Genome identification of NAC gene family and its gene expression patterns in responding to salt and drought stresses in Rhododendron delavayi

Abstract

Background: The NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs), which play a vital role in plant growth and development, stress response, and disease resistance, have been extensively analyzed in various plant species. However, there is limited knowledge regarding the NAC family in Rhododendron delavayi, an important ornamental flower.

Result: In this study, a total of 102 RdNAC genes were identified from the R. delavayi genome. Phylogenetic analysis divided these genes into seven subfamilies, each characterized by similar conserved motifs. Chromosomal mapping revealed an uneven distribution of RdNACs across all 13 chromosomes, with gene family expansion driven primarily by dispersed duplication (110 gene pairs) and, to a lesser extent, tandem duplication (17 pairs). Intraspecific synteny analysis detected 26 pairs of duplicated RdNAC genes, while interspecific collinearity with Arabidopsis thaliana uncovered 83 orthologous pairs, indicating both lineage-specific diversification and conserved evolutionary relationships. Ka/Ks ratio calculations for both intra-RdNAC duplicates and RdNAC-AtNAC orthologs yielded values below 0.5, reflecting strong purifying selection. Conserved-motif and domain analyses identified ten distinct motifs and 35 structural domains, with the NAM and MIT CorA-like superfamily domains being the most prevalent. Promoter analysis of 2 kb upstream regions revealed a high abundance of abiotic stress-related cis-acting elements (e.g., ABRE, ARE, CGTCA-motif, LTR). Finally, qRT-PCR demonstrated that under drought (20% PEG) and salt (200 mM NaCl) treatments, multiple RdNACs, particularly RdNAC022 and RdNAC099, were significantly upregulated, underscoring their potential roles in stress response.

Conclusion: This study provides a comprehensive identification of the RdNAC TF family in R. delavayi, contributing to a better understanding of these transcription factors in this species. The findings also serve as a reference for analyzing stress responses, particularly concerning drought and salt stress in R. delavayi.

Keywords: NAC gene family; Rhododendron Delavayi; Abiotic stresses; Gene expression.

Fig. 1

Construction of phylogenetic tree of NAC in A. thaliana and R. delavayi and distribution of RdNACs on chromosomes. A ML phylogenetic tree of NAC proteins from R. delavayi and A. thaliana. The tree was constructed using IQ-TREE2 with 1,000 bootstrap replicates. RdNACs are indicated with red stars. B The distribution of RdNACs across 13 chromosomes in R. delavayi. The background heatmap reflects gene density, with blue indicating low density and red indicating high density regions

Fig. 2

The intraspecific and interspecific collinearity analysis of NAC genes. A The intraspecific collinearity RdNACs gene pairs within R. delavayi. B The interspecific NAC gene collinearity pairs between A. thaliana and R. delavay

Fig. 3

Ka/Ks values of NAC genes in R. delavay. Statistical significance was assessed using the non-parametric Mann–Whitney U test, which was applied due to the non-normal distribution of Ka/Ks values. Significance levels: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)

Fig. 4

Detailed structures of NAC family proteins in R. delavay. A conserved motif, B conserved domain, C gene structure

Fig. 5

The prediction results of the cis-acting elements of the RdNAC gene family in R. delavay. The horizontal bar chart on the left indicates the total number of each cis-element type found across all RdNAC gene promoters. The vertical bars on the top represent the number of RdNAC genes containing specific combinations of cis-elements, as denoted by the dots in the matrix below. Each row of the matrix corresponds to a distinct cis-element type, and connected filled dots represent co-occurring elements in the same gene promoters

Fig. 6

The relative expression of selected RdNACs under the treatment of PEG (A) and high NaCl solution (B). Differences among the various groups were assessed using One-way ANOVA followed by Tukey’s post-hoc test