Genome-Wide Identification and Analysis of the Aux/IAA Gene Family in Panax ginseng: Evidence for the Role of PgIAA02 in Lateral Root Development

Abstract

Panax ginseng C. A. Meyer (Ginseng) is one of the most used traditional Chinese herbal medicines, with its roots being used as the main common medicinal parts; its therapeutic potential has garnered significant attention. AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) is a family of early auxin-responsive genes capable of regulating root development in plants through the auxin signaling pathway. In the present study, 84 Aux/IAA genes were identified from the ginseng genome and their complexity and diversity were determined through their protein domains, phylogenetic relationships, gene structures, and cis-acting element predictions. Phylogenetic analyses classified PgIAA into six subgroups, with members in the same group showing greater sequence similarity. Analyses of interspecific collinearity suggest that segmental duplications likely drove the evolution of PgIAA genes, followed by purifying selection. An analysis of cis-regulatory elements suggested that PgIAA family genes may be involved in the regulation of plant hormones. RNA-seq data show that the expression pattern of Aux/IAA genes in Ginseng is tissue-specific, and PgIAA02 and PgIAA36 are specifically highly expressed in lateral, fibrous, and arm roots, suggesting their potential function in root development. The PgIAA02 overexpression lines exhibited an inhibition of lateral root growth in Ginseng. In addition, yeast two-hybrid and subcellular localization experiments showed that PgIAA02 interacted with PgARF22/PgARF36 (ARF: auxin response factor) in the nucleus and participated in the biological process of root development. The above results lay the foundation for an in-depth study of Aux/IAA and provide preliminary information for further research on the role of the Aux/IAA gene family in the root development of Ginseng.

Keywords: Aux/IAA gene family; Panax ginseng; auxin-responsive genes; evolution; expression pattern analysis.

Figure 1

Distribution of gene density on 24 chromosomes of Panax ginseng and chromosomal localization of PgIAA family genes. The red region indicates a high density of gene number regions on the chromosome.

Figure 2

Phylogenetic tree of Panax ginseng, Oryza sativa, and Arabidopsis thaliana Aux/IAA proteins. A phylogenetic tree was built based on the JTTDCMut+F+R4 model using the maximum likelihood method IQ-TREE. I–VI are different subgroups.

Figure 3

Conserved motifs and structural distribution of ginseng Aux/IAA proteins. Domain I is called the ethylene response factor (ERF)-associated amphiphilic repression (EAR) motif. The “GWPPV” motif in domain II is a degron, which controls the turnover of Aux/lAA proteins. Domain III and IV together form type I/Ⅱ Phox and Bem1p (PB1) domains.

Figure 4

Predicted cis-regulatory elements of PgIAA genes. The heat map shows the number of cis-elements; the right side contains the species statistics.

Figure 5

Chromosomal localization and collinearity analysis of the PgIAA genes. The gray lines in the background represent blocks of collinearity within Panax ginseng species. Red lines highlight PgIAA gene pairs with covariates.

Figure 6

Gene expression pattern of PgIAA genes. (A) Expression levels of PgIAA genes in different tissues; (B) Expression levels of PgIAA genes under different hormone treatments. Gene expression levels were reflected as normalized TPM values in Log2 (TPM+1).

Figure 7

qRT-PCR analysis of tissue-specific expression of PgIAA genes. Relative mRNA expression levels of individual genes were normalized to the expression level of β-actin. The standard error of the mean of three replicates is shown. Letters above the error line mark the significance of the expression level. Main root (Mr), main root periderm (Mrp), main root cortex (Mrc), main root steles (Mrs), rhizome (Rh), leg root (Lr), fiber root (Fr), and arm root (Ar).

Figure 8

Subcellular localization analysis of PgIAA02/42/58 in Nicotiana tabacum leaves; 35S-GFP is the negative control, and the blue fluorescent signal of DAPI indicates the nucleus. Scale bar: 20 µm.

Figure 9

Wild type (WT) and 35S plants: PgIAA02 transgenic plant seedlings were grown on 1/2 MS medium for 10 days. (A) Growth phenotype. Scale bar = 2 mm. (B) Length of main root, number of lateral roots, and length of lateral roots. ** represents an extremely significant difference.

Figure 10

The interaction between PgIAA02 and PgARF22/36 was detected using the yeast two-hybrid system. In the system, BD (pGBKT7) and AD (pGADT7) were used as bait and prey constructs, respectively. The positive control and negative control were pGBKT7-53 + pGADT7-T and pGBKT7-Lam + pGADT7-T, respectively.