resistance is of great significance for grain production and food security. Hexokinases (HXKs) play a role in sugar signal transduction and are involved in abiotic stress responses in wheat. To clarify the relationship between HXKs and drought stress in wheat, we used the rice active oxygen induction gene OsHXK1 as a reference sequence and the homologously cloned wheat TaHXK7-1A gene. TaHXK7-1A was localized in the nucleus and cell membrane. Under drought stress, over-expression of TaHXK7-1A increased the contents of O2·ï¼ and malondialdehyde (MDA) and significantly up-regulated the respiratory burst oxidative homologue (RBOHs) genes in transgenic Arabidopsis. In addition, the over-expression of TaHXK7-1A inhibited the growth of Arabidopsis seedlings and increased ROS accumulation under 6 % exogenous glucose treatment. Gene silencing of TaHXK7-1 decreased the contents of O2·ï¼ and MDA in wheat leaves under drought stress, and the RBOHs was significantly down-regulated, which improved the drought resistance of wheat. The results of yeast one-hybrid, EMSA, and dual-luciferase assays showed that TabHLH148-5A bound to the E-box motif of the TaHXK7-1A promoter and inhibited the expression of TaHXK7-1A. In addition, yeast two-hybrid and luciferase complementation imaging assays showed that TaHXK7-1A interacted with TaGRF3-4A. These results indicate that the glucose sensor TaHXK7-1A was negatively regulated by TabHLH148-5A, interacted with TaGRF3-4A, and negatively regulated wheat drought resistance by regulating RBOHs expression and inducing ROS production, thus providing a theoretical basis for revealing the molecular mechanism of wheat drought resistance.
Assuntos
Arabidopsis , Resistência à Seca , Triticum/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/metabolismo , Saccharomyces cerevisiae/metabolismo , Secas , Luciferases/genética , Luciferases/metabolismo , Glucose/metabolismo , Estresse Fisiológico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Regulação da Expressão Gênica de PlantasRESUMO
R2R3-MYB transcription factors play an important role in the synthesis of phenylpropanoid-derived compounds, which in turn provide salt tolerance in plant. In this study, we found that the expression of foxtail millet R2R3-MYB factor SiMYB16 can be induced by salt and drought. SiMYB16 is localized in the nucleus and acts as a transcriptional activator. Phylogenetic analysis indicates that SiMYB16 belongs to the R2R3-MYB transcription factor family subgroup 24. Transgenic rice expressing SiMYB16 (OX16) had a higher survival rate, lower malondialdehyde content, and heavier fresh weight compared with type (WT) under salt stress conditions. The transgenic plants also had a higher germination rate in salt treatment conditions and higher yield in the field compared with wild-type plants. Transcriptome analysis revealed that the up-regulated differential expression genes in the transgenic rice were mainly involved in phenylpropanoid biosynthesis, fatty acid elongation, phenylalanine metabolism, and flavonoid biosynthesis pathways. Quantitative real-time PCR analysis also showed that the genes encoding the major enzymes in the lignin and suberin biosynthesis pathways had higher expression level in SiMYB16 transgenic plants. Correspondingly, the content of flavonoid and lignin, and the activity of fatty acid synthase increased in SiMYB16 transgenic rice compared with wild-type plants under salt stress treatment. These results indicate that SiMYB16 gene can enhance plant salt tolerance by regulating the biosynthesis of lignin and suberin.
Assuntos
Oryza , Setaria (Planta) , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tolerância ao Sal/genética , Setaria (Planta)/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Filogenia , Lignina/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Flavonoides/metabolismo , SecasRESUMO
Allosteric activators are generally believed to shift the equilibrium distribution of enzyme conformations to favor a catalytically productive structure; the kinetics of conformational exchange is seldom addressed. Several observations suggested that the usual allosteric mechanism might not apply to the activation of IMP dehydrogenase (IMPDH) by monovalent cations. Therefore, we investigated the mechanism of K(+) activation in IMPDH by delineating the kinetic mechanism in the absence of monovalent cations. Surprisingly, the K(+) dependence of k(cat) derives from the rate of flap closure, which increases by ≥65-fold in the presence of K(+). We performed both alchemical free energy simulations and potential of mean force calculations using the orthogonal space random walk strategy to computationally analyze how K(+) accelerates this conformational change. The simulations recapitulate the preference of IMPDH for K(+), validating the computational models. When K(+) is replaced with a dummy ion, the residues of the K(+) binding site relax into ordered secondary structure, creating a barrier to conformational exchange. K(+) mobilizes these residues by providing alternate interactions for the main chain carbonyls. Potential of mean force calculations indicate that K(+) changes the shape of the energy well, shrinking the reaction coordinate by shifting the closed conformation toward the open state. This work suggests that allosteric regulation can be under kinetic as well as thermodynamic control.
Assuntos
Regulação Alostérica/efeitos dos fármacos , IMP Desidrogenase/química , Potássio/farmacologia , Simulação por Computador , Cryptosporidium parvum/enzimologia , Ativação Enzimática , IMP Desidrogenase/efeitos dos fármacos , IMP Desidrogenase/metabolismo , Cinética , Simulação de Dinâmica Molecular , Conformação Proteica/efeitos dos fármacosRESUMO
Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the biosynthesis of guanine nucleotides. This reaction involves two different chemical transformations, an NAD-linked redox reaction and a hydrolase reaction, that utilize mutually exclusive protein conformations with distinct catalytic residues. How did Nature construct such a complicated catalyst? Here we employ a "Wang-Landau" metadynamics algorithm in hybrid quantum mechanical/molecular mechanical (QM/MM) simulations to investigate the mechanism of the hydrolase reaction. These simulations show that the lowest energy pathway utilizes Arg418 as the base that activates water, in remarkable agreement with previous experiments. Surprisingly, the simulations also reveal a second pathway for water activation involving a proton relay from Thr321 to Glu431. The energy barrier for the Thr321 pathway is similar to the barrier observed experimentally when Arg418 is removed by mutation. The Thr321 pathway dominates at low pH when Arg418 is protonated, which predicts that the substitution of Glu431 with Gln will shift the pH-rate profile to the right. This prediction is confirmed in subsequent experiments. Phylogenetic analysis suggests that the Thr321 pathway was present in the ancestral enzyme, but was lost when the eukaryotic lineage diverged. We propose that the primordial IMPDH utilized the Thr321 pathway exclusively, and that this mechanism became obsolete when the more sophisticated catalytic machinery of the Arg418 pathway was installed. Thus, our simulations provide an unanticipated window into the evolution of a complex enzyme.
Assuntos
Aminoácidos/metabolismo , IMP Desidrogenase/química , Modelos Biológicos , Água/metabolismo , Substituição de Aminoácidos , Catálise , Simulação por Computador , Hidrolases/metabolismo , IMP Desidrogenase/metabolismo , Filogenia , Teoria Quântica , TermodinâmicaRESUMO
MADS-box transcription factors play vital roles in multiple biological processes in plants. At present, a comprehensive investigation into the genome-wide identification and classification of MADS-box genes in foxtail millet (Setaria italica L.) has not been reported. In this study, we identified 72 MADS-box genes in the foxtail millet genome and give an overview of the phylogeny, chromosomal location, gene structures, and potential functions of the proteins encoded by these genes. We also found that the expression of 10 MIKC-type MADS-box genes was induced by abiotic stresses (PEG-6000 and NaCl) and exogenous hormones (ABA and GA), which suggests that these genes may play important regulatory roles in response to different stresses. Further studies showed that transgenic Arabidopsis and rice (Oryza sativa L.) plants overexpressing SiMADS51 had reduced drought stress tolerance as revealed by lower survival rates and poorer growth performance under drought stress conditions, which demonstrated that SiMADS51 is a negative regulator of drought stress tolerance in plants. Moreover, expression of some stress-related genes were down-regulated in the SiMADS51-overexpressing plants. The results of our study provide an overall picture of the MADS-box gene family in foxtail millet and establish a foundation for further research on the mechanisms of action of MADS-box proteins with respect to abiotic stresses.