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MYB-related transcription factors (TFs), named for their highly conserved 52 amino acid MYB domain, are predominant and critical members of the MYB TF family in plants (Riechmann and Ratcliffe, 2000; Dubos et al., 2010). MYB TFs can be generally divided into four subfamilies based on the number of MYB domains present: 1R-MYB (MYB-related), R2R3-MYB, 3R(R1R2R3)-MYB, and 4R-MYB proteins (Rosinski and Atchley, 1998). The 1R-MYB are collectively called MYB-related proteins and include proteins with a single or a partial MYB repeat (Dubos et al., 2010). Recently, many studies performing systematic identification and genome-wide characterization of the MYB family have reported genes in various plants, such as Petunia hybrida, potato (Solanum tuberosum), peach (Amygdalus persica), and Chinese jujube (Ziziphus jujuba) (Zhang et al., 2018; Ji et al., 2019; Liu et al., 2020; Chen et al., 2021). The MYB-related and R2R3-MYB subfamily contain the highest quantity of genes within the MYB family (Liu et al., 2020). To date, R2R3-MYB has been comprehensively characterized and reported to have diverse functions (Nesi et al., 2001; Koes et al., 2005; Ambawat et al., 2013), particularly in enhancing volatile aroma production, promoting anthocyanin accumulation, and responding to abiotic stresses. In contrast, several MYB-related proteins control diverse physiological processes, particularly the regulation of the anthocyanin biosynthesis pathway and the amelioration of multiple abiotic stresses. The first MYB-related TF identified in plants was in potato (MYBSt1) (Baranowskij et al., 1994), and since then, the MYB family has gradually gained more attention. However, to date, the function of MYB-related genes in regulating the synthesis of floral aroma remains largely unknown.

According to the most recent classification based on the highly conserved motif, MYB-related TFs are divided into three subgroups: the CPC-like subgroup of R3-type single-MYB proteins consisting of CPC (CAPRICE)-like, TRY (TRIPTYCHON)-like, ETC (Enhancer of TRY and CPC)-like and MYBL2; the CCA1-like/R-R subgroup, including the CCA1 (Circadian Clock Associated1)-like, TBP (Telomeric DNA-binding Protein)-like, RAD (RADIALIS)-like, TRF-like, LHY (Late Elongated Hypocotyl), and RVE (Early Phytochrome Responsive1)-like proteins; and the GARP subgroup, which is further classified into the LUX, MYB_CC, KANADI, ARR, HHO, GLK (GOLDEN2-LIKE), and GARP-like subfamilies. The MYB-related genes were subsequently confirmed to play fundamental roles as transcriptional regulators, associated repressors, circadian clock, and telomeric repeat-binding proteins in various biological processes. AtMYBD, a member of the CCA1-like/R-R subgroup in Arabidopsis thaliana, regulates anthocyanin biosynthesis in a circadian clock-related manner (Nguyen and Lee, 2016). SlTRY, an R3 MYB member of the CPC-like subgroup, has been confirmed to be involved in anthocyanin biosynthesis and is considered a core regulator of root-hair development and the plant trichome (Rumi et al., 2013). The grape TF AQUILO, a member of the GARP subgroup, increases cold tolerance by promoting the accumulation of raffinose family oligosaccharides (Sun et al., 2018). In recent years, MYB-related TFs have been found to be involved in regulating anthocyanin biosynthesis in various pathways and products. The proteins MdLUX and MdPCL-like, containing a single MYB-like repeat, promote anthocyanin accumulation through DNA hypomethylation in apple (Malus domestica) (Li et al., 2021). The functions of MYB-related proteins in metabolite biosynthesis have been characterized in various land plants, for instance, Arabidopsis, soybean (Glycine max), grapevine (Vitis vinifera), lily (Lilium spp.), and tiger lily (Lilium lancifolium) (Matsui et al., 2008; Niu et al., 2016; Sakai et al., 2019; Yong et al., 2019).

Sweet Osmanthus (Osmanthus fragrans) is an evergreen woody flowering member of the Oleaceae. Because of its pleasing floral fragrance and multitudinous flowers, sweet osmanthus is widely cultivated in China as a common landscape tree species (Huang et al., 2015). Sweet osmanthus is relevant to several industries, including tea, food, and perfume production (Li et al., 2020a). The floral fragrance of sweet osmanthus has been comprehensively studied in terms of compound identification and classification. Studies have identified that β-ionone is an essential substance that strongly affects the floral fragrance of sweet osmanthus, and CCD4 is a vital enzyme regulating the formation of β-ionone (Han et al., 2016). Previous studies have confirmed that OfCCD4 increases the production of β-ionone, based on the existence of β-carotene, and both OfWRKY3 and OfERF61 are positive regulators of the OfCCD4 gene and enable the synthesis of β-ionone in petals of sweet osmanthus (Han et al., 2016, 2019). According to data on the sweet osmanthus genome, we found that the OfCCD4 promoter region contains numerous cis-acting regulatory elements, thus indicating that OfCCD4 might be regulated by other TFs (Yang et al., 2018). However, functional identification of related TFs and examination of the transcriptional regulation mechanism of floral volatile organic compounds (VOCs) have been limited. Therefore, studies on the MYB-related TFs’ regulation of enhancing floral VOC production are needed to improve understanding of the mechanism of VOC production and the genetic quality of sweet osmanthus.

Here, we conducted a comprehensive analysis of sweet osmanthus’ MYB-related TF family members, including their potential roles in regulating the formation of floral VOCs. In this study, 212 MYB-related members were identified from the genome of sweet osmanthus, and their chromosomal locations, conserved motifs, and expression patterns were analyzed and visualized. Additionally, we selected three candidate MYB-related genes that had flower organ/development stage-preferential expression patterns and then performed detailed gas chromatography-mass spectrometry (GC-MS) analysis of floral VOCs between transient expression plants and empty vector plants.

To date, MYB TFs, mainly comprising MYB-related and R2R3 MYB, modulate the transcription levels of numerous essential genes in secondary metabolism biosynthetic pathways, thereby regulating the production of diverse floral VOCs (Jian et al., 2019). Several MYB members have been identified as a conserved module regulating terpene synthesis. MsMYB, a novel negative regulator in Mentha spicata associated with the promoter region of geranyl diphosphate synthase (GPPS), decreases the production of monoterpenes by inhibiting the activity of GPPS (Reddy et al., 2017). Recently, the function of R2R3MYBs in floral aroma formation in Hedychium coronarium has been extensively reported (Abbas et al., 2021). However, further studies of MYB-related proteins, particularly the close relationship between the function of MYB-related TFs and floral aroma synthesis, remain unclear.

The number of OfMYB-related genes was more significant than that observed in other plants reported over the recent years (Table 1; Jin et al., 2017), such as 109 genes in peach (Zhang et al., 2018). In our study, we identified 212 MYB-related genes in O. fragrans, a value close to the number of R2R3-MYB genes in O. fragrans (243 genes) (Li et al., 2020a), thus representing the largest subfamily of MYB genes, which play critical roles in various processes of growth, development, and responses to abiotic or biotic stress. While high, the number of R2R3-MYB subfamily members did not exceed the number of 1R-MYB subfamily members. The amount of 1R-MYBs in Ganoderma was twice the number of R2R3-MYBs. Moreover, previous studies have performed genome-wide analyses of MYB-related proteins from 16 species of flowering plants, moss, Selaginella, and algae (Du et al., 2013). In contrast to the detailed studies on the R2R3-MYB subfamily, little attention has been paid to the MYB-related (1R-MYB) subfamily, which has been reported only in a few common crops in recent years: potato, sunflower (Helianthus annuus), radish (Raphanus sativus), and oil palm (Elaeis guineensis) (Li et al., 2020b; Liu et al., 2020; Zhou et al., 2020; Muleke et al., 2021).

As an important part of the MYB family, MYB-related genes were initially classified into five subgroups: the CCA1-like, CPC-like, TBP-like, I-box-binding-like, R-R-type (Zhang et al., 2018). However, after detailed studies on the GARP family, MYB-related genes were divided into three subgroups: CCA1-like/R-R, CPC-like, and GARP-like (Liu et al., 2020). After extensive further studies, a merger occurred in the Circadian Clock Associated 1-like and R-R-type, equivalent to the CCA1-like/R-R subgroups in this study; the results are consistent with those of the most recent studies (Liu et al., 2020). In previous work, CCA1-like proteins have been found to be closely associated with R-R type MYB repeats, thus implying that R-R-type gene loss events may directly lead to CCA1-like proteins. In this classification, 44 members of the CCA1-like/R-R group contained the conserved motif SHAQK (motif 2 in Supplementary Figure 1) in the MYB repeat, in agreement with previous studies in Arabidopsis (Ji et al., 2019). The CAPR-like subgroups initially comprised maize G2-like and Arabidopsis ARR-B, which were newly derived in angiosperms. In agreement with previous research, the GARP-like subgroups were classified into six clades in this identification (Liu et al., 2020).

As a typical MYB member, the CPC-like subgroup has been reported to be involved in the basic function of regulating root-hair development and the plant trichome. It has been confirmed to regulate the synthesis of secondary metabolites in recent years. For instance, AtCPC, characterized as a repressor of anthocyanin production, has provided novel insight into the transcriptional regulation of flavonoid biosynthesis. SlTRY and SlGL3, MYB-related homologs of tomato, also influence synthesis of anthocyanin (Jian et al., 2019). In contrast, AtMYBL2, an R3-MYB protein, has been found to restrain flavonoid synthesis. Interestingly, MYBL2 inhibits flavonoid biosynthesis and trichome development (Matsui et al., 2008). LlMYB3, a MYB related homolog from tiger lily, positively regulates the stress tolerance of plants and is involved in the anthocyanin biosynthesis pathway, thereby regulating stress tolerance (Yong et al., 2019).

In this expression analysis, OfMYB1R70, OfMYB1R114, and OfMYB1R201, which were preferentially expressed in floral organs, had higher RPKM values than other OfMYB1Rs, particularly OfMYB1R70 and OfMYB1R114, which had the top two RPKM values. Moreover, subcellular localization prediction indicated that they were mainly located in the nucleus, thus suggesting that these candidate genes may play important roles in regulating floral aroma synthesis during the bloom-stage of sweet osmanthus; therefore, they were selected for follow-up experiments. However, no prior studies have focused on the functions of MYB-related TFs in floral aroma biosynthesis. The relationship with the synthesis of floral VOCs has been reported only for 2RMYB (Reddy et al., 2017), although 2RMYB and MYB-related TFs jointly compose the main family in plants and are involved in many biological processes, particularly secondary metabolism, and responses to environmental factors, in divergent ways (Jian et al., 2019; Zhao et al., 2019).

Transcription factors have been reported to regulate the synthesis of floral VOCs by directly binding the promoter regions of target enzyme genes and controlling their expression levels. In H. coronarium, HcMYB genes directly bind the promoters of key structural volatile synthesis genes and regulate the biosynthesis of terpenoids and benzenoids (Abbas et al., 2021). In O. fragrans, OfERF61 up-regulates the expression of OfCCD4 and influences the synthesis of β-ionone (Han et al., 2019). In petunia, PhMYB4 controls the production of floral volatile benzenoid/phenylpropanoid by suppressing the downstream gene transcription (Thomas et al., 2011). PhERF6 binds the promoters of EOBI protein, which influences floral scent, thus negatively regulating the production of volatile compounds (Liu et al., 2017). In spearmint, MsMYB represses the expression of the GPPS gene, thus modifying secondary terpene metabolism (Reddy et al., 2017). In this study, we found that OfMYB1R201 is nuclearly located with self-activation activity its transient overexpression decreases the synthesis of β-ionone (Figure 7) and other VOCs in tobacco leaves (Supplementary Figure 6). The expression of NbCCD4.3 was significantly diminished in transformed plants with transient expression of the OfMYB1R201 vector (Figure 7). These results indicated that OfMYB1R201 could participate in negatively regulating VOC synthesis.

Remarkably, OfMYB1R70 and OfMYB1R114 were found to have nuclear localization (Supplementary Figure 5), and their expression during flower development was highly correlated (Figure 4). The transient transformation results showed that OfMYB1R70 and OfMYB1R114 were both involved in the regulation of aroma component synthesis, as confirmed by the markedly greater production of β-ionone and other emitted metabolites than that in the empty vector plants (Supplementary Figure 6). The expression of NbCCD4.1 and NbCCD4.3 was elevated in plants with transient expression of the OfMYB1R114 vector, and the expression of NbCCD4.1 and NbCCD4.2 was markedly elevated in plants with transient expression of the OfMYB1R70 vector (Figure 7). However, we found that OfMYB1R70 and OfMYB1R114 had no transcriptional activity, thus indicating that they do not directly activate the transcription of their downstream genes. Previous studies have reported that TFs interact with other proteins in regulating the synthesis of secondary metabolites by the MBW complex (Koes et al., 2005). For example, FhMYBx, containing a bHLH-binding motif in the R3 domains, suppresses the formation of the MYB-bHLH-WD40 (MBW) complex via an association with bHLH proteins, thus down-regulating anthocyanin metabolism (Li et al., 2020c). SmelMYBL1, an R3 MYB repressor in eggplant, acts as an inhibitor of the MBW complex by competing with MYB activators (SmelANT1 and SmelAN2) for the bHLH binding site involved in anthocyanin biosynthesis (Andrea et al., 2020). Hence, we concluded that OfMYB1R70/114/201 regulates aroma component formation by interacting with other TFs or acting as activators in competing pathways that alter the availability of precursors. The molecular mechanism of OfMYB1R70/114/201 in regulating the aroma metabolite synthesis will be further explored in following studies.

In this study, to provide novel insight into the regulation of floral aroma by all MYB-related genes in sweet osmanthus, we comprehensively identified 212 putative MYB-related members by genome-wide analysis. We categorized them into three subgroups, including 12 categories related to 132 MYB-related members of A. thaliana in the phylogenetic tree. Additionally, the information on conserved motifs in genes was visualized in figures, and the expression patterns indicated that three candidate OfMYB1Rs might be associated with the development and growth of flowers. Notably, members of the CC1-like/R-R subgroup in sweet osmanthus, OfMYB1R70/114/201, localized primarily in the nucleus. Remarkably, transient expression studies demonstrated that all those genes were associated with the regulation of floral VOC synthesis, and OfMYB1R114 had an apparent ability to positively regulate the synthesis of floral VOCs, such as β-ionone and nonanal, whereas OfMYB1R201, as a TF, negatively regulated floral VOCs. Together, our findings provide insight into OfMYB-related genes, explaining their roles in plants as regulators of the production of volatile aroma compounds. Our future efforts will focus on modulating the functions of OfMYB1R70/114/201 in regulating the production of floral aroma and investigating the roles of upstream regulatory factors.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

YY and XnY conceived the original idea. XnY and XW performed the experimental work. XnY and WD contributed to the data analysis. XnY wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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