RESUMO
Drought is one of the main climate threats limiting crop production. Potato is one of the four most important food crop species worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect Arabidopsis and potato responses to drought by regulating the level of microRNA159 and, consequently, the levels of the MYB33 and MYB101 transcription factors (TFs). Here, we show that three MYB TFs, MYB33, MYB65, and MYB101, are involved in plant responses to water shortage. Their downregulation in Arabidopsis causes stomatal hyposensitivity to abscisic acid (ABA), leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing these genes, with a mutated recognition site in miR159, show hypersensitivity to ABA and relatively high tolerance to drought conditions. Thus, the MYB33, MYB65, and MYB101 genes may be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Solanum tuberosum , Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Secas , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácido Abscísico/farmacologia , Regulação da Expressão Gênica de Plantas/genética , MicroRNAs/genética , Água/metabolismo , Transdução de Sinais/genéticaRESUMO
Potato is one of the four most important food crop plants worldwide and is strongly affected by drought. The following two pairs of potato cultivars, which are related in ancestry but show different drought tolerances, were chosen for comparative gene expression studies: Gwiazda/Oberon and Tajfun/Owacja. Comparative RNA-seq analyses of gene expression differences in the transcriptomes obtained from drought-tolerant versus drought-sensitive plants during water shortage conditions were performed. The 23 top-ranking genes were selected, 22 of which are described here as novel potato drought-responsive genes. Moreover, all but one of the potato genes selected have homologues in the Arabidopsis genome. Of the seven tested A. thaliana mutants with altered expression of the selected homologous genes, compared to the wild-type Arabidopsis plants, six showed an improved tolerance to drought. These genes encode carbohydrate transporter, mitogen-activated protein kinase kinase kinase 15 (MAPKKK15), serine carboxypeptidase-like 19 protein (SCPL19), armadillo/beta-catenin-like repeat-containing protein, high-affinity nitrate transporter 2.7 and nonspecific lipid transfer protein type 2 (nsLPT). The evolutionary conservation of the functions of the selected genes in the plant response to drought confirms the importance of these identified potato genes in the ability of plants to cope with water shortage conditions. Knowledge regarding these gene functions can be used to generate potato cultivars that are resistant to unfavourable conditions. The approach used in this work and the obtained results allowed for the identification of new players in the plant response to drought.
Assuntos
Secas , Solanum tuberosum/metabolismo , Solanum tuberosum/fisiologia , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Solanum tuberosum/genéticaRESUMO
The use of artificial microRNAs (amiRNAs) is still a relatively new technique in molecular biology with a wide range of applications in life sciences. Here, we describe the silencing of the CBP80/ABH1 gene in Solanum tuberosum with the use of amiRNA. The CBP80/ABH1 protein is part of the Cap Binding Complex (CBC), which is involved in plant responses to drought stress conditions. Transformed plants with a decreased level of CBP80/ABH1 display increased tolerance to water shortage conditions. We describe how to design amiRNA with the Web MicroRNA Designer platform in detail. Additionally, we explain how to perform all steps of a procedure aiming to obtain transgenic potato plants with the use of designed amiRNA, through callus tissue regeneration and Agrobacterium tumefaciens strain LBA4404 as a transgene carrier.
Assuntos
Secas , MicroRNAs/genética , Solanum tuberosum/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Solanum tuberosum/fisiologiaRESUMO
Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants.
Assuntos
Secas , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/genética , Solanum tuberosum/genéticaRESUMO
In bryophytes (lower plants), sex determination is manifested in the gametophyte generation by the production of egg- and sperm-forming gametangia. We identified four genes specifically expressed in the male thalli of dioecious liverwort Pellia endiviifolia species B using RDA-cDNA method. These are: PenB_TUA1 coding for an α-tubulin family protein, PenB_Raba1/11 coding for a Rab family protein, PenB_HMG-box coding for an HMG-box family protein and PenB_MT coding for an unknown transcript that contains an ORF of 295 amino acid residues. The expression of identified genes shows developmental and environmental regulation. PenB_TUA1 and PenB_Raba1/11 are expressed in the male thalli, regardless of whether they develop antheridia. PenB_HMG-box and PenB_MT are exclusively expressed in the male thalli-producing antheridia while growing in the field. Moreover, two genes PenB_TUA1 and PenB_Raba1/11 are encoded only in the male genome of P. endiviifolia sp B. Our studies show for the first time the specific contribution of identified genes in the liverwort male gametophyte development. In higher plants, correct regulation of α-tubulin and Rab family genes activity is essential for tip-focused membrane trafficking and growth of the male gametophyte. Thus these genes are critical to the reproductive success of these plants. Plant HMG-box proteins bind DNA and may affect chromatin structure, promoting the assembly of nucleoprotein complexes that control DNA-dependent processes including transcription. Our results show that genes connected with the gametogenesis processes are evolutionarily conserved from the liverworts - the oldest living land plants, to higher plants.