RESUMO
Drought tolerance is an important trait being pursued by the agbiotech industry. Abscisic acid (ABA) is a stress hormone that mediates a multitude of processes in growth and development, water use efficiency (WUE) and gene expression during seed development and in response to environmental stresses. Arabidopsis B3-domain transcription factor Related to ABA-Insensitive3 (ABI3)/Viviparous1 (namely AtRAV2) and basic leucine zipper (bZIPs) AtABI5 or AtABF3 transactivated ABA-inducible promoter:GUS reporter expression in a maize mesophyll protoplast transient assay and showed synergies in reporter transactivation when coexpressed. Transgenic cotton (Gossypium hirsutum) expressing AtRAV1/2 and/or AtABI5 showed resistance to imposed drought stress under field and greenhouse conditions and exhibited improved photosynthesis and WUEs associated with absorption through larger root system and greater leaf area. We observed synergy for root biomass accumulation in the greenhouse, intrinsic WUE in the field and drought tolerance in stacked AtRAV and AtABI5 double-transgenic cotton. We assessed AtABI5 and AtRAV1/2 involvement in drought stress adaptations through reactive oxygen species scavenging and osmotic adjustment by marker gene expression in cotton. Deficit irrigation-grown AtRAV1/2 and AtABI5 transgenics had 'less-stressed' molecular and physiological phenotypes under drought, likely due to improved photoassimilation and root and shoot sink strengths and enhanced expression of endogenous GhRAV and genes for antioxidant and osmolyte biosynthesis. Overexpression of bZIP and RAV TFs could impact sustainable cotton agriculture and potentially other crops under limited irrigation conditions.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Arabidopsis/genética , Secas , Regulação da Expressão Gênica de Plantas , Gossypium/genética , Gossypium/fisiologia , Estresse Fisiológico/genética , Ácido Abscísico/metabolismo , Irrigação Agrícola , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Biomassa , Cruzamentos Genéticos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células do Mesofilo/metabolismo , Fotossíntese , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Protoplastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transformação Genética , Transgenes , Água , Zea mays/genéticaRESUMO
The 14,200 available full length Arabidopsis thaliana cDNAs in the universal plasmid system (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a "functional map-space" of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , DNA de Plantas/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Ácido Abscísico/metabolismo , DNA Complementar/genética , Perfilação da Expressão Gênica , Vetores Genéticos , Filogenia , Plantas Geneticamente Modificadas , Proteína Fosfatase 2C , Protoplastos/metabolismo , Transformação Genética , Zea mays/genética , Zea mays/metabolismoRESUMO
Abscisic acid-responsive gene expression is regulated by numerous transcription factors, including a subgroup of basic leucine zipper factors that bind to the conserved cis-acting sequences known as ABA-responsive elements. Although one of these factors, ABA-insensitive 5 (ABI5), was identified genetically, the paucity of genetic data for the other family members has left it unclear whether they perform unique functions or act redundantly to ABI5 or each other. To test for potential redundancy with ABI5, we identified the family members with most similar effects and interactions in transient expression systems (ABF3 and ABF1), then characterized loss-of-function lines for those loci. The abf1 and abf3 monogenic mutant lines had at most minimal effects on germination or seed-specific gene expression, but the enhanced ABA- and stress-resistance of abf3 abi5 double mutants revealed redundant action of these genes in multiple stress responses of seeds and seedlings. Although ABI5, ABF3, and ABF1 have some overlapping effects, they appear to antagonistically regulate each other's expression at specific stages. Consequently, loss of any one factor may be partially compensated by increased expression of other family members.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Ácido Abscísico/metabolismo , Relação Dose-Resposta a Droga , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter , Germinação/efeitos dos fármacos , Família Multigênica , Regiões Promotoras Genéticas , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Cloreto de Sódio/farmacologia , Sorbitol/farmacologia , Ativação TranscricionalRESUMO
Brassinosteroid (BR) homeostasis and signaling are crucial for normal growth and development of plants. BR signaling through cell-surface receptor kinases and intracellular components leads to dephosphorylation and accumulation of the nuclear protein BZR1. How BR signaling regulates gene expression, however, remains unknown. Here we show that BZR1 is a transcriptional repressor that has a previously unknown DNA binding domain and binds directly to the promoters of feedback-regulated BR biosynthetic genes. Microarray analyses identified additional potential targets of BZR1 and illustrated, together with physiological studies, that BZR1 coordinates BR homeostasis and signaling by playing dual roles in regulating BR biosynthesis and downstream growth responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Esteroides/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Retroalimentação Fisiológica , Genes de Plantas , Genes Reporter , Homeostase , Luz , Mutação , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reguladores de Crescimento de Plantas/biossíntese , Reguladores de Crescimento de Plantas/farmacologia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Esteroides/biossíntese , Esteroides/farmacologia , Transcrição GênicaAssuntos
Ácido Abscísico/farmacologia , Arabidopsis/genética , Reguladores de Crescimento de Plantas/farmacologia , Sementes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação/efeitos dos fármacos , Germinação/genética , Mutação , Fosforilação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Sementes/crescimento & desenvolvimento , Transdução de Sinais/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Abscisic acid (ABA) regulates seed maturation, germination, and adaptation of vegetative tissues to environmental stresses. The mechanisms of ABA action and the specificity conferred by signaling components in overlapping pathways are not completely understood. The ABI5 gene (ABA insensitive 5) of Arabidopsis encodes a basic leucine zipper factor required for ABA response in the seed and vegetative tissues. Using transient gene expression in rice protoplasts, we provide evidence for the functional interactions of ABI5 with ABA signaling effectors VP1 (viviparous 1) and ABI1 (ABA insensitive 1). Co-transformation experiments with ABI5 cDNA constructs resulted in specific transactivation of the ABA-inducible wheat Em, Arabidopsis AtEm6, bean beta-Phaseolin, and barley HVA1 and HVA22 promoters. Furthermore, ABI5 interacted synergistically with ABA and co-expressed VP1, indicating that ABI5 is involved in ABA-regulated transcription mediated by VP1. ABI5-mediated transactivation was inhibited by overexpression of abi1-1, the dominant-negative allele of the protein phosphatase ABI1, and by 1-butanol, a competitive inhibitor of phospholipase D involved in ABA signaling. Lanthanum, a trivalent ion that acts as an agonist of ABA signaling, potentiated ABI5 transactivation. These results demonstrate that ABI5 is a key target of a conserved ABA signaling pathway in plants.
