RESUMO
Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the Arabidopsis thaliana TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF OBP3-RESPONSIVE GENE 3 (ORG3). While grf9 knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing GRF9 (GRF9ox). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that GRF9 restricts cell proliferation in the early developing leaf. Performing in vitro binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified ORG3 as a direct downstream, and positively regulated target of GRF9. Genetic analysis of grf9 org3 and GRF9ox org3 double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Folhas de Planta/crescimento & desenvolvimento , Proteínas 14-3-3/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Proliferação de Células/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Redes Reguladoras de Genes/fisiologia , Folhas de Planta/citologia , Plantas Geneticamente Modificadas , Ligação Proteica/genéticaRESUMO
Salinity stress limits plant growth and has a major impact on agricultural productivity. Here, we identify NAC transcription factor SlTAF1 as a regulator of salt tolerance in cultivated tomato (Solanum lycopersicum). While overexpression of SlTAF1 improves salinity tolerance compared with wild-type, lowering SlTAF1 expression causes stronger salinity-induced damage. Under salt stress, shoots of SlTAF1 knockdown plants accumulate more toxic Na+ ions, while SlTAF1 overexpressors accumulate less ions, in accordance with an altered expression of the Na+ transporter genes SlHKT1;1 and SlHKT1;2. Furthermore, stomatal conductance and pore area are increased in SlTAF1 knockdown plants during salinity stress, but decreased in SlTAF1 overexpressors. We identified stress-related transcription factor, abscisic acid metabolism and defence-related genes as potential direct targets of SlTAF1, correlating it with reactive oxygen species scavenging capacity and changes in hormonal response. Salinity-induced changes in tricarboxylic acid cycle intermediates and amino acids are more pronounced in SlTAF1 knockdown than wild-type plants, but less so in SlTAF1 overexpressors. The osmoprotectant proline accumulates more in SlTAF1 overexpressors than knockdown plants. In summary, SlTAF1 controls the tomato's response to salinity stress by combating both osmotic stress and ion toxicity, highlighting this gene as a promising candidate for the future breeding of stress-tolerant crops.
Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Estresse Salino/fisiologia , Solanum lycopersicum/metabolismo , Técnicas de Silenciamento de Genes , Homeostase , Transporte de Íons/genética , Transporte de Íons/fisiologia , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Raízes de Plantas , Brotos de Planta , Potássio , Estresse Salino/genética , Sódio , Cloreto de Sódio/toxicidadeRESUMO
Leaf senescence is an essential physiological process in plants that supports the recycling of nitrogen and other nutrients to support the growth of developing organs, including young leaves, seeds, and fruits. Thus, the regulation of senescence is crucial for evolutionary success in wild populations and for increasing yield in crops. Here, we describe the influence of a NAC transcription factor, SlNAP2 (Solanum lycopersicum NAC-like, activated by Apetala3/Pistillata), that controls both leaf senescence and fruit yield in tomato (S. lycopersicum). SlNAP2 expression increases during age-dependent and dark-induced leaf senescence. We demonstrate that SlNAP2 activates SlSAG113 (S. lycopersicum SENESCENCE-ASSOCIATED GENE113), a homolog of Arabidopsis (Arabidopsis thaliana) SAG113, chlorophyll degradation genes such as SlSGR1 (S. lycopersicum senescence-inducible chloroplast stay-green protein 1) and SlPAO (S. lycopersicum pheide a oxygenase), and other downstream targets by directly binding to their promoters, thereby promoting leaf senescence. Furthermore, SlNAP2 directly controls the expression of genes important for abscisic acid (ABA) biosynthesis, S. lycopersicum 9-cis-epoxycarotenoid dioxygenase 1 (SlNCED1); transport, S. lycopersicum ABC transporter G family member 40 (SlABCG40); and degradation, S. lycopersicum ABA 8'-hydroxylase (SlCYP707A2), indicating that SlNAP2 has a complex role in establishing ABA homeostasis during leaf senescence. Inhibiting SlNAP2 expression in transgenic tomato plants impedes leaf senescence but enhances fruit yield and sugar content likely due to prolonged leaf photosynthesis in aging tomato plants. Our data indicate that SlNAP2 has a central role in controlling leaf senescence and fruit yield in tomato.
Assuntos
Frutas/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Ácido Abscísico/genética , Ácido Abscísico/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Escuridão , Dioxigenases/genética , Dioxigenases/metabolismo , Frutas/genética , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Fatores de Transcrição/genéticaRESUMO
Calcium (Ca2+ ) is a universal messenger that mediates intracellular responses to extracellular stimuli in living organisms. Calmodulin (CaM) and calmodulin-like (CML) proteins are the important Ca2+ sensors in plants that decode Ca2+ -signatures to execute downstream intracellular level responses. Several studies indicate the interlinking of Ca2+ and sugar signaling in plants; however, no genes have been functionally characterized to provide molecular evidence. Our study found that expression of TaCML20 was significantly correlated with water soluble carbohydrate (WSC) concentrations in recombinant inbred lines in wheat. TaCML20 has four EF-hand motifs that may facilitate the binding of Ca2+ . To explore the role of CML20, we generated TaCML20 overexpressing transgenic lines in wheat. These lines accumulated higher WSC concentrations in the shoots, and we also found a significantly increased transcript level of sucrose:sucrose-1-fructosyltransferase (1-SST) in the internodes compared with the control plants. In addition, TaCML20 overexpressing plants showed significantly increased tillers per plant and also increased about 19% of grain weight per plant compared with control plants. The results also suggested a role for TaCML20 in drought stress, as its transcripts significantly increased in the shoots of wild-type plants under water deficit. These results uncovered the role of CML20 in determining multiple traits in wheat.
Assuntos
Calmodulina/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Triticum/metabolismo , Água/metabolismo , Carboidratos , Grão Comestível/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genéticaRESUMO
Leaf senescence is a key process in plants that culminates in the degradation of cellular constituents and massive reprogramming of metabolism for the recovery of nutrients from aged leaves for their reuse in newly developing sinks. We used molecular-biological and metabolomics approaches to identify NAC transcription factor (TF) RD26 as an important regulator of metabolic reprogramming in Arabidopsis thaliana. RD26 directly activates CHLOROPLAST VESICULATION (CV), encoding a protein crucial for chloroplast protein degradation, concomitant with an enhanced protein loss in RD26 overexpressors during senescence, but a reduced decline of protein in rd26 knockout mutants. RD26 also directly activates LKR/SDH involved in lysine catabolism, and PES1 important for phytol degradation. Metabolic profiling revealed reduced γ-aminobutyric acid (GABA) in RD26 overexpressors, accompanied by the induction of respective catabolic genes. Degradation of lysine, phytol and GABA is instrumental for maintaining mitochondrial respiration in carbon-limiting conditions during senescence. RD26 also supports the degradation of starch and the accumulation of mono- and disaccharides during senescence by directly enhancing the expression of AMY1, SFP1 and SWEET15 involved in carbohydrate metabolism and transport. Collectively, during senescence RD26 acts by controlling the expression of genes across the entire spectrum of the cellular degradation hierarchy.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Escuridão , Fatores de Transcrição/metabolismo , Aminoácidos/metabolismo , Arabidopsis/genética , Sítios de Ligação , Proteínas de Cloroplastos/metabolismo , Ciclo do Ácido Cítrico , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Metaboloma , Modelos Biológicos , Fitol/metabolismo , Plantas Geneticamente Modificadas , Proteólise , Plântula/genética , Plântula/crescimento & desenvolvimento , Açúcares/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
High temperature at grain filling can severely reduce wheat yield. Heat shock factors (Hsfs) are central regulators in heat acclimation. This study investigated the role of TaHsfC2a, a member of the monocot-specific HsfC2 subclass, in the regulation of heat protection genes in Triticum aestivum. Three TaHsfC2a homoeologous genes were highly expressed in wheat grains during grain filling and showed only transient up-regulation in the leaves by heat stress but were markedly up-regulated by drought and abscisic acid (ABA) treatment. Overexpression of TaHsfC2a-B in transgenic wheat resulted in up-regulation of a suite of heat protection genes (e.g. TaHSP70d and TaGalSyn). Most TaHsfC2a-B target genes were heat, drought and ABA inducible. Transactivation analysis of two representative targets (TaHSP70d and TaGalSyn) showed that TaHsfC2a-B activated expression of reporters driven by these target promoters. Promoter mutagenesis analyses revealed that heat shock element is responsible for transactivation by TaHsfC2a-B and heat/drought induction. TaHsfC2a-B-overexpressing wheat showed improved thermotolerance but not dehydration tolerance. Most TaHsfC2a-B target genes were co-up-regulated in developing grains with TaHsfC2a genes. These data suggest that TaHsfC2a-B is a transcriptional activator of heat protection genes and serves as a proactive mechanism for heat protection in developing wheat grains via the ABA-mediated regulatory pathway.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Triticum/crescimento & desenvolvimento , Triticum/metabolismo , Sequência de Bases , Secas , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Temperatura Alta , Estresse Oxidativo/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Sintase do Amido/metabolismo , Estresse Fisiológico/genética , Termotolerância/genética , Ativação Transcricional/genética , Triticum/genética , Regulação para Cima/genéticaRESUMO
Responses to pathogens, including host transcriptional reprogramming, require partially antagonistic signalling pathways dependent on the phytohormones salicylic (SA) and jasmonic (JA) acids. However, upstream factors modulating the interplay of these pathways are not well characterized. Here, we identify the transcription factor ANAC032 from Arabidopsis thaliana as one such regulator in response to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). ANAC032 directly represses MYC2 activation upon Pst attack, resulting in blockage of coronatine-mediated stomatal reopening which restricts entry of bacteria into plant tissue. Furthermore, ANAC032 activates SA signalling by repressing NIMIN1, a key negative regulator of SA-dependent defence. Finally, ANAC032 reduces expression of JA-responsive genes, including PDF1.2A Thus, ANAC032 enhances resistance to Pst by generating an orchestrated transcriptional output towards key SA- and JA-signalling genes coordinated through direct binding of ANAC032 to the MYC2, NIMIN1 and PDF1.2A promoters.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas syringae/patogenicidade , Ácido Salicílico/metabolismo , Transativadores/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Defensinas/genética , Defensinas/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Transdução de Sinais/genética , Transativadores/genética , Fatores de TranscriçãoRESUMO
KEY MESSAGE: TaRNAC1 is a constitutively and predominantly root-expressed NAC transcription factor. TaRNAC1 overexpression in wheat roots confers increased root length, biomass and drought tolerance and improved grain yield under water limitation. A large and deep root system is an important trait for yield sustainability of dryland cereal crops in drought-prone environments. This study investigated the role of a predominantly root-expressed NAC transcription factor from wheat (TaRNAC1) in the root growth. Expression analysis showed that TaRNAC1 was a constitutively expressed gene with high level expression in the roots and was not drought-upregulated. Overexpression of TaRNAC1 in wheat using a predominantly root-expressed promoter resulted in increased root length and biomass observed at the early growth stage and a marked increase in the maturity root biomass with dry root weight of > 70% higher than that of the wild type plants. Analysis of some root growth-related genes revealed that the expression level of GA3-ox2, which encodes GIBBERELLIN 3-OXIDASE catalysing the conversion of inactive gibberellin (GA) to active GA, was elevated in the roots of transgenic wheat. TaRNAC1 overexpressing transgenic wheat showed more dehydration tolerance under polyethylene glycol (PEG) treatment and produced more aboveground biomass and grain under water-limited conditions than the wild type plants. These data suggest that TaRNAC1 may play a role in root growth and be used as a molecular tool for potential enlargement of root system in wheat.
Assuntos
Biomassa , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/genética , Fatores de Transcrição/genética , Triticum/genética , Adaptação Fisiológica/genética , Secas , Giberelinas/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/metabolismo , Triticum/crescimento & desenvolvimento , Triticum/metabolismoRESUMO
A well-known physiological adaptation process of plants encountering drying soil is to achieve water balance by reducing shoot growth and maintaining or promoting root elongation, but little is known about the molecular basis of this process. This study investigated the role of a drought-up-regulated Triticum aestivum NAC69-1 (TaNAC69-1) in the modulation of root growth in wheat. TaNAC69-1 was predominantly expressed in wheat roots at the early vegetative stage. Overexpression of TaNAC69-1 in wheat roots using OsRSP3 (essentially root-specific) and OsPIP2;3 (root-predominant) promoters resulted in enhanced primary seminal root length and a marked increase in maturity root biomass. Competitive growth analysis under water-limited conditions showed that OsRSP3 promoter-driven TaNAC69-1 transgenic lines produced 32% and 35% more above-ground biomass and grains than wild-type plants, respectively. TaNAC69-1 overexpression in the roots down-regulated the expression of TaSHY2 and TaIAA7, which are from the auxin/IAA (Aux/IAA) transcriptional repressor gene family and are the homologs of negative root growth regulators SHY2/IAA3 and IAA7 in Arabidopsis. The expression of TaSHY2 and TaIAA7 in roots was down-regulated by drought stress and up-regulated by cytokinin treatment, which inhibited root growth. DNA binding and transient expression analyses revealed that TaNAC69-1 bound to the promoters of TaSHY2 and TaIAA7, acted as a transcriptional repressor and repressed the expression of reporter genes driven by the TaSHY2 or TaIAA7 promoter. These data suggest that TaNAC69-1 is a transcriptional repressor of TaSHY2 and TaIAA7 homologous to Arabidopsis negative root growth regulators and is likely to be involved in promoting root elongation in drying soil.
Assuntos
Biomassa , Secas , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Transcrição Gênica , Triticum/genética , Triticum/fisiologia , Regulação para Cima/genética , Biotinilação , Núcleo Celular/metabolismo , Citocininas/farmacologia , DNA de Plantas/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/metabolismo , Estresse Fisiológico/genética , Transcrição Gênica/efeitos dos fármacos , Triticum/anatomia & histologia , Triticum/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Senescence represents a fundamental process of late leaf development. Transcription factors (TFs) play an important role for expression reprogramming during senescence; however, the gene regulatory networks through which they exert their functions, and their physiological integration, are still largely unknown. Here, we identify the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)- and hydrogen peroxide-activated TF Arabidopsis thaliana activating factor1 (ATAF1) as a novel upstream regulator of senescence. ATAF1 executes its physiological role by affecting both key chloroplast maintenance and senescence-promoting TFs, namely GOLDEN2-LIKE1 (GLK1) and ORESARA1 (Arabidopsis NAC092), respectively. Notably, while ATAF1 activates ORESARA1, it represses GLK1 expression by directly binding to their promoters, thereby generating a transcriptional output that shifts the physiological balance toward the progression of senescence. We furthermore demonstrate a key role of ATAF1 for ABA- and hydrogen peroxide-induced senescence, in accordance with a direct regulatory effect on ABA homeostasis genes, including nine-CIS-epoxycarotenoid dioxygenase3 involved in ABA biosynthesis and ABC transporter G family member40, encoding an ABA transport protein. Thus, ATAF1 serves as a core transcriptional activator of senescence by coupling stress-related signaling with photosynthesis- and senescence-related transcriptional cascades.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Cloroplastos/genética , Proteínas Repressoras/metabolismo , Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Escuridão , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Homeostase/efeitos dos fármacos , Homeostase/genética , Peróxido de Hidrogênio/farmacologia , Modelos Biológicos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Proteínas Repressoras/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
MYB transcription factors (TFs) are important regulators of flavonoid biosynthesis in plants. Here, we report MYB112 as a formerly unknown regulator of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana). Expression profiling after chemically induced overexpression of MYB112 identified 28 up- and 28 down-regulated genes 5 h after inducer treatment, including MYB7 and MYB32, which are both induced. In addition, upon extended induction, MYB112 also positively affects the expression of PRODUCTION OF ANTHOCYANIN PIGMENT1, a key TF of anthocyanin biosynthesis, but acts negatively toward MYB12 and MYB111, which both control flavonol biosynthesis. MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative polymerase chain reaction, we show that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters, revealing them as direct downstream target genes. We further show that MYB112 expression is up-regulated by salinity and high light stress, environmental parameters that both require the MYB112 TF for anthocyanin accumulation under these stresses. In contrast to several other MYB TFs affecting anthocyanin biosynthesis, MYB112 expression is not controlled by nitrogen limitation or an excess of carbon. Thus, MYB112 constitutes a regulator that promotes anthocyanin accumulation under abiotic stress conditions.
Assuntos
Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Luz , Regiões Promotoras Genéticas/genética , Salinidade , Fatores de Transcrição/genéticaRESUMO
In rosette plants, root flooding (waterlogging) triggers rapid upward (hyponastic) leaf movement representing an important architectural stress response that critically determines plant performance in natural habitats. The directional growth is based on localized longitudinal cell expansion at the lower (abaxial) side of the leaf petiole and involves the volatile phytohormone ethylene (ET). We report the existence of a transcriptional core unit underlying directional petiole growth in Arabidopsis thaliana, governed by the NAC transcription factor speedy hyponastic growth (SHYG). Overexpression of SHYG in transgenic Arabidopsis thaliana enhances waterlogging-triggered hyponastic leaf movement and cell expansion in abaxial cells of the basal petiole region, while both responses are largely diminished in shyg knockout mutants. Expression of several expansin and xyloglucan endotransglycosylase/hydrolase genes encoding cell wall-loosening proteins was enhanced in SHYG overexpressors but lowered in shyg. We identified ACC oxidase5 (ACO5), encoding a key enzyme of ET biosynthesis, as a direct transcriptional output gene of SHYG and found a significantly reduced leaf movement in response to root flooding in aco5 T-DNA insertion mutants. Expression of SHYG in shoot tissue is triggered by root flooding and treatment with ET, constituting an intrinsic ET-SHYG-ACO5 activator loop for rapid petiole cell expansion upon waterlogging.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Estresse Fisiológico , Fatores de Transcrição/fisiologia , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Crescimento Celular , Regulação da Expressão Gênica de Plantas , Mutagênese Insercional , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , ÁguaRESUMO
KEY MESSAGE: A strong, stable and root-specific expression system was developed from a rice root-specific GLYCINE - RICH PROTEIN 7 promoter for use as an enabling technology for genetic manipulation of wheat root traits. Root systems play an important role in wheat productivity. Genetic manipulation of wheat root traits often requires a root-specific or root-predominant expression system as an essential enabling technology. In this study, we investigated promoters from rice root-specific or root-predominant expressed genes for development of a root expression system in bread wheat. Transient expression analysis using a GREEN FLUORESCENT PROTEIN (GFP) reporter gene driven by rice promoters identified six promoters that were strongly expressed in wheat roots. Extensive organ specificity analysis of three rice promoters in transgenic wheat revealed that the promoter of rice GLYCINE-RICH PROTEIN 7 (OsGRP7) gene conferred a root-specific expression pattern in wheat. Strong GFP fluorescence in the seminal and branch roots of wheat expressing GFP reporter driven by the OsGRP7 promoter was detected in epidermal, cortical and endodermal cells in mature parts of the root. The GFP reporter driven by the promoter of rice METALLOTHIONEIN-LIKE PROTEIN 1 (OsMTL1) gene was mainly expressed in the roots with essentially no expression in the leaf, stem or seed. However, it was also expressed in floral organs including glume, lemma, palea and awn. In contrast, strong expression of rice RCg2 promoter-driven GFP was found in many tissues. The GFP expression driven by these three rice promoters was stable in transgenic wheat plants through three generations (T1-T3) examined. These data suggest that the OsGRP7 promoter can provide a strong, stable and root-specific expression system for use as an enabling technology for genetic manipulation of wheat root traits.
Assuntos
Expressão Gênica , Técnicas Genéticas , Raízes de Plantas/genética , Transgenes/genética , Triticum/genética , Raízes de Plantas/metabolismo , Triticum/metabolismoRESUMO
Heat stress is a significant environmental factor adversely affecting crop yield. Crop adaptation to high-temperature environments requires transcriptional reprogramming of a suite of genes involved in heat stress protection. This study investigated the role of TaHsfA6f, a member of the A6 subclass of heat shock transcription factors, in the regulation of heat stress protection genes in Triticum aestivum (bread wheat), a poorly understood phenomenon in this crop species. Expression analysis showed that TaHsfA6f was expressed constitutively in green organs but was markedly up-regulated during heat stress. Overexpression of TaHsfA6f in transgenic wheat using a drought-inducible promoter resulted in up-regulation of heat shock proteins (HSPs) and a number of other heat stress protection genes that included some previously unknown Hsf target genes such as Golgi anti-apoptotic protein (GAAP) and the large isoform of Rubisco activase. Transgenic wheat plants overexpressing TaHsfA6f showed improved thermotolerance. Transactivation assays showed that TaHsfA6f activated the expression of reporter genes driven by the promoters of several HSP genes (TaHSP16.8, TaHSP17, TaHSP17.3, and TaHSP90.1-A1) as well as TaGAAP and TaRof1 (a co-chaperone) under non-stress conditions. DNA binding analysis revealed the presence of high-affinity TaHsfA6f-binding heat shock element-like motifs in the promoters of these six genes. Promoter truncation and mutagenesis analyses identified TaHsfA6f-binding elements that were responsible for transactivation of TaHSP90.1-A1 and TaGAAP by TaHsfA6f. These data suggest that TaHsfA6f is a transcriptional activator that directly regulates TaHSP, TaGAAP, and TaRof1 genes in wheat and its gene regulatory network has a positive impact on thermotolerance.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Choque Térmico/genética , Proteínas de Plantas/genética , Triticum/genética , Sequência de Aminoácidos , Sequência de Bases , Genes Reporter , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Alinhamento de Sequência , Triticum/química , Triticum/metabolismoRESUMO
In land plants, the NAD(P)H dehydrogenase (NDH) complex reduces plastoquinones and drives cyclic electron flow (CEF) around PSI. It also produces extra ATP for photosynthesis and improves plant fitness under conditions of abiotic environmental stress. To elucidate the role of CEF in salt tolerance of the photosynthetic apparatus, Na(+) concentration, chlorophyll fluorescence, and expression of NDH B and H subunits, as well as of genes related to cellular and vacuolar Na(+) transport, were monitored. The salt-tolerant Glycine max (soybean) variety S111-9 exhibited much higher CEF activity and ATP accumulation in light than did the salt-sensitive variety Melrose, but similar leaf Na(+) concentrations under salt stress. In S111-9 plants, ndhB and ndhH were highly up-regulated under salt stress and their corresponding proteins were maintained at high levels or increased significantly. Under salt stress, S111-9 plants accumulated Na(+) in the vacuole, but Melrose plants accumulated Na(+) in the chloroplast. Compared with Melrose, S111-9 plants also showed higher expression of some genes associated with Na(+) transport into the vacuole and/or cell, such as genes encoding components of the CBL10 (calcineurin B-like protein 10)-CIPK24 (CBL-interacting protein kinase 24)-NHX (Na(+)/H(+) antiporter) and CBL4 (calcineurin B-like protein 4)-CIPK24-SOS1 (salt overly sensitive 1) complexes. Based on the findings, it is proposed that enhanced NDH-dependent CEF supplies extra ATP used to sequester Na(+) in the vacuole. This reveals an important mechanism for salt tolerance in soybean and provides new insights into plant resistance to salt stress.
Assuntos
Trifosfato de Adenosina/biossíntese , Glycine max/fisiologia , Tolerância ao Sal , Sódio/metabolismo , Transporte de Elétrons , Células do Mesofilo/metabolismo , NADPH Desidrogenase/genética , NADPH Desidrogenase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Glycine max/genética , Estresse Fisiológico , Vacúolos/metabolismoRESUMO
The transition from juvenility through maturation to senescence is a complex process that involves the regulation of longevity. Here, we identify JUNGBRUNNEN1 (JUB1), a hydrogen peroxide (H(2)O(2))-induced NAC transcription factor, as a central longevity regulator in Arabidopsis thaliana. JUB1 overexpression strongly delays senescence, dampens intracellular H(2)O(2) levels, and enhances tolerance to various abiotic stresses, whereas in jub1-1 knockdown plants, precocious senescence and lowered abiotic stress tolerance are observed. A JUB1 binding site containing a RRYGCCGT core sequence is present in the promoter of DREB2A, which plays an important role in abiotic stress responses. JUB1 transactivates DREB2A expression in mesophyll cell protoplasts and transgenic plants and binds directly to the DREB2A promoter. Transcriptome profiling of JUB1 overexpressors revealed elevated expression of several reactive oxygen species-responsive genes, including heat shock protein and glutathione S-transferase genes, whose expression is further induced by H(2)O(2) treatment. Metabolite profiling identified elevated Pro and trehalose levels in JUB1 overexpressors, in accordance with their enhanced abiotic stress tolerance. We suggest that JUB1 constitutes a central regulator of a finely tuned control system that modulates cellular H(2)O(2) level and primes the plants for upcoming stress through a gene regulatory network that involves DREB2A.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Sítios de Ligação , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Peróxido de Hidrogênio/farmacologia , Metaboloma , Mutagênese Insercional , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Regiões Promotoras Genéticas , Estresse Fisiológico , Fatores de Transcrição/genética , TranscriptomaRESUMO
Heat shock factors (Hsfs) play a central regulatory role in acquired thermotolerance. To understand the role of the major molecular players in wheat adaptation to heat stress, the Hsf family was investigated in Triticum aestivum. Bioinformatic and phylogenetic analyses identified 56 TaHsf members, which are classified into A, B, and C classes. Many TaHsfs were constitutively expressed. Subclass A6 members were predominantly expressed in the endosperm under non-stress conditions. Upon heat stress, the transcript levels of A2 and A6 members became the dominant Hsfs, suggesting an important regulatory role during heat stress. Many TaHsfA members as well as B1, C1, and C2 members were also up-regulated during drought and salt stresses. The heat-induced expression profiles of many heat shock protein (Hsp) genes were paralleled by those of A2 and A6 members. Transactivation analysis revealed that in addition to TaHsfA members (A2b and A4e), overexpression of TaHsfC2a activated expression of TaHsp promoter-driven reporter genes under non-stress conditions, while TaHsfB1b and TaHsfC1b did not. Functional heat shock elements (HSEs) interacting with TaHsfA2b were identified in four TaHsp promoters. Promoter mutagenesis analysis demonstrated that an atypical HSE (GAACATTTTGGAA) in the TaHsp17 promoter is functional for heat-inducible expression and transactivation by Hsf proteins. The transactivation of Hsp promoter-driven reporter genes by TaHsfC2a also relied on the presence of HSE. An activation motif in the C-terminal domain of TaHsfC2a was identified by amino residue substitution analysis. These data demonstrate the role of HsfA and HsfC2 in regulation of Hsp genes in wheat.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Família Multigênica , Estresse Fisiológico/genética , Triticum/genética , Triticum/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Secas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Genes Reporter , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/classificação , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Dados de Sequência Molecular , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Transativadores/metabolismo , Triticum/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Plastid casein kinase 2 (CK2) is a major Ser/Thr-specific enzyme for protein phosphorylation in the chloroplast stroma and its kinase activity is regulated by redox signals. To understand the role of CK2 phosphorylation of chloroplast proteins in abiotic stress signalling, an Arabidopsis plastid CK2 (CKA4) knockout mutant was investigated in terms of the plant response to abscisic acid (ABA) and heat stress. CKA4 expression was upregulated by ABA and heat treatment. The cka4 mutant showed reduced sensitivity to ABA during seed germination and seedling growth, and increased stomatal aperture and leaf water loss with a slightly reduced leaf ABA level. The cka4 mutant was more sensitive to heat stress than the wild-type Columbia-0. The expression levels of a number of genes in the ABA regulatory network were reduced in the cka4 mutant. Many heat-upregulated genes (heat-shock factors and heat-shock proteins) were also reduced in the cka4 mutant. The cka4 mutant showed reduced expression levels of plastid-encoded RNA polymerase target genes (atpB and psbA). CKA4 knockout mutation also resulted in a reduction in expression of some critical genes (PTM, ABI4, and PRS1) involved in retrograde signalling from the chloroplast to the nucleus. Similar results were observed in mutant plants with the knockout mutation in both CKA4 and CKA3, which encodes a nuclear CK2 α3 subunit. CKA3 expression was not responsive to ABA and heat stress. These results suggest that CKA4 is an enhancing factor in abiotic stress signalling through modulating the expression of some molecular players in retrograde signalling.
Assuntos
Ácido Abscísico/fisiologia , Arabidopsis/enzimologia , Caseína Quinase II/metabolismo , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Caseína Quinase II/genética , Técnicas de Inativação de Genes , Temperatura Alta , Malondialdeído/metabolismo , Estômatos de Plantas/fisiologia , Prolina/metabolismo , Água/fisiologiaRESUMO
Fructans represent the major component of water soluble carbohydrates (WSCs) in the maturing stem of temperate cereals and are an important temporary carbon reserve for grain filling. To investigate the importance of source carbon availability in fructan accumulation and its molecular basis, we performed comparative analyses of WSC components and the expression profiles of genes involved in major carbohydrate metabolism and photosynthesis in the flag leaves of recombinant inbred lines from wheat cultivars Seri M82 and Babax (SB lines). High sucrose levels in the mature flag leaf (source organ) were found to be positively associated with WSC and fructan concentrations in both the leaf and stem of SB lines in several field trials. Analysis of Affymetrix expression array data revealed that high leaf sucrose lines grown in abiotic-stress-prone environments had high expression levels of a number of genes in the leaf involved in the sucrose synthetic pathway and photosynthesis, such as Calvin cycle genes, antioxidant genes involved in chloroplast H(2)O(2) removal and genes involved in energy dissipation. The expression of the majority of genes involved in fructan and starch synthetic pathways were positively correlated with sucrose levels in the leaves of SB lines. The high level of leaf fructans in high leaf sucrose lines is likely attributed to the elevated expression levels of fructan synthetic enzymes, as the mRNA levels of three fructosyltransferase families were consistently correlated with leaf sucrose levels among SB lines. These data suggest that high source strength is one of the important genetic factors determining high levels of WSC in wheat.
Assuntos
Frutanos/metabolismo , Triticum/genética , Triticum/metabolismo , Trifosfato de Adenosina/biossíntese , Antioxidantes/metabolismo , Metabolismo dos Carboidratos , Cloroplastos/metabolismo , Genes de Plantas , Variação Genética , Genótipo , Peróxido de Hidrogênio/metabolismo , Redes e Vias Metabólicas , Modelos Biológicos , Fotossíntese/genética , Folhas de Planta/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Solubilidade , Amido/biossíntese , Sacarose/metabolismo , TranscriptomaRESUMO
Fructans are the major component of temporary carbon reserve in the stem of temperate cereals, which is used for grain filling. Three families of fructosyltransferases are directly involved in fructan synthesis in the vacuole of Triticum aestivum. The regulatory network of the fructan synthetic pathway is largely unknown. Recently, a sucrose-upregulated wheat MYB transcription factor (TaMYB13-1) was shown to be capable of activating the promoter activities of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT) in transient transactivation assays. This work investigated TaMYB13-1 target genes and their influence on fructan synthesis in transgenic wheat. TaMYB13-1 overexpression resulted in upregulation of all three families of fructosyltransferases including fructan:fructan 1-fructosyltransferase (1-FFT). A γ-vacuolar processing enzyme (γ-VPE1), potentially involved in processing the maturation of fructosyltransferases in the vacuole, was also upregulated by TaMYB13-1 overexpression. Multiple TaMYB13 DNA-binding motifs were identified in the Ta1-FFT1 and Taγ-VPE1 promoters and were bound strongly by TaMYB13-1. The expression profiles of these target genes and TaMYB13-1 were highly correlated in recombinant inbred lines and during stem development as well as the transgenic and non-transgenic wheat dataset, further supporting a direct regulation of these genes by TaMYB13-1. TaMYB13-1 overexpression in wheat led to enhanced fructan accumulation in the leaves and stems and also increased spike weight and grain weight per spike in transgenic plants under water-limited conditions. These data suggest that TaMYB13-1 plays an important role in coordinated upregulation of genes necessary for fructan synthesis and can be used as a molecular tool to improve the high fructan trait.