The Stilbene Synthase Family in Arachis: A Genome-Wide Study and Functional Characterization in Response to Stress

Abstract

Peanut (Arachis hypogaea) and its wild relatives are among the few species that naturally synthesize resveratrol, a well-known stilbenoid phytoalexin that plays a crucial role in plant defense against biotic and abiotic stresses. Resveratrol has received considerable attention due to its health benefits, such as preventing and treating various human diseases and disorders. Chalcone (CHS) and Stilbene (STS) Synthases are plant-specific type III Polyketide Synthases (PKSs) that share the same substrates and are key branch enzymes in the biosynthesis of flavonoids and stilbenoids, respectively. Although resveratrol accumulation in response to external stimulus has been described in peanut, there are no comprehensive studies of the CHS and STS gene families in the genus Arachis. In the present study, we identified and characterized 6 CHS and 46 STS genes in the tetraploid peanut and an average of 4 CHS and 22 STS genes in three diploid wild species (Arachis duranensis, Arachis ipaënsis and Arachis stenosperma). The CHS and STS gene and protein structures, chromosomal distributions, phylogenetic relationships, conserved amino acid domains, and cis-acting elements in the promoter regions were described for all Arachis species studied. Based on gene expression patterns of wild A. stenosperma STS genes in response to different biotic and abiotic stresses, we selected the candidate AsSTS4 gene, which is strongly induced by ultraviolet (UV) light exposure, for further functional investigation. The AsSTS4 overexpression in peanut hairy roots significantly reduced (47%) root-knot nematode infection, confirming that stilbene synthesis activation in transgenic plants can increase resistance to pathogens. These findings contribute to understanding the role of resveratrol in stress responses in Arachis species and provide the basis for genetic engineering for improved production of valuable secondary metabolites in plants.

Keywords: abiotic stress; biotic stress; chalcone synthase; functional analysis; gene expression; peanut; resveratrol.

Figure 1

Exon–intron gene structure of CHS (A) and STS in Arachis duranensis (B); A. ipaënsis (C); A. stenosperma (D); and A. hypogaea (E).

Figure 2

Genomic distribution and syntenic relationships of the CHS and STS genes in Arachis spp. The ten chromosomes of A. duranensis is represented as Aradu (A01 to A10) and A. ipaënsis as Araip (B01 to B10), and the 20 chromosomes of A. hypogaea as Arahy (chr01 to chr20). The syntenic relationships between the genes are represented by lines.

Figure 3

Schematic representation of STS gene clusters organization on chromosomes 04 and 14 of Arachis spp. Right and left arrows indicate whether genes or transposable elements are located on the + or − strand, respectively.

Figure 4

Phylogenetic tree of STS (Groups A and B) and CHS (Groups C, D, E, and F) S amino acid sequences from Arachis duranensis; A. ipaënsis; A. stenosperma; and A. hypogaea.

Figure 5

Protein structure of CHS and STS in Arachis duranensis; A. ipaënsis; A. stenosperma; and A. hypogaea.

Figure 6

Protein organization of CHS and STS in Arachis duranensis; A. ipaënsis; A. stenosperma; and A. hypogaea. (A) Percentage of occurrence and (B) organization of conserved protein domains in predicted proteins.

Figure 7

Heatmap of cis-acting elements associated with responses to hormones (HRE), light (LRE), and stress (STRE) and related to tissue specificity and development (TS&DE) in the promoter regions of STS (A) and CHS (B) genes of A. duranensis (Ad), A. ipaënsis (Ai), A. stenosperma (As), and A. hypogaea (Ah). The heatmap colors range from red to blue scale, where darker colors indicate increasing and decreasing values in the numbers of cis-elements.

Figure 8

Distribution in the HRE, LRE, STRE, and TS&DEV categories of cis-acting elements found in the putative promoter sequences of STS (A) and CHS (B) genes from A. duranensis, A. ipaënsis, A. stenosperma and A. hypogaea.

Figure 9

Heatmap of the in silico expression patterns of 20 Arachis stenosperma STS genes in response to different types of stresses: nematode infection (at 3, 6, 7, and 9 days after infection; DAI); ultraviolet (UV) exposure; drought treatments (dry-down and dehydration); and combined drought imposition and nematode infection (cross). The color key represents differential gene expression magnitude in Log2 fold change (FC) values.

Figure 10

Peanut hairy roots transformed with A. rhizogenes harboring pPZP-AsSTS4 vector and observed under a stereomicroscope using bright field (A) and epifluorescence (B) 30 days after A. rhizogenes transformation. Transgenic peanut hairy roots transformed with pPZP- AsSTS4 (C) and pPZP-empty vectors (D), showing gall formation 60 days after M. arenaria inoculation. Mean number of galls per gram of transgenic hairy roots (E) and relative quantification of AsSTS4 gene in transgenic peanut hairy roots transformed with pPZP-empty, pPZP-ASSTS4, and in non-transformed control roots (F). Arrows indicate nematode galls. Error bars mean the standard error of samples, and asterisks mean significant differences between samples (p < 0.05; Student’s t-test).