RESUMO
Plant expansins are structural cell wall-loosening proteins implicated in several developmental processes and responses to environmental constraints and pathogen infection. To date, there is limited information about the biological function of expansins-like B (EXLBs), one of the smallest and less-studied subfamilies of plant expansins. In the present study, we conducted a functional analysis of the wild Arachis AdEXLB8 gene in transgenic tobacco (Nicotiana tabacum) plants to clarify its putative role in mediating defense responses to abiotic and biotic stresses. First, its cell wall localization was confirmed in plants expressing an AdEXLB8:eGFP fusion protein, while nanomechanical assays indicated cell wall reorganization and reassembly due to AdEXLB8 overexpression without compromising the phenotype. We further demonstrated that AdEXLB8 increased tolerance not only to isolated abiotic (drought) and biotic (Sclerotinia sclerotiorum and Meloidogyne incognita) stresses but also to their combination. The jasmonate and abscisic acid signaling pathways were clearly favored in transgenic plants, showing an activated antioxidative defense system. In addition to modifications in the biomechanical properties of the cell wall, we propose that AdEXLB8 overexpression interferes with phytohormone dynamics leading to a defense primed state, which culminates in plant defense responses against isolated and combined abiotic and biotic stresses.
Assuntos
Arachis/genética , Nicotiana/fisiologia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Ácido Abscísico/metabolismo , Animais , Ascomicetos/patogenicidade , Fenômenos Biomecânicos , Parede Celular/genética , Parede Celular/metabolismo , Ciclopentanos/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Células Vegetais/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Nicotiana/citologia , Nicotiana/genética , Nicotiana/microbiologia , Tylenchoidea/patogenicidadeRESUMO
Arachis stenosperma is a wild peanut relative exclusive to South America that harbors high levels of resistance against several pathogens, including the peanut root-knot nematode (RKN) Meloidogyne arenaria. In this study, a proteomic survey of A. stenosperma-M. arenaria interaction using 2-DE and LC-MS/MS identified approximately 1400 proteins, out of which 222 were differentially abundant (DAPs) when RKN inoculated root samples were compared to the control. Most of these DAPs were assigned to functional categories related to plant responses to pathogens including stress, glycolysis, redox and tricarboxylic acid cycle. The comparison between the transcriptome (RNA-Seq) and proteome expression changes, showed that almost 55% of these DAPs encode genes with a similar expression trend to their protein counterparts. Most of these genes were induced during RKN infection and some were related to plant defense, such as MLP-like protein 34 (MLP34), cinnamoyl-CoA reductase 1 (CCR1), enolase (ENO), alcohol dehydrogenase (ADH) and eukaryotic translation initiation factor 5A (eIF5A). The overexpression of AsMLP34 in Agrobacterium rhizogenes transgenic roots in a susceptible peanut cultivar showed a reduction in the number of M. arenaria galls and egg masses, indicating that AsMLP34 is a promising candidate gene to be exploited in breeding programs for RKN control in peanut. SIGNIFICANCE: The use of an integrated approach to compare plant-nematode transcriptional and translational data enabled the identification of a new gene, AsMLP34, for Meloidogyne resistance.
Assuntos
Tylenchoidea , Agrobacterium , Animais , Arachis/genética , Cromatografia Líquida , Resistência à Doença/genética , Melhoramento Vegetal , Doenças das Plantas/genética , Raízes de Plantas , Proteômica , América do Sul , Espectrometria de Massas em TandemRESUMO
Peanut wild relatives (Arachis spp.) have high genetic diversity and are important sources of resistance to biotic and abiotic stresses. In this study, proteins were analyzed in root tissues of A. duranensis submitted to a progressive water deficit in soil and the differential abundance was compared to transcript expression profiles obtained by RNA-seq and qRT-PCR. Using a 2-DE approach, a total of 31 proteins were identified, most of which were associated with stress response and drought perception. These comprised a chitinase-2 (unique to stressed condition), an MLP-like protein, a glycine-rich protein DOT1-like, a maturase K and heat shock-related proteins (HSP70 - an isoform unique to the control, and HSP17.3). Other proteins unique to the control condition comprised a transcription initiation factor IIF subunit alpha-like protein, a SRPBCC ligand-binding domain superfamily protein, an Adenine phosphoribosyl transferase, a Leo1-like protein, a Cobalamine-independent methionine synthase and a Transmembrane emp24 domain-containing protein p24delta9-like. Correlation of mRNA expression and corresponding protein abundance was observed for 15 of the identified proteins, with genes encoding the majority of proteins (14) negatively regulated in stressed roots. Proteins identified in this study offer potential for the genetic improvement of cultivated peanut for drought tolerance. SIGNIFICANCE: The comparison of protein abundance and corresponding transcript expression levels (RNA-seq and qRT-PCR) revealed that 15 of the identified proteins showed similar expression behavior, with the majority (14 proteins) negatively regulated in stressed roots. Chitinase-2 (Cht2) was the only protein with an upregulation behavior in all approaches. These proteins appear to play an important role in drought tolerance in A. duranensis and may be further explored in peanut genetic breeding programs.
Assuntos
Arachis/metabolismo , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/biossíntese , Raízes de Plantas/metabolismo , Arachis/genética , Desidratação/genética , Desidratação/metabolismo , Perfilação da Expressão Gênica , Proteínas de Plantas/genética , Raízes de Plantas/genética , ProteômicaRESUMO
Peanut, Arachis hypogaea (Linnaeus, 1753) is an allotetraploid cultivated plant with two subgenomes derived from the hybridization between two diploid wild species, A. duranensis (Krapovickas & W. C. Gregory, 1994) and A. ipaensis (Krapovickas & W. C. Gregory, 1994), followed by spontaneous chromosomal duplication. To understand genome changes following polyploidy, the chromosomes of A. hypogaea, IpaDur1, an induced allotetraploid (A. ipaensis × A. duranensis)4x and the diploid progenitor species were cytogenetically compared. The karyotypes of the allotetraploids share the number and general morphology of chromosomes; DAPI+ bands pattern and number of 5S rDNA loci. However, one 5S rDNA locus presents a heteromorphic FISH signal in both allotetraploids, relative to corresponding progenitor. Whilst for A. hypogaea the number of 45S rDNA loci was equivalent to the sum of those present in the diploid species, in IpaDur1, two loci have not been detected. Overall distribution of repetitive DNA sequences was similar in both allotetraploids, although A. hypogaea had additional CMA3+ bands and few slight differences in the LTR-retrotransposons distribution compared to IpaDur1. GISH showed that the chromosomes of both allotetraploids had preferential hybridization to their corresponding diploid genomes. Nevertheless, at least one pair of IpaDur1 chromosomes had a clear mosaic hybridization pattern indicating recombination between the subgenomes, clear evidence that the genome of IpaDur1 shows some instability comparing to the genome of A. hypogaea that shows no mosaic of subgenomes, although both allotetraploids derive from the same progenitor species. For some reasons, the chromosome structure of A. hypogaea is inherently more stable, or, it has been at least, partially stabilized through genetic changes and selection.
RESUMO
BACKGROUND AND AIMS: Peanut (Arachis hypogaea) is an allotetraploid (AABB-type genome) of recent origin, with a genome of about 2·8 Gb and a high repetitive content. This study reports an analysis of the repetitive component of the peanut A genome using bacterial artificial chromosome (BAC) clones from A. duranensis, the most probable A genome donor, and the probable consequences of the activity of these elements since the divergence of the peanut A and B genomes. METHODS: The repetitive content of the A genome was analysed by using A. duranensis BAC clones as probes for fluorescence in situ hybridization (BAC-FISH), and by sequencing and characterization of 12 genomic regions. For the analysis of the evolutionary dynamics, two A genome regions are compared with their B genome homeologues. KEY RESULTS: BAC-FISH using 27 A. duranensis BAC clones as probes gave dispersed and repetitive DNA characteristic signals, predominantly in interstitial regions of the peanut A chromosomes. The sequences of 14 BAC clones showed complete and truncated copies of ten abundant long terminal repeat (LTR) retrotransposons, characterized here. Almost all dateable transposition events occurred <3·5 million years ago, the estimated date of the divergence of A and B genomes. The most abundant retrotransposon is Feral, apparently parasitic on the retrotransposon FIDEL, followed by Pipa, also non-autonomous and probably parasitic on a retrotransposon we named Pipoka. The comparison of the A and B genome homeologous regions showed conserved segments of high sequence identity, punctuated by predominantly indel regions without significant similarity. CONCLUSIONS: A substantial proportion of the highly repetitive component of the peanut A genome appears to be accounted for by relatively few LTR retrotransposons and their truncated copies or solo LTRs. The most abundant of the retrotransposons are non-autonomous. The activity of these retrotransposons has been a very significant driver of genome evolution since the evolutionary divergence of the A and B genomes.
Assuntos
Arachis/genética , DNA Intergênico , Evolução Molecular , Genoma de Planta , Cromossomos Artificiais Bacterianos/genética , Hibridização in Situ Fluorescente , Filogenia , Sequências Repetitivas de Ácido Nucleico , Retroelementos/fisiologiaRESUMO
Root-knot nematodes constitute a constraint for important crops, including peanut (Arachis hypogaea L.). Resistance to Meloidogyne arenaria has been identified in the peanut wild relative Arachis stenosperma Krapov. & W. C. Greg., in which the induction of feeding sites by the nematode was inhibited by an early hypersensitive response (HR). Here, the transcription expression profiles of 19 genes selected from Arachis species were analysed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), during the early phases of an A. stenosperma-M. arenaria interaction. Sixteen genes were significantly differentially expressed in infected and non-infected roots, in at least one of the time points analysed: 3, 6, and 9 days after inoculation. These genes are involved in the HR and production of secondary metabolites related to pathogen defence. Seven genes encoding a resistance protein MG13, a helix-loop helix protein, an ubiquitin protein ligase, a patatin-like protein, a catalase, a DUF538 protein, and a resveratrol synthase, were differentially expressed in all time points analysed. Transcripts of two genes had their spatial and temporal distributions analysed by in situ hybridisation that validated qRT-PCR data. The identification of candidate resistance genes involved in wild peanut resistance to Meloidogyne can provide additional resources for peanut breeding and transgenic approaches.
RESUMO
BACKGROUND: Cultivated peanut (Arachis hypogaea) is one of the most widely grown grain legumes in the world, being valued for its high protein and unsaturated oil contents. Worldwide, the major constraints to peanut production are drought and fungal diseases. Wild Arachis species, which are exclusively South American in origin, have high genetic diversity and have been selected during evolution in a range of environments and biotic stresses, constituting a rich source of allele diversity. Arachis stenosperma harbors resistances to a number of pests, including fungal diseases, whilst A. duranensis has shown improved tolerance to water limited stress. In this study, these species were used for the creation of an extensive databank of wild Arachis transcripts under stress which will constitute a rich source for gene discovery and molecular markers development. RESULTS: Transcriptome analysis of cDNA collections from A. stenosperma challenged with Cercosporidium personatum (Berk. and M.A. Curtis) Deighton, and A. duranensis submitted to gradual water limited stress was conducted using 454 GS FLX Titanium generating a total of 7.4 x 10(5) raw sequence reads covering 211 Mbp of both genomes. High quality reads were assembled to 7,723 contigs for A. stenosperma and 12,792 for A. duranensis and functional annotation indicated that 95% of the contigs in both species could be appointed to GO annotation categories. A number of transcription factors families and defense related genes were identified in both species. Additionally, the expression of five A. stenosperma Resistance Gene Analogs (RGAs) and four retrotransposon (FIDEL-related) sequences were analyzed by qRT-PCR. This data set was used to design a total of 2,325 EST-SSRs, of which a subset of 584 amplified in both species and 214 were shown to be polymorphic using ePCR. CONCLUSIONS: This study comprises one of the largest unigene dataset for wild Arachis species and will help to elucidate genes involved in responses to biological processes such as fungal diseases and water limited stress. Moreover, it will also facilitate basic and applied research on the genetics of peanut through the development of new molecular markers and the study of adaptive variation across the genus.