Comprehensive Analysis of NAC Transcription Factors Reveals Their Evolution in Malvales and Functional Characterization of AsNAC019 and AsNAC098 in Aquilaria sinensis

Abstract

<i>NAC</i> is a class of plant-specific transcription factors that are widely involved in the growth, development and (a)biotic stress response of plants. However, their molecular evolution has not been extensively studied in Malvales, especially in <i>Aquilaria sinensis</i>, a commercial and horticultural crop that produces an aromatic resin named agarwood. In this study, 1502 members of the <i>NAC</i> gene family were identified from the genomes of nine species from Malvales and three model plants. The macroevolutionary analysis revealed that whole genome duplication (WGD) and dispersed duplication (DSD) have shaped the current architectural structure of <i>NAC</i> gene families in Malvales plants. Then, 111 <i>NAC</i> genes were systemically characterized in <i>A. sinensis</i>. The phylogenetic analysis suggests that <i>NAC</i> genes in <i>A. sinensis</i> can be classified into 16 known clusters and four new subfamilies, with each subfamily presenting similar gene structures and conserved motifs. RNA-seq analysis showed that <i>AsNACs</i> presents a broad transcriptional response to the agarwood inducer. The expression patterns of 15 <i>AsNACs</i> in <i>A. sinensis</i> after injury treatment indicated that <i>AsNAC019</i> and <i>AsNAC098</i> were positively correlated with the expression patterns of four polyketide synthase (PKS) genes. Additionally, <i>AsNAC019</i> and <i>AsNAC098</i> were also found to bind with the <i>AsPKS07</i> promoter and activate its transcription. This comprehensive analysis provides valuable insights into the molecular evolution of the <i>NAC</i> gene family in Malvales plants and highlights the potential mechanisms of <i>AsNACs</i> for regulating secondary metabolite biosynthesis in <i>A. sinensis</i>, especially for the biosynthesis of 2-(2-phenyl) chromones in agarwood.