Genome-Wide Analysis Reveals the Potential Role of MYB Transcription Factors in Floral Scent Formation in Hedychium coronarium

Abstract

The MYB gene family is one of the largest groups of transcription factors (TFs) playing diverse roles in several biological processes. Hedychium coronarium (white ginger lily) is a renowned ornamental plant both in tropical and subtropical regions due to its flower shape and strong floral scent mainly composed of terpenes and benzenoids. However, there is no information available regarding the role of the MYB gene family in H. coronarium. In the current study, the MYB gene family was identified and extensively analyzed. The identified 253 HcMYB genes were unevenly mapped on 17 chromosomes at a different density. Promoter sequence analysis showed numerous phytohormones related to cis-regulatory elements. The majority of HcMYB genes contain two to three introns and motif composition analysis showed their functional conservation. Phylogenetic analysis revealed that HcMYBs could be classified into 15 distinct clades, and the segmental duplication events played an essential role in the expansion of the HcMYB gene family. Tissue-specific expression patterns of HcMYB genes displayed spatial and temporal expression. Furthermore, seven HcMYB (HcMYB7/8/75/79/145/238/248) were selected for further investigation. Through RT-qPCR, the response of candidates HcMYB genes toward jasmonic acid methyl ester (MeJA), abscisic acid (ABA), ethylene, and auxin was examined. Yeast one-hybrid (Y1H) assays revealed that candidate genes directly bind to the promoter of bottom structural volatile synthesis genes (HcTPS1, HcTPS3, HcTPS10, and HcBSMT2). Moreover, yeast two-hybrid (Y2H) assay showed that HcMYB7/8/75/145/248 interact with HcJAZ1 protein. In HcMYB7/8/79/145/248-silenced flowers, the floral volatile contents were decreased and downregulated the expression of key structural genes, suggesting that these genes might play crucial roles in floral scent formation in H. coronarium by regulating the expression of floral scent biosynthesis genes. Collectively, these findings indicate that HcMYB genes might be involved in the regulatory mechanism of terpenoids and benzenoid biosynthesis in H. coronarium.

Keywords: Hedychium coronarium; MYB; floral scent; structural genes; terpenes.

FIGURE 1

Phylogenetic analysis of MYB proteins among H. coronarium, Arabidopsis, tomato, and rice. Full-length amino acid sequences were aligned using ClustalX 2.1 program and unrooted NJ phylogenetic tree was generated using MEGA X with 1,000 bootstrap values. The bootstrap values of less than 50 were omitted. All MYB proteins are clustered into 15 subgroups (G1–G15) indicated by different colors.

FIGURE 2

Synteny and collinearity analysis of MYB gene family. (A) Circos plot of MYB genes in H. coronarium genome. Segmental duplication of MYB gene pairs is indicated by red color in H. coronarium. (B) Colinearity plot of HcMYB genes between Musa acuminata and three other representative plant species. Gray lines in the background showed collinear blocks within H. coronarium and other plant genomes, while red lines indicate syntenic MYB gene pairs.

FIGURE 3

The total volatile contents and expression levels of candidates HcMYB genes. (A) The total volatile contents emitted from flower, leaf, and rhizome. (B) The total floral volatiles released during different flower developmental stages (D1: squaring stage; D2: half open stage; D3: full-bloom; D4: senescence stage). (C) Differential expression patterns of selected candidate HcMYB genes during four different flower developmental stages. The heat map was generated with the relative expression RT-qPCR data of candidate HcMYB genes. The red (upregulated expression levels) and green (downregulated expression levels) color represent the log² transformed expression values.

FIGURE 4

Relative expression level of candidate HcMYB genes and emission of total floral volatiles under phytohormone stresses. (A) Total floral volatiles contents emitted during phytohormone treatments. (B) mRNA levels of key structural volatile biosynthesis genes upon phytohormone stresses. (C) IAA: (Inode-3-acetic acid), (D) ABA: (abscisic acid), (E) ethylene, (F) MeJA: jasmonic acid methyl ester. Error bars indicate SD of three biological replicates and asterisk represents significant differences (^∗p < 0.05).

FIGURE 5

Nuclear localization of HcMYB7/8/145/248 proteins in Arabidopsis protoplasts. GFP; green fluorescent protein, mCheyy; nuclear marker, merged; combined mCheryy and GFP and bright field. Bars, 10 μm.

FIGURE 6

HcMYB7/8/79/145/248 silencing changes the amount of main floral volatile contents and expression of key structure genes in H. coronarium flowers. (A) RT-qPCR analysis of HcMYB7/8/79/145/248 mRNA levels in BSMV silenced and control flowers. (B) mRNA levels of bottom structural volatile synthesis genes in BSMV silenced flowers compared with control. (C) GC-MS analysis of volatile contents of eucalyptol, (D) ocimene, (E) methyl benzoate, and (F) linalool after silencing of HcMYB7/8/79/145/248 genes. Data are presented as the mean ± SEM (n = 3). Asterisk indicates significant differences [Student’s t-test, ^∗(P ≤ 0.05)] between treatments and control.

FIGURE 7

Candidate HcMYB proteins binds to the promoters of bottom structural genes. (A) The interaction was determined on SD medium lacking leucine (Leu) in the presence of aureobasidin A (-Leu + AbA). AD was used as a control. (B) HcMYB7/8/75/79/145/238/248 interacts with HcJAZ1 protein by yeast two-hybrid assay. The vectors were transformed into yeast strain Y2HGold followed by the screening of transformants via SD/-Trp/-His/-Ade + X-α-gal media. Yeast cells transformed with pGBKT7–53 + pGADT7-T, pGBKT7-Lamin + pGADT7-T, or pGADT7-T- pGBKT7-HcJAZ1 were included as negative or positive controls.