Expansins are plant cell wall-loosening proteins involved in adaptive responses to environmental stimuli and various developmental processes. The first genome-wide analysis of the expansin superfamily in the Arachis genus identified 40 members in A. duranensis and 44 in A. ipaënsis, the wild progenitors of cultivated peanut (A. hypogaea). These expansins were further characterized regarding their subfamily classification, distribution along the genomes, duplication events, molecular structure, and phylogeny. A RNA-seq expression analysis in different Arachis species showed that the majority of these expansins are modulated in response to diverse stresses such as water deficit, root-knot nematode (RKN) infection, and UV exposure, with an expansin-like B gene (AraEXLB8) displaying a highly distinct stress-responsive expression profile. Further analysis of the AraEXLB8 coding sequences showed high conservation across the Arachis genotypes, with eight haplotypes identified. The modulation of AraEXLB8 expression in response to the aforementioned stresses was confirmed by qRT-PCR analysis in distinct Arachis genotypes, whilst in situ hybridization revealed transcripts in different root tissues according to the stress imposed. The overexpression of AraEXLB8 in soybean (Glycine max) composite plants remarkably decreased the number of galls in transformed hairy roots inoculated with RKN. This study improves the current understanding of the molecular evolution, divergence, and gene expression of expansins in Arachis, and provides molecular and functional insights into the role of expansin-like B, the less-studied plant expansin subfamily.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-017-0594-8) contains supplementary material, which is available to authorized users.

BackgroundPeanut (Arachis hypogaea) production is largely affected by a variety of abiotic and biotic stresses, including the root-knot nematode (RKN) Meloidogyne arenaria that causes yield losses worldwide. Transcriptome studies of wild Arachis species, which harbor resistance to a number of pests and diseases, disclosed several candidate genes for M. arenaria resistance. Peanut is recalcitrant to genetic transformation, so the use of Agrobacterium rhizogenes-derived hairy roots emerged as an alternative for in-root functional characterization of these candidate genes.ResultsThe present report describes an ex vitro methodology for hairy root induction in detached leaves based on the well-known ability of peanut to produce roots spontaneously from its petiole, which can be maintained for extended periods under high-humidity conditions. Thirty days after infection with the A. rhizogenes ‘K599’ strain, 90% of the detached leaves developed transgenic hairy roots with 5 cm of length in average, which were then inoculated with M. arenaria. For improved results, plant transformation, and nematode inoculation parameters were adjusted, such as bacterial cell density and growth stage; moist chamber conditions and nematode inoculum concentration. Using this methodology, a candidate gene for nematode resistance, AdEXLB8, was successfully overexpressed in hairy roots of the nematode-susceptible peanut cultivar ‘Runner’, resulting in 98% reduction in the number of galls and egg masses compared to the control, 60 days after M. arenaria infection.ConclusionsThis methodology proved to be more practical and cost-effective for functional validation of peanut candidate genes than in vitro and composite plant approaches, as it requires less space, reduces analysis costs and displays high transformation efficiency. The reduction in the number of RKN galls and egg masses in peanut hairy roots overexpressing AdEXLB8 corroborated the use of this strategy for functional characterization of root expressing candidate genes. This approach could be applicable not only for peanut–nematode interaction studies but also to other peanut root diseases, such as those caused by fungi and bacteria, being also potentially extended to other crop species displaying similar petiole-rooting competence.

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.