Over-expression of CcMYB24, encoding a R2R3-MYB transcription factor from a high-leaf-number mutant of Cymbidium, increases the number of leaves in Arabidopsis

Introduction

As an important organ for photosynthesis, leaves are usually exposed to a variety of ecological and environmental factors such as light, temperature and herbivores, and play a critical role in the growth and development of plants. The morphology of leaf and the pattern of shoot branching largely determine the growth habit of seed plants. In recent years, several genes involved in leaf development have been identified and characterized in plants. Most of them are transcription factors (TFs) that form a complex regulatory network to control the development of various leaf traits (Du, Guan & Jiao, 2018; Wang, Kong & Zhou, 2021). A deeper understanding of the molecular basis of leaf development is needed, not only to improve crop production but also to deepen our overall comprehension of plant biology.

The v-myb avian myeloblastosis viral oncogene homolog (MYB) genes represent one of the largest gene superfamilies encoding TFs in eukaryotes (Rosinski & Atchley, 1998; Jiang & Rao, 2020). A number of studies has shown that the MYB gene family underwent sizable functional diversification during evolution, to form different subfamilies or groups (Jiang & Rao, 2020; Li et al., 2020; Wu et al., 2022). Based on the number of conserved MYB domain at the N-terminus, which contains one to four imperfect repeats (R) associated with DNA-binding and protein–protein interactions, MYB TFs can be classified into four different groups, namely the 1R, R2R3, 3R, and 4R classes, which contain one to four repeats, respectively (Dai et al., 2020; Wang et al., 2020). Among them, the R2R3-MYB proteins form the largest subfamily of MYB TFs, although they only exist in terrestrial plants (Dai et al., 2020; Wang et al., 2020). The R2R3-MYB proteins can act as transcriptional activators as well as repressors in plants, and most of them are activators which function by binding cis-elements in gene promoters to recruit transcriptional machinery and activate gene expression (Wu et al., 2022). A series of biological functions of R2R3-MYBs have been verified in model plants and non-model crops in recent years with the development of approaches which focus on gene functional verification. In summary, the R2R3-MYBs have been shown to be involved in regulating specialized metabolic pathways, such as anthocyanin and phenylpropanoid biosynthesis pathways, response to biotic and abiotic stresses, such as fungal pathogens and drought or salt stresses, participation in plant development and cell differentiation, and responses to hormone signaling (Stracke, Werber & Weisshaar, 2001; Wang, Niu & Zheng, 2021; Ambawat et al., 2013; Wu et al., 2022). Therefore, MYBs are a class of important TFs necessary for the basic life cycle of plants, and more research is needed to determine their functions in basic developmental processes.

Leaf differentiation and morphogenesis are highly flexible processes that are regulated by MYB genes. Previous studies had shown that the MYB domain proteins play a key role in leaf polarity establishment, organ initiation and the subsequent developmental processes (Müller, Schmitz & Theres, 2006; Ge & Chen, 2014). Three RAX genes in Arabidopsis thaliana, RAX1, RAX 2 and RAX 3, are members of the R2R3-Myb gene family. These genes control the formation of axillary meristems at different developmental times (Müller, Schmitz & Theres, 2006). There is also some evidence showing that sub- or neo-functionalization of A. thaliana RAX-1 orthologs regulates the architecture of leaves and shoots in Solanum lycopersicum (Busch et al., 2011). Some MYB genes, such as RS2 and PHAN, participate in leaf development by negatively regulating KNOX genes that determine the acquisition of founder cell identity in the meristem during leaf initiation (Tsiantis et al., 1999). Another case involves the MYB gene ARP, which not only plays an important role in establishment of proper polarities along the adaxial-abaxial, proximodistal, and medial-lateral axes, but also participates in the regulation of leaf petiole identity, as indicated by evidence showing that suppression of ARP expression led to a series of abnormal leaf developmental processes in Medicago truncatula and Nicotiana benthamiana (Huang et al., 2013; Ge & Chen, 2014). Such findings indicate that MYB transcription factors may influence leaf traits in multiple pathways. With the development of gene function verification technology, it can be expected that more functions of MYB transcription factors in relation to the regulation of leaf traits will be revealed in different plant taxa in the future.

Cymbidium is one of the most economically important genera of the Orchidaceae and is highly popular around the world for its beautiful flowers and leaf variegation (Jiang et al., 2022). A mutagenesis-based approach is the main way to produce various flower and leaf color mutants in different Cymbidium species (Jiang et al., 2022). Leaf traits, such as color, variegation patterning, shape and number of leaves, are important in determining the quality of ornamental foliage plants. In China, the variation in leaf traits in ornamental foliage plants has been called ‘leaf art’, and novel variation in leaf traits is one of the goals in ornamental plant breeding. In our previous study, we obtained a new orchid mutant, temporarily named ‘TRIR-2’, through radiation mutation breeding on a hybrid between two Cymbidium species (Su et al., 2016). A comparative cytological study between the non-mutant Cymbidium inter-specific hybrid ‘TRIR-1’ and ‘TRIR-2’, which was not induced by irradiation, revealed significant differences. The study found that ‘TRIR-2’ had fewer chloroplasts, mitochondria, and other organelles, and some leaf cells of ‘TRIR-2’ contained only minimal cytoplasm. Consequently, the leaves of ‘TRIR-2’ appeared albino, with green color only visible at the edges and tips (Su et al., 2016). Furthermore, it was obvious that ‘TRIR-2’ generated more leaves than ‘TRIR-1’. According to our unpublished data, the result of RNA sequencing had revealed that expression of a MYB gene CcMYB24 was significantly upregulated in ‘TRIR-2’, compared with ‘TRIR-1’ as the control, indicating that this gene may contribute to the morphological differences in ‘TRIR-2’.

In the present study, we describe the cloning and characterization of CcMYB24 from the ornamental foliage mutant line ‘TRIR-2’. The sequence analysis and protein structure prediction showed that CcMYB24 belongs to the R2R3-MYB subfamily. Overexpression of CcMYB24 in transgenic Arabidopsis seedlings significantly increased the number of leaves. These results indicate that this gene plays a positive role in increasing the number of leaves in ‘TRIR-2’, providing a basis for exploring the molecular mechanism of CcMYB24 in leaf development, and identifying a potential gene target for the molecular breeding of new cultivars in ornamental foliage plants.

Materials and Methods

Results

Variation in morphological traits between ‘TRIR-2’ and ‘TRIR-1’

Individuals of mutant ‘TRIR-2’ showed distinct variation with respect to several morphological traits in comparison with wild-type ‘TRIR-1’. Whereas ‘TRIR-1’ has normal green leaves, the leaves of TRIR-2 are mostly white, with green color appearing only at the edges and tip of each leaf (Fig. 1A). The ‘TRIR-1’ plants were significantly taller than ‘TRIR-2’ plants of the same age (p = 0.03, Fig. 1B). Although the length of leaves was significantly longer in ‘TRIR-1’ than in ‘TRIR-2’ (p < 0.01, Fig. 1C), ‘TRIR-2’ produced significantly more leaves than TRIR-1 did (p = 0.02, Fig. 1D), and the leaves of ‘TRIR-2’ were wider than those of ‘TRIR-1’ (p = 0.04, Fig. 1E).

Cloning and sequence analysis of CcMYB24

The complete sequence of CcMYB24 was amplified and sequenced from ‘TRIR-1’ and ‘TRIR-2’. The full-length sequence of CcMYB24 was 1389 bp, and the length of the open reading frame (ORF) was 906 bp, encoding 301 amino acids (File S1). The amplified sequence of ‘TRIR-1’ was determined to be 861 base pairs in length. Upon sequence alignment with the corresponding sequence of ‘TRIR-2’, it was observed that ‘TRIR-1’ exhibited a segmental variation of 95 base pairs, including three gaps with a deletion of 45 base pairs, and 50 single nucleotide polymorphisms (SNPs) when compared to ‘TRIR-2’. These observed variations led to a loss of 15 amino acids and a substitution of 24 amino acids in the peptide chain of ccMYB24 in ‘TRIR-1’. The alignment results are provided in File S2. Analysis of conserved domains showed that the protein encoded by CcMYB24 had a conserved Myb DNA-binding domain (Fig. 2A). According to the results of BLAST, the ten sequences with the highest amino acid similarity to CcMYB24 were obtained, including CeRAX2-like (from Cymbidium ensifolium, 95.99% similarity), DcRAX2 (from Dendrobium catenatum, 86.15% similarity), PeRAX2-like (from Phalaenopsis equestris, 76.49% similarity), EgRAX2-like (from Elaeis guineensis, 60.80% similarity), PdMYB36 (from Phoenix dactylifera, 64.17% similarity), MeRAX2 (from Manihot esculenta, 52.51% similarity), JrRAX2-like (from Juglans regia, 51.39% similarity), QlRAX2-like (from Quercus lobata, 45.12% similarity), QsRAX2-like (from Quercus suber 50.53% similarity), NtRAX (from Nicotiana tabacum, 45.85% similarity) and AtRAX2 (from A. thaliana, 39.60% similarity). All of these genes belong to the R2R3-MYB family which includes most MYB TFs (Kelemen et al., 2015). The result of sequence alignments showed that all these sequences had conserved domains of the Myb superfamily proteins, such as the PLN03091 superfamily, REB1 superfamily, and two MYB DNA-binding domains (Fig. 2B). Phylogenetic analysis showed that the CcMYB24 protein is closely related to the CeRAX2-like protein and the DcRAX2 protein (Fig. 2C).

The physicochemical properties and structure of CcMYB24 protein

The ProtParam ExPaSy program was used to analyze the amino acid sequence and chemical properties of the CcMYB24 protein. The result showed that CcMYB24 possesses the molecular formula C₁₄₅₇H₂₂₆₃N₄₀₇S₁₅, and the molecular weight was calculated to be 33,242.35 Da. The theoretical isoelectric point (pI) was 8.59, and the instability index was 63.02, indicating that the protein is unstable. The grand average value of the hydropathy index (GRAVY) was calculated as the sum of the hydropathy values of all amino acids, and the estimated value of GRAVY was −0.483 (Fig. 3A). This finding suggests that the CcMYB24 protein is likely to be largely hydrophilic. The result of the transmembrane helix region and signal peptide prediction analysis showed that neither transmembrane structure nor signal peptide could be found in the CcMYB24 protein, indicating that it is not a transmembrane protein, nor a secretory protein. The secondary structure of the protein was predicted using the SOPMA server and shown in Fig. 3B. The result showed that CcMYB24 protein was mainly composed of random coil (52.49%), alpha-helix (24.58%), extended strand (16.28%) and beta turn (6.64%). The prediction of the tertiary protein structure of CcMYB24 (Fig. 3C) by the SWISS-MODEL analysis platform showed a similar structure with a R2R3-MYB transcription factor from A. thaliana which has six alpha-helices belonging to R2 and R3 repeats (Wang et al., 2020).

Subcellular localization of CcMYB24

Most R2R3-MYB proteins are exclusively localized in the nucleus, where they function as TFs (Stracke, Werber & Weisshaar, 2001). For CcMYB24, the result of localization prediction using the pSORT web server predicted that it was located in the nucleus. We further determined the subcellular localization of CcMYB24 via transient expression of a translation fusion with GFP in tobacco leaves. The result showed that the fluorescence signal of the GFP control was localized to the cytoplasm and the nucleus in N. benthamiana leaves, whereas the CcMYB24-GFP was located exclusively in the nucleus (Fig. 4), a finding similar to that of other R2R3-MYB TFs, suggesting that CcMYB24 also functions in the nucleus as a TF.

Expression pattern of CcMYB24 in ‘TRIR-1’, ‘TRIR-2’ and transgenic Arabidopsis

The expression pattern of CcMYB24 was examined in wild-type Cymbidium ‘TRIR-1’, mutant Cymbidium ‘TRIR-2’ and transgenic Arabidopsis by using quantitative real-time PCR (qPCR). As shown in Fig. 5A, the relative expression of CcMYB24 transcripts was about 1.4 time higher in ‘TRIR-2’ than in ‘TRIR-1’, indicating that more transcripts were generated in leaves of ‘TRIR-2’ than in those of the wild-type parent. In the case of Arabidopsis, the expression level was evaluated in terms of log2 fold change between transgenic and WT individuals. The result showed that CcMYB24 expression was much higher in the stem, leaf and flower of transgenic Arabidopsis than in the corresponding organs of the WT (Fig. 5B).

CcMYB24 induced production of more leaves when overexpressed in Arabidopsis

To investigate the function of CcMYB24 in ‘TRIR-2’, the coding region was expressed in A. thaliana Col-0. We observed some differences between the transgenic and WT individuals (Figs. 6A–6D). Despite a p-value of 0.06, statistical analysis revealed no significant difference in the number of branches between WT and transgenic individuals (Fig. 6E). The mean number of leaves in transgenic individuals was significantly higher than that in WT plants (p < 0.01, Fig. 6F).

CcMYB24 markedly affected the accumulation of photosynthetic pigments and total flavonoids

The influence of CcMYB24 on a number of physiological traits, namely photosynthetic pigments, total flavonoids, and soluble sugar and protein, were examined and compared between transgenic and WT Arabidopsis. For photosynthetic pigments, the results showed that chlorophyll a (p < 0.01), chlorophyll b (p = 0.02), and total chlorophyll concentrations (p < 0.01) were significantly higher in transgenic Arabidopsis (Fig. 7A), whereas total carotenoid concentration was significantly lower in transgenic Arabidopsis plants than in WT individuals (p < 0.01, Fig. 7A). The total flavonoid concentration was also significantly lower in transgenic Arabidopsis than in WT individuals (p < 0.01; Fig. 7B), but soluble sugar and protein concentrations did not differ significantly between transgenic and WT plants (Figs. 7C and 7D).

Discussion

Cymbidium plants enjoy a prime position in the floriculture industry. The ornamental value of Cymbidiums is conferred not only by the large and colorful flowers and extended flower longevity, but also by its foliage and floral fragrance (Jiang et al., 2022). Some Cymbidium varieties have long been cultivated as foliage plants which are grown for attractive leaves, and the combined character of leaf variegation and colorful flowers have an added advantage in the ornamental plant market. Variations in leaf traits, including color, shape and number of leaves, are vital for the quality of foliage plant. In this study, a new foliage variant of Cymbidium was generated by combining inter-specific hybridization with radiation mutation breeding. The new variant, ‘TRIR-2’, has significantly broader but shorter leaves compared with the parent, ‘TRIR-1’, and showed an increased number of leaves and a part-albino leaf phenotype, with a large area of white in the middle of the leaves with green only at the edges and tip of the leaf (Fig. 1A), making it an attractive ornamental plant.

Several researches have explored mutation in leaf development including shape development, photosynthetic mechanisms, chloroplast development and other mechanisms of genetic control on color variation of leaves. These studies mainly focused on model plants or crops such as A. thaliana (Moon et al., 2008), Zea mays (Lonosky et al., 2004), Nicotiana tabacum (Wu et al., 2020), and Oryza sativa (Zheng et al., 2019). The knowledge of genetic control of number of leaves is still limited, especially in ornamental foliage plants which commercial value strongly influenced by the number of leaves. Recent studies have shown that transcription factors control many aspects of leaf developmental processes (Liu et al., 2018; Zhao et al., 2020; Heisler et al., 2022). The MYB transcription factors are versatile in regulation of development, stress responses as well as in leaf development (Jiang & Rao, 2020; Wang, Niu & Zheng, 2021; Wu et al., 2022), but the knowledge about the role it plays in control of number of leaves is still limited.

In the present study, a R2R3-MYB gene, CcMYB24, was reported to participate in producing more leaves in ‘TRIR-2’, a high-leaf-number mutant of Cymbidium. The expression analysis showed that CcMYB24 exhibited a higher expression level in ‘TRIR-2’ than in ‘TRIR-1’. The mechanism of expression level increasing is complex and remain elusive. It may be due the mutation of upstream and/or regulatory gene, or the difference in epigenetic control between ‘TRIR-1’ and ‘TRIR-2’. More works are needed in the future to discover the reason why there were more transcripts of CcMYB24 in ‘TRIR-2’. We plan to conduct comparative transcriptome and gene co-expression network analysis between ‘TRIR-1’ and ‘TRIR-2’ and further integrated the results with the analysis of epigenetic control of CcMYB24.

According to the results of sequence similarity analysis, CcMYB24 appeared to be a RAX2-like gene which belongs to the R2R3-MYB transcription factor gene family. Many studies have proved that the RAX homolog genes play important roles in determination of axillary meristems by generating a tissue environment conducive to the establishment of meristems to control the spatial pattern of axillary meristems development (reviewed by Zhang et al., 2022), thus participate in branching and leaf development. In Arabidopsis, transcripts of RAX genes accumulated in the axils of young leaf primordia and controlled a very early step of axillary meristem initiation, with rax mutants showing new phenotypes characterized by defects in lateral bud formation in overlapping zones along the shoot axis (Müller, Schmitz & Theres, 2006). Much evidence has also been reported that RAX genes control axillary meristem formation and shoot branching in Arabidopsis by collaborating in gene modules with other transcriptional factors, such as bHLH and WRKY (Yang et al., 2012; Guo & Qin, 2016). In Chrysanthemum morifolium, CmRAX2 was expressed in the lateral branches and roots, with overexpression of CmRAX2 leading to increases in lateral branch number and plant height (Song et al., 2022). RAX genes were also be found to participate in shoot branching and leaf dissection in tomato (Busch et al., 2011; Zhang et al., 2022). Based on these evidences, it could be inferred that the CcMYB24 may be responsible for the variation in foliage traits shown by ‘TRIR-2’.

The current studies showed the regulatory role played by RAX2 genes in shoot branching and leaf development. The results of transgenic experiment in this study showed that overexpression of CcMYB24 in Arabidopsis stimulated production of more leaves (Fig. 6E) and more branches (Fig. 6F), although the latter effect was not significant (but close with p-value of 0.06), indicating that CcMYB24 may contribute to the phenotype of more leaves in ‘TRIR-2’ compared with ‘TRIR-1’. Orchid plants grow by only one or two growth patterns, namely sympodial or monopodial. Both types grow by leaves rather than obvious branches. Therefore, it can be inferred that the phenotype of more leaves in ‘TRIR-2’ may be related to the branching regulation nature of RAX2 genes. However, the factors contributing to the higher expression level of MYB24 in ‘TRIR-2’ than that in ‘TRIR-1’ are still unknown. More research focusing on the expression pattern and regulatory role at different leaf developmental stages is needed to explore the relationship between CcMYB24 and the formation of novel leaf traits in ‘TRIR-2’.

The content of pigments strongly influences the color of leaves, and in this study, carotenoid concentration was found to be significantly lower in transgenic Arabidopsis than in WT plants (Fig. 7A). It is known that carotenoid mutants in Arabidopsis can cause developmental defects in plastids, resulting in albino patches on leaves (Bartley & Scolnik, 1995). However, the higher concentrations of chlorophyll a, chlorophyll b, and total chlorophyll in transgenic Arabidopsis indicated that the expression of CcMYB24 was not the direct cause of the albino phenotype in ‘TRIR-2’ leaves, as chlorophylls a and b are the primary sources of green color.

Several studies have shown that MYB TFs work as regulators of primary and secondary metabolism in plants, and are essential for regulating pathways involved in plant growth, development, and responses to biotic and abiotic stress (Wang, Niu & Zheng, 2021). Plant metabolites include many types of compounds, such as sugars, proteins, flavonoids etc. Evidence has shown that the MYB TFs participate in flavonoid biosynthesis, and that the expression of some MYB transcription factor genes could promote flavonoid accumulation in plants, which could result in increased tolerance to environmental stresses (Jin et al., 2022). In the present study, accumulation of total flavonoids in transgenic Arabidopsis was significantly lower than in WT individuals (Fig. 7B), indicating that CcMYB24 may negatively influence the accumulation of total flavonoids. Some MYB genes could also influence sugar transport and promote accumulation of soluble sugars and proteins. For instance, two MYB proteins, CSA2 and CSA, were proved to regulate sugar transport in rice (Wang et al., 2021). Overexpression of MYB1 in Arabidopsis resulted in accumulation of greater concentrations of soluble sugars in transgenic individuals than in the WT plants (Cheng et al., 2013). However, the concentration of soluble sugars and proteins was not affected by overexpression of CcMYB24 in Arabidopsis (Figs. 7C and 7D), indicating that CcMYB24 may not be involved in the determination of the concentrations of soluble sugars and proteins. In addition, there were differences in traits exhibited by other plant organs between ‘TRIR-1’ and ‘TRIR-2’, such as root number and plant height (Fig. 1A). As MYB TFs play roles in many aspects of plant development (Ambawat et al., 2013; Li et al., 2019; Jiang & Rao, 2020), more research is needed to explore the function of CcMYB24 in ‘TRIR-2’.

Conclusions

In this study, a R2R3-MYB transcription factor CcMYB24 was cloned from a new foliage variant of Cymbidium, ‘TRIR-2’. ‘TRIR-2’ was an induced mutant from ‘TRIR-1’, an inter-specific Cymbidium hybrid, and it showed a series of trait variations on the leaf organ. Specifically, ‘TRIR-2’ plants produce more leaves than ‘TRIR-1’ plants. Overexpression of the CcMYB24 gene significantly increased the number of leaves in transgenic Arabidopsis plants, indicating it may play a positive role in controlling the number of leaves in ‘TRIR-2’. The number of leaves is an important trait that directly affects the quality of ornamental foliage plants, with the ornamental value of foliage plants usually being increased by increasing the number of leaves. Therefore, CcMYB24 can be considered as a potential target gene to regulate the number of leaves in molecular breeding of ornamental foliage plants.

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