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Article

The Regulatory Role of R2R3-MYB Family Genes in Trichome Formation in Solanum aculeatissimum

by
Fan Yang
1,2,†,
Yanbo Yang
1,2,†,
Wanqi Li
1,2,†,
Qihang Cai
1,2,†,
Man Miao
1,2,
Zhenghai Sun
1,2,* and
Liping Li
3,*
1
College of Landscape and Horticulture, Southwest Forestry University, Kunming 650224, China
2
Yunnan International Joint R&D Center for Intergrated Utilization of Ornamental Grass, Kunming 650224, China
3
College of Ecology and Environment (College of Wetlands), Southwest Forestry University, Kunming 650224, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(11), 2637; https://doi.org/10.3390/agronomy15112637
Submission received: 30 September 2025 / Revised: 6 November 2025 / Accepted: 14 November 2025 / Published: 18 November 2025
(This article belongs to the Topic Plant Breeding, Genetics and Genomics, 2nd Edition)
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Abstract

Solanum aculeatissimum is a medicinally and economically significant crop characterized by its aerial organs, which are densely covered with trichomes and spines. Trichomes serve as crucial sites for the synthesis of secondary metabolites in medicinal plants and represent important structural adaptations for resisting biotic and abiotic stresses. Elucidating the molecular mechanisms underlying trichome formation in S. aculeatissimum holds significant implications for enhancing both its medicinal value and stress resistance. The R2R3-MYB subfamily, the largest within the MYB transcription factor family, plays a pivotal role in regulating trichome development. Here, we present the first genome-wide identification of the R2R3-MYB gene family in S. aculeatissimum, characterizing 99 members. Phylogenetic analysis classified these SaMYBs into 10 groups. Cis-element predictions in their promoter regions revealed an abundance of light-responsive, phytohormone-responsive, and abiotic stress-responsive elements, suggesting roles in environmental adaptation. Furthermore, synteny analysis identified 25 segmentally duplicated gene pairs, and the purifying selection has been the dominant evolutionary force. Through comparative transcriptomic analysis of leaves from wild-type and sparse-trichome plants, we identified 16 differentially expressed SaMYB genes, comprising 3 upregulated and 13 downregulated genes. Subsequent qRT-PCR analysis showed that SaMYB1, SaMYB59, and SaMYB36 were highly expressed during early leaf development. Virus-induced gene silencing (VIGS) targeting these candidates demonstrated that silencing SaMYB59 significantly reduced trichome density, whereas silencing SaMYB1 or SaMYB36 produced no observable phenotypic change, confirming SaMYB59 as a key positive regulator of trichome formation. Our findings provide crucial molecular targets for elucidating the mechanisms of trichome development in S. aculeatissimum and establish a theoretical foundation for genetic improvement of this medicinal plant through regulation of the SaMYB59 gene.

1. Introduction

Solanum aculeatissimum, a wild species within the Solanaceae family, is noted for its medicinal properties and resistance to Verticillium wilt, Ralstonia solanacearum, and high temperatures. Owing to these traits, it is extensively utilized both as a rootstock for eggplant (Solanum melongena) and as a germplasm resource in hybridization programs, aiming to enhance disease resistance and abiotic stress tolerance in Solanaceous crops [1,2,3,4]. This species is also valued for its medicinal compounds, with solanine being the primary component. Notably, S. aculeatissimum contains the highest levels of solanine among commonly cultivated Solanaceae species [5,6], which underpins its broad pharmacological activities, such as anti-inflammatory, antibacterial, antioxidant, anti-obesity, and anti-cancer effects [7]. The significant medicinal and economic value of S. aculeatissimum has led to its cultivation across approximately 4000 hectares in various regions of India [7]. Furthermore, studies on eggplant grafting have demonstrated that using S. aculeatissimum as a rootstock not only effectively improves host resistance but also influences fruit quality [2,8].
Trichomes play important roles in the biosynthesis of medicinal compounds and stress resistance in plants. Their density and morphology thereby have an impact on the accumulation efficiency of these compounds and the plant’s defensive capabilities [9,10,11,12,13]. Trichome also exhibits remarkable resilience against drought, cold, and intense light radiation [14]. Plant trichome not only has important protective effect on itself but also has high application value and economic value [15,16,17]. Plant trichomes are the source of substances like menthol, ephedrine, and artemisinin, and the trichome-derived compounds are frequently of great worth [18,19]. Phytohormones and transcriptional regulators play a role in controlling the formation of trichomes in plants. The MBW complex consisting of MYB, bHLH, and WD40 gene families plays a central regulatory role in this process [20], with the R2R3-MYB subfamily a member of the MYB family, which plays a major role in the formation of trichome.
As one of the most extensive transcription factor families in higher plants, MYB transcription factors have been extensively studied across numerous plant species. Due to the different number of amino acid tandem repeats, the MYB family can be divided into four subfamilies: 1R-MYB, R2R3-MYB, R1R2R3-MYB, and 4R-MYB [21]. R2R3-MYB is the subfamily with the largest number in the MYB family [22]. Previous studies have demonstrated that R2R3-MYB transcription factors exert complex regulatory effects on the cell cycle [23], cell differentiation [24], abiotic stress response [14], secondary metabolite pathways [25], and the biosynthesis of anthocyanin and lignin in plants [26,27]. Consequently, R2R3-MYB is essential for plant growth, as well as its ability to withstand stress [26]. Multiple studies have demonstrated that members of the R2R3-MYB family play a role in the creation and control of plant trichomes. In Arabidopsis thaliana, MYB23, GLABRA1, and MYB5 form complexes with the transcription factors of the WD40 family and bHLH family to control the formation of trichome [28,29,30]. GhMYB2, GhMYB212, GhMYB109, and GhMYB25 play positive regulatory roles in fiber formation in cotton [31]. R2R3-MYB transcription factor AaMIXTA1 of Artemisia annul forms a complex with AaHD8 to promote the initiation of trichome [32].
In summary, S. aculeatissimum is an important medicinal plant. Its trichomes play a vital role in the biosynthesis of medicinal compounds and in stress resistance. The core regulatory role of R2R3-MYB transcription factors in glandular trichome development has been established in plants like Arabidopsis and cotton. However, in S. aculeatissimum, the identification of its R2R3-MYB family members and their specific functions in trichome formation remain unclear. Therefore, an in-depth analysis of the family characteristics and regulatory functions of R2R3-MYB transcription factors in S. aculeatissimum is key to revealing the molecular mechanisms underlying its high solanine production and stress resistance. It also provides an important theoretical basis and genetic resources for improving the quality and resistance of Solanaceae crops through genetic engineering approaches.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

In this research, the seeds of S. aculeatissimum were selected as experimental materials and grown in a commercial potting mix with peat and vermiculite (3:1) under controlled conditions: temperature 25/20 °C, light intensity 10,000 lx, and a photoperiod of 16 h light/8 h dark.

2.2. Identification of the R2R3-MYB Gene Family in S. aculeatissimum

The hidden Markov model (HMM) profile for the MYB gene family (PF00249) was obtained from the Pfam database (version 38.0; http://pfam.xfam.org/; accessed on 25 July 2025). Based on the previously available whole-genome sequencing data from the research group, the Simple HMM Search function in TBtools software (version 2.371) was used to identify MYB members across the entire genome. The sequences of A. thaliana R2R3-MYB transcription factors were acquired from the PlantTFDB (http://planttfdb.gao-lab.org/, accessed on 26 July 2025) and TAIR databases (https://www.arabidopsis.org/; accessed on 26 July 2025). BLASTp was performed to search for homologous sequences of A. thaliana R2R3-MYB in S. aculeatissimum. The candidate genes were confirmed by screening for the presence of the characteristic MYB domain using the NCBI CD-Search (https://www.ncbi.nlm.nih.gov/, accessed on 26 July 2025) and Pfam databases (http://pfam-legacy.xfam.org/; accessed on 26 July 2025). After removing genes that encoded truncated proteins or contained incomplete MYB DNA-binding domains, the final set of 99 R2R3-MYB transcription factors in S. aculeatissimum was obtained and numbered SaMYB1 to SaMYB99. The sequences of the SaMYB proteins are provided in the Supplementary File S1. The physicochemical properties of the SaMYB proteins, including molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY), were predicted using the ExPASy ProtParam tool (https://www.expasy.org/, accessed on 28 July 2025).

2.3. Phylogenetic Analysis of SaMYB Members

Multiple sequence alignment of the SaMYB protein sequences from S. aculeatissimum and A. thaliana was performed using the MUSCLE algorithm in MEGA 11 (Supplementary File S2). An unrooted phylogenetic tree was constructed from the alignment using the neighbor-joining (NJ) method with 1000 bootstrap replicates.

2.4. Analysis of Conserved Motifs and Cis-Acting Elements

Conserved motifs of SaMYB members were identified using the MEME online tool (https://meme-suite.org/; accessed on 28 July 2025) with the motif count set to 10. To characterize the DNA-binding domain of SaMYB members, R2 and R3 repeat sequences were extracted from respective proteins and visualized as sequence logos using TBtools [33]. Promoter regions (2000 bp upstream of the transcription start site) of SaMYB members were subjected to Cis-acting element prediction via PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 28 July 2025). All predicted elements identified with the default parameters were recorded.

2.5. Synteny and Ka/Ks Analysis of SaMYB Members

The synteny of SaMYB members was analyzed using the MCScanX tool in TBtools (version 2.371) with its default settings. The chromosomal positions and synteny graphs of the transcription factors were visualized using TBtools software, and the ratio of nonsynonymous to synonymous substitution rates (Ka/Ks) was calculated based on the synteny relationships.

2.6. SaMYBs Expression Pattern Analysis

The analysis of expression patterns was based on transcriptome sequencing of wild-type leaves (WT) and sparse-trichome individual leaves (SC) of S. aculeatissimum. The transcriptome sequencing was conducted by Applied Protein Technology Co., Ltd. (Shanghai, China), utilizing the Illumina HiSeq platform for paired-end (PE) sequencing with a read length of 150 bp. Adapter sequences and low-quality reads were filtered out using Fastp (version 0.23.2). The resulting clean reads were then aligned to the reference genome of S. aculeatissimum assembled by our research group using HISAT2 software (version 2.2.1). The quality control reports for the sequencing data are provided in the Supplementary File S3. Differentially expressed genes were identified by applying a threshold of adjusted p-value < 0.05 and |log2FC| > 1. qRT-PCR was performed to analyze the expression of potential positive regulatory trichome genes in S. aculeatissimum leaves at different growth stages (Figure S1).

2.7. Functional Analysis of SaMYB Based on VIGS

Referring to Zhou’s method [34], SaMYB36, SaMYB1, and SaMYB59 were subjected to gene silencing by VIGS. Specific 300-bp fragments for VIGS were designed using the SGN VIGS Tool (https://vigs.solgenomics.net/, accessed on 21 August 2025). The details of these target sequences are available in Supplementary File S4. The recombinant silencing vector containing each target fragment was constructed using the ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China). The experimental treatments included the following groups: an experimental group (TRV2-target gene + TRV1), a negative control group (TRV2 + TRV1), and a blank control (H2O). Whole plants of 15-day-old seedlings were infected via vacuum infiltration. Following infection, the plants were maintained in darkness at 22 °C for 24 h. Following the dark incubation, plants were returned to a 16/8 h photoperiod. Newly developed leaves were collected 20 days post-infection for DNA and RNA extraction. PCR detection was performed by TRV2 primers, verifying that the virus was inoculated successfully. For scanning electron microscopy (SEM) observation, leaf samples were mounted on aluminum stubs and sputter-coated with a thin layer of gold using an ion sputter coater. The leaf surface was then observed under a HITACHI SU8100 cold field emission scanning electron microscope at an accelerating voltage of 5.0 kV.

2.8. Amplification of Silencing Fragments and qRT-PCR

Specific primers were designed to flank the silencing fragments (Table S1). The objective fragment was amplified using MegaFiTM Fidelity 2X PCR MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada). The reaction system (20 μL) contained 10 μL of MegaFiTM Fidelity 2X PCR MasterMix, 1 μL of upstream and downstream primers, and 1 μL template cDNA and 7 μL ddH2O. The amplification procedure was initial denaturation at 98 °C for 30 s, denaturation at 98 °C for 10 s, annealing at 60 °C for 20 s, extension at 72 °C for 15 s for 30 cycles, and final extension at 72 °C for 2 min.
Total RNA from leaves was extracted with RNA extraction kits (Shanghai Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China) following the manufacturer’s instructions. The integrity of the RNA was confirmed by 1% agarose gel electrophoresis, and only samples with clear ribosomal RNA bands were used for subsequent library preparation. Subsequently, cDNA was synthesized from 500 ng of total RNA using the Goldenstar RT6 cDNA Synthesis Mix kit (Tsingke Biotechnology Co., Ltd., Beijing, China). The amplification of qRT-PCR was performed using ArtiCanCEO SYBR qPCR Mix (Tsingke Biotechnology Co., Ltd.). The mRNA transcript levels of the target genes were detected by qRT-PCR, and the primers used are listed in Table S1. The reaction system (20 μL) contained 10 μL of ArtiCanCEO SYBR qPCR Mix, 1 μL of each of the upstream and downstream primers, 1 μL of template cDNA, and 7 μL of ddH2O. The amplification procedure was initial denaturation at 95 °C for 5 min, denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 20 s for 40 cycles. The dissolution procedure was at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. The qRT-PCR analysis was performed with three biological replicates using TUA as the reference gene, and the relative gene expression was calculated using the 2−ΔΔCt method [35].

3. Results

3.1. Identification and Chromosomal Localization of the SaMYB Genes

A total of 149 MYB transcription factors were identified in the genome of S. aculeatissimum via Hidden Markov Model (HMM) search and homologous sequence alignment. Following quality control and removal of duplicates, 99 were classified as R2R3-MYB members, while the remaining 50 belonged to other subfamilies (R1, R1R2R3-MYB, and 4R-MYB). This confirmed that the R2R3-MYB subfamily is the largest group of MYB transcription factors in S. aculeatissimum. As shown in Figure 1, these 99 R2R3-MYB transcription factors are distributed across all 12 chromosomes (designated Chr1 to Chr12), and they were designated SaMYB1 to SaMYB99 based on their sequential order along the chromosomes. Their distribution is uneven across chromosomes, with no immediate obvious pattern, and they are predominantly located near the chromosome ends. Chr7, Chr11, and Chr12 harbor the highest numbers of SaMYB genes (11, 11, and 13, respectively), while Chr9 contains the fewest (only 5). Gene density analysis revealed that SaMYB genes are frequently located in chromosomal regions exhibiting high gene density.

3.2. Physiochemical Properties Analysis of SaMYB Proteins

Physiochemical properties showed that SaMYB39 had the longest amino acid sequence of 1030 aa, SaMYB99 had the shortest amino acid sequence of 173 aa, and the molecular weights were from 19.96 kDA (SaMYB99) to 114.25 kDA (SaMYB29). The theoretical pI ranging from 4.88 (SaMYB62) to 9.65 (SaMYB27), there are 49 acidic proteins with pI < 6.5, 35 basic proteins with pI > 7.5, and 15 neutral proteins. The hydrophilicity of each transcription factor is less than 0, indicating that they are all hydrophilic proteins. It can be seen that the physicochemical properties of the SaMYB proteins are full of diversity. This diversity suggests the potential for significant structural and functional variations among its members (Table S2).

3.3. Phylogeny and Classification of R2R3-MYB Genes

To further analyze the phylogenetic relationship and roles of SaMYB members, we constructed a neighbor-joining (NJ) phylogenetic tree of S. aculeatissimum and A. thaliana R2R3-MYB genes (Figure 2). The phylogenetic analysis showed that the SaMYB members can be divided into 10 main groups (shown by the background color of gene numbers in Figure 2). To facilitate functional annotation, and based on the established classification of R2R3-MYB genes in A. thaliana, the phylogenetic tree was further divided into 36 subgroups [21]. The transcription factors not specifically grouped in previous studies were numbered as M1~M13. The fact that 93 SaMYB members could be matched into the A. thaliana subgroups indicates a strong evolutionary conservation and suggests potential functional similarities. Interestingly, subgroup M11 is unique to S. aculeatissimum, which may represent a lineage-specific expansion with specialized functions, and no SaMYB members were found in the S12 subgroup, implying possible gene loss or functional divergence in this species.

3.4. Gene Conserved Structures and Cis-Acting Elements Prediction Analysis of SaMYB Genes

Motif and conserved structure analysis was conducted on the SaMYB genes (Figure 3). The results showed that members within the same subgroup exhibited similar distribution patterns of these structures. Most of the SaMYB members had similar Motif compositions and had the main structure of Motif 3 + Motif 2/Motif 6 + Motif 1 + Motif 4, indicating that these conservative motifs play an important role in SaMYB members, where Motif 2 is often replaced by Motif 6. Motif 8 was observed only in M11 subgroup members (SaMYB30, SaMYB31, SaMYB32, and SaMYB33) (Figure 3b). All members had R2R3-MYB-associated PLN03091 and PLN03212 structures, suggesting that SaMYB members were identified accurately (Figure 3c).
In order to further research the sequence characteristics, the structural characteristic sequences of R2 and R3 of the SaMYB proteins were subjected to multiple sequence alignment, and seqlogo visualization analysis was carried out on the conservation of each domain (Figure S2). The results showed that the R2-MYB structure in S. aculeatissimum formed by Motif 3 and Motif 2/Motif 6 and contained three conservative tryptophans (W, Trp), which were located at 4, 24, and 44, respectively, with 19 amino acids spaced therebetween; the R3-MYB structure consists of Motif1 and Motif4, involving two conservative tryptophans (W, Trp), at 19 and 38, respectively, with 18 amino acids therebetween. The hydrophobic core formed by these tryptophans is a typical sign of plant MYB transcription factors, and it is consistent with the structural characteristics of R2R3-MYB in A. thaliana and other plants.
Promoter analysis was performed in the upstream 2000-bp region to investigate the potential function of members of the SaMYB members (Figure 3d). The results indicate that SaMYB genes contain a large number of hormone responsiveness elements, optical responsiveness elements, anaerobic production elements, and tissue expression elements. The counting of cis-elements found that all members have multiple light responsiveness elements, 77 members have ABA responsiveness elements, 56 members have GA responsiveness elements, 36 members have auxin responsiveness elements, 61 members have MeJA responsiveness elements, and 41 members have SA responsiveness elements. In addition, SaMYB members also have a large number of stress responsiveness elements. The results showed that the expression of these members may be regulated by phytohormones, environmental factors, biotic or abiotic stress, and defense signaling transduction during the growth of S. aculeatissimum.

3.5. Synteny Analysis of SaMYB Genes

To investigate the expansion and functional evolution mechanisms of the SaMYB members, we analyzed their syntenic relationships and calculated the Ka/Ks ratios for each gene pair (Table S3). A total of 25 pairs of syntenic gene pairs were identified, driven by fragment replication expansion, forming cross-chromosomal or long distance (>10 Mb) syntenic relationships (Figure 4). Purifying selection was the predominant evolutionary force. Among the syntenic gene pairs, 16 pairs were under strong purifying selection (Ka/Ks < 0.3), while 6 pairs experienced moderate purifying selection (0.3 ≤ Ka/Ks < 0.5). Three gene pairs exhibited synonymous site saturation (pS > 0.75), and their high non-synonymous substitution rates (Ka > 0.28) suggested potential functional divergence. SaMYB84 exhibited syntenic relationships with SaMYB15, SaMYB50, and SaMYB77, with Ka/Ks ratios < 0.25 indicating extreme evolutionary conservation of this gene. Notably, the Ks values showed significant divergence across these gene pairs (SaMYB84–SaMYB15: 4.2085; SaMYB84–SaMYB50: 2.2672; SaMYB84–SaMYB77: 1.1164), demonstrating that the synteny originated from segmental duplications at distinct evolutionary times rather than a single whole-genome duplication (WGD) event.

3.6. Analysis of the Expression Patterns of SaMYB Genes

To explore the potential regulatory roles of R2R3-MYB transcription factors in trichome formation on S. aculeatissimum leaves, SaMYB members with significantly differential expression were identified based on transcriptome data from wild-type (WC) and sparse-trichome individuals (SC). As shown in Figure 5A, 16 SaMYB members with significant expression differences were found in total. Three members were significantly lower in WT leaves than in SC leaves, indicating that these SaMYB genes may be involved in the negative regulation of trichome formation. Thirteen members were notably elevated in WT leaves compared to SC leaves, indicating the potential involvement of these SaMYB genes in the favorable regulation of trichome.
To further screen SaMYB genes involved in regulating trichomes in S. aculeatissimum, we analyzed the expression patterns of potential positive regulatory genes using qRT-PCR. The results showed that these genes exhibited different expression patterns in leaves at different growth stages (Figure 5B). SaMYB1, SaMYB36, and SaMYB59 exhibit significantly high expression levels during the early stages of leaf development (YC1 stage), with a gradual decrease as growth progresses, suggesting their potential role in early leaf morphogenesis through dosage-dependent regulation; SaMYB2, SaMYB9, SaMYB23, SaMYB25, SaMYB46, and SaMYB55 exhibit higher expression levels during the YC1 and YC3 stages, while their expression is suppressed during the mid-stage (YC2 stage), suggesting that they may regulate the initial expansion and maturation of leaves; the expression peaks of SaMYB61, SaMYB78, and SaMYB94 are concentrated in the late developmental stage (YC3 stage), suggesting that they may participate in the leaf maturation process.

3.7. SaMYB59 Silencing Decreases Trichome Density in S. aculeatissimum

To further investigate the function of SaMYB genes in affecting the development of trichome, we employed the VIGS method to silence three SaMYB genes (SaMYB1, SaMYB36, and SaMYB59), which have potential regulatory functions for early trichome development. The results of PCR testing of new leaves showed that the virus was detected in plants inoculated with empty vector TRV2 and TRV2 target genes but not in the H2O treatment group. The length of the amplified product of the TRV2 target gene treatment group was about 300 bp longer than that of the TRV2 treatment group, indicating that the silencing target fragment was successfully propagated, along with the TRV (Figure 6A). qRT-PCR was used to detect the mRNA transcript levels of target genes in different treatment groups (Figure 6B). The results showed that the gene expression abundance of SaMYB36, SaMYB1, and SaMYB59 in the TRV2 target gene treatment group was significantly reduced compared with the empty TRV2 and H2O treatment, indicating that the VIGS experiment successfully silenced these three transcription factors. In order to further observe the effect of target gene silencing on the trichome formation of the leaves of S. aculeatissimum, the mature leaves of the determined silenced plants and the leaves of the control group (empty TRV2 and H2O) were taken for morphological observation by scanning electron microscope (Figure 6C). The results showed that no significant change in the trichomes of the leaves of the SaMYB1- and SaMYB36-silenced plants compared with that of the control group, indicating that these genes are not essential for trichome initiation. Compared with the control group, the number of trichomes on the leaves of SaMYB59 gene-silenced plants was significantly reduced, indicating that SaMYB59 is essential for trichome initiation in S. aculeatissimum.

4. Discussion

Previous research has shown that R2R3-MYB transcription factors play an important role in diverse plants. At present, genome studies on A. thaliana [36], rice [37], and poplar [38], it has been found that the MYB subfamily members of such plants have the largest number of transcription factors in the R2R3-MYB subfamily. In A. thaliana, researchers divided A. thaliana R2R3-MYB transcription factors into 25 subgroups, and each subgroup was with a unique function [21]. For example, R2R3-MYB transcription factors in the S6 subgroup can regulate anthocyanin biosynthesis in nutritive tissues [39]; the S2 subgroup may control the seed coat color of A. thaliana [40]; and the S1, S22, S20, and S2 subgroups are involved in the adaptation of A. thaliana to abiotic stresses [41,42]. Building upon the established knowledge of R2R3-MYB transcription factors in the model plant A. thaliana [21], we classified the R2R3-MYB genes of S. aculeatissimum into 36 phylogenetic subgroups. The S12 subgroup lacked any SaMYB members, while the M11 subgroup was specifically identified in S. aculeatissimum. Motif analysis revealed that motif 8 was exclusively present in the M11 subgroup, suggesting its potential role in conferring distinct evolutionary features and functional specialization to this clade.
Trichome is an important character of plants with high research value in plant resistance to biotic and abiotic stress, development of medicinal ingredients, and improvement of horticultural traits [43,44]. During the development of plant trichomes, the apical meristem shrinks and the hardness of the trichomes increases, accompanied by the increase in lignin and cellulose contents [45]. In A. thaliana, it was found that AtMYB0/GL1, AtMYB23, AtMYB5, and AtMYB66 in S15 subgroup had complex regulation on the growth and development of trichome [46,47,48]. S9 subgroup AtMYB106 had a negative regulation on the growth of trichomes [49]. To further understand the function of SaMYB members, the cis-elements of R2R3-MYB were analyzed, and the hormone response elements such as ABA, GA, MeJA, SA, and auxin and stress response elements of low temperature, drought, and the like were identified. It has been reported that gibberellic acid (GA) has promoted trichomes initiation and morphogenesis [50,51], and jasmonic acid (JA) was involved in the induction of trichome development in tomatoes and A. thaliana [52,53], while salicylic acid (SA) antagonized JA to inhibit the development of trichomes [54]. We found that 41 members of SaMYB genes had at least one SA response element, and this may be responsible for the potential function of these genes to inhibit trichomes; however, the specific gene function needs to be further verified by experiments.
To explore the expression of R2R3-MYB transcription factors in the process of trichome formation of S. aculeatissimum, wild-type (WT) leaves and sparse-trichome (SC) leaves were selected for transcriptome analysis. Results showed that the expression of 16 SaMYB members were significantly different in WT and SC, potentially regulating trichome formation, and of these, 3 were negatively regulated and 13 were positively regulated. It is noteworthy that several SaMYB genes, including SaMYB1, SaMYB36, and SaMYB23, exhibit highly consistent expression patterns with SaMYB59. Further cis-regulatory element analysis revealed that the promoter regions of these co-expressed genes are significantly enriched with MeJA and gibberellin (GA) responsive elements. This co-expression pattern, combined with their shared key cis-regulatory basis, suggests that SaMYB59 may not function in isolation but rather forms a core regulatory module with these genes. They may integrate upstream signals and promote trichome development through mechanisms such as coordinated responses to MeJA/GA hormonal signals or protein–protein interactions [55,56]. Through qRT-PCR analysis, it was discovered that the expression of positive regulatory members in leaves at various growth stages displays intricate regulatory patterns. Virus-induced gene silencing (VIGS) is a powerful tool for verifying gene functions [57], and Zhou successfully verified the role of the SacMi gene in the root nematode resistance process of S. aculeatissimum through VIGS [34]. To verify the function of SaMYB genes in the formation of trichomes, we selected SaMYB1, SaMYB36, and SaMYB59 for VIGS verification with high expression in early leaf morphogenesis.
In A. thaliana, AtMYB5 (homologous gene of SaMYB1) and AtMYB23 redundantly regulate trichome morphogenesis by controlling trichome-related genes, including AtTTG1 and AtGL2 [58]. In this study, silencing SaMYB1 did not alter trichome phenotypes in S. aculeatissimum, potentially due to functional compensation by redundant homologs that mitigated the loss-of-function effects. This genetic redundancy is a common phenomenon in large plant transcription factor families. It enhances the stability of key traits through functional backup mechanisms, representing a conserved strategy during evolution [59]. AtMYB2 (homologous gene of SaMYB1) functions as a central positive regulator in the abscisic acid (ABA) signaling pathway, exhibiting dynamic expression patterns across developmental stages [60,61]. The silencing of SaMYB36 did not produce an observable effect on plant trichome development. This lack of phenotypic impact could possibly stem from inherent functional redundancy within the R2R3-MYB transcription factor family or limitations arising during the initial candidate gene selection process, potentially influenced by the gene’s pronounced stage-specific expression dynamics [58]. It is noteworthy that AtTT2 forms the MBW complex with the bHLH transcription factor TT8 and the WD40 protein TTG1. This complex activates downstream target genes, executing multitiered regulation of flavonoid metabolism in A. thaliana [62,63]. Silencing of SaMYB59 resulted in a significant reduction in leaf trichome density. Building on established research regarding the A. thaliana gene AtTT2, it is speculated that SaMYB59 may influence trichome formation by modulating the expression levels of core trichome developmental pathway components, such as TTG1 or its downstream target genes. Furthermore, considering its phylogenetic position and the functions of related genes in A. thaliana, it is hypothesized that SaMYB59 in S. aculeatissimum might regulate TTG1 or its core downstream trichome developmental targets by modifying the function of the MBW complex.
In conclusion, this research identified S. aculeatissimum R2R3-MYB members, and comprehensive analysis of these members was performed through physicochemical property analysis, sequence feature analysis, phylogenetic analysis, expression pattern analysis, synteny analysis, and cis-elements. The virus-derived gene silencing experiment shows that SaMYB59 plays an important role in trichome formation in S. aculeatissimum. This research provides the basis for understanding the R2R3-MYB transcription factor family of S. aculeatissimum as a reference to analyze the mechanism of plant trichome formation so as to provide suitable breeding candidate genes of the trichome-free variety of S. aculeatissimum. Future research can combine ChIP-seq, yeast hybridization, and phytohormone profiling to systematically analyze how SaMYB59 regulates downstream gene networks during trichome development. This work will provide a theoretical basis for understanding the biosynthesis of medicinal compounds and the enhancement of stress resistance in Solanaceous crops.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15112637/s1. Supplementary File S1: SaMYB members protein sequence; Supplementary File S2: Protein multiple sequence alignment; Supplementary File S3: Transcriptome quality control document; Supplementary File S4: VIGS target sequence; Figure S1: Illustration of leaf at different growth stages of S. aculeatissimum. The YC1 is tender leaves, the YC2 is growing leaves, and the YC3 is mature leaves; Figure S2: Seqlogo picture of R2 and R3 repeats, asterisk represent conservative tryptophams (W, Trp); Table S1: The characteristics of R2R3-MYB genes of S. aculeatissimum; Table S2: Synteny analysis of SaMYB genes and the KA/KS ratios for each gene pair; Table S3: The primer sequences used in this research.

Author Contributions

Conceptualization, F.Y., Y.Y., W.L., Q.C., and Z.S.; Formal analysis, F.Y., Y.Y., W.L., and Q.C.; Investigation, F.Y., Y.Y., W.L., Q.C., and M.M.; Methodology, F.Y., Y.Y., W.L., Q.C., M.M., Z.S., and L.L.; Project administration, F.Y. and Y.Y.; Resources, Z.S. and L.L.; Supervision, Z.S. and L.L.; Validation, F.Y., Y.Y., W.L., and Q.C.; Visualization, F.Y. and Y.Y.; Writing—original draft, F.Y.; Writing—review and editing, Y.Y., Z.S., and L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by The Key Research and Development Program of Yunnan Province (202403AP140045; 202403AP140026). The Basic Research and Development Program of Yunnan Province (202501BD070001-019).

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Wu, S.; Sun, Z.; Li, L. Transcriptome Analysis Reveals Key Genes and Pathways Associated with Cadmium Stress Tolerance in Solanum aculeatissimum C.B. Clarke. Agriculture 2024, 14, 1686. [Google Scholar] [CrossRef]
  2. Yan, Y.; Wang, W.; Hu, T.; Hu, H.; Wang, J.; Wei, Q.; Bao, C. Metabolomic and Transcriptomic Analyses Reveal the Effects of Grafting on Nutritional Properties in Eggplant. Foods 2023, 12, 3082. [Google Scholar] [CrossRef] [PubMed]
  3. Yang, X.; Liu, F.; Zhang, Y.; Wang, L.; Cheng, Y.-f. Cold-responsive miRNAs and their target genes in the wild eggplant species Solanum aculeatissimum. BMC Genom. 2017, 18, 1000. [Google Scholar] [CrossRef] [PubMed]
  4. Jarald, E.E.; Edwin, S.; Saini, V.; Deb, L.; Gupta, V.; Wate, S.; Busari, K. Anti-inflammatory and anthelmintic activities of Solanum khasianum Clarke. Nat. Prod. Res. 2008, 22, 269–274. [Google Scholar] [CrossRef]
  5. Lal, M.; Munda, S.; Bhandari, S.; Saikia, S.; Begum, T.; Pandey, S.K. Molecular genetic diversity analysis using SSR marker amongst high solasodine content lines of Solanum khasianum C.B. Clarke, an industrially important plant. Ind. Crops Prod. 2022, 184, 115073. [Google Scholar] [CrossRef]
  6. Begum, T.; Munda, S.; Pandey, S.K.; Lal, M. Estimation of selection criteria through multi-year assessment of variability parameters, association studies and genetic diversity of Solanum khasianum CB Clarke. Sci. Hortic. 2022, 297, 110923. [Google Scholar] [CrossRef]
  7. Gogoi, R.; Sarma, N.; Pandey, S.; Lal, M. Phytochemical constituents and pharmacological potential of Solanum khasianum C.B. Clarke., extracts: Special emphasis on its skin whitening, anti-diabetic, acetylcholinesterase and genotoxic activities. Trends Phytochem. Res. 2021, 5, 47–61. [Google Scholar]
  8. Srinivas, M.; Krishnan, R. Effect of Grafting on Solasodine Content in Solanum viarum. Planta Med. 1996, 62, 360–361. [Google Scholar] [CrossRef]
  9. Hao, N.; Yao, H.; Suzuki, M.; Li, B.; Wang, C.; Cao, J.; Fujiwara, T.; Wu, T.; Kamiya, T. Novel lignin-based extracellular barrier in glandular trichome. Nat. Plants 2024, 10, 381–389. [Google Scholar] [CrossRef]
  10. Tissier, A.; Morgan, J.A.; Dudareva, N. Plant Volatiles: Going ‘In’ but not ‘Out’ of Trichome Cavities. Trends Plant Sci. 2017, 22, 930–938. [Google Scholar] [CrossRef]
  11. Xie, L.; Yan, T.; Li, L.; Chen, M.; Hassani, D.; Li, Y.; Qin, W.; Liu, H.; Chen, T.; Fu, X.; et al. An HD-ZIP-MYB complex regulates glandular secretory trichome initiation in Artemisia annua. New Phytol. 2021, 231, 2050–2064. [Google Scholar] [CrossRef]
  12. Chen, R.; Bu, Y.; Ren, J.; Pelot, K.A.; Hu, X.; Diao, Y.; Chen, W.; Zerbe, P.; Zhang, L. Discovery and modulation of diterpenoid metabolism improves glandular trichome formation, artemisinin production and stress resilience in Artemisia annua. New Phytol. 2021, 230, 2387–2403. [Google Scholar] [CrossRef]
  13. Xie, Z.; Mi, Y.; Kong, L.; Gao, M.; Chen, S.; Chen, W.; Meng, X.; Sun, W.; Chen, S.; Xu, Z. Cannabis sativa: Origin and history, glandular trichome development, and cannabinoid biosynthesis. Hortic. Res. 2023, 10, uhad150. [Google Scholar] [CrossRef]
  14. Liu, H.; Liu, S.; Jiao, J.; Lu, T.J.; Xu, F. Trichomes as a natural biophysical barrier for plants and their bioinspired applications. Soft Matter 2017, 13, 5096–5106. [Google Scholar] [CrossRef] [PubMed]
  15. Schilmiller, A.; Shi, F.; Kim, J.; Charbonneau, A.L.; Holmes, D.; Daniel Jones, A.; Last, R.L. Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines. Plant J. 2010, 62, 391–403. [Google Scholar] [CrossRef] [PubMed]
  16. Bryant, L.; Patole, C.; Cramer, R. Proteomic analysis of the medicinal plant Artemisia annua: Data from leaf and trichome extracts. Data Brief 2016, 7, 325–331. [Google Scholar] [CrossRef] [PubMed]
  17. Wang, Q.; Reddy, V.A.; Panicker, D.; Mao, H.-Z.; Kumar, N.; Rajan, C.; Venkatesh, P.N.; Chua, N.-H.; Sarojam, R. Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata). Plant Biotechnol. J. 2016, 14, 1619–1632. [Google Scholar] [CrossRef]
  18. Tiwari, P. Recent advances and challenges in trichome research and essential oil biosynthesis in Mentha arvensis L. Ind. Crops Prod. 2016, 82, 141–148. [Google Scholar] [CrossRef]
  19. Akhtar, M.Q.; Qamar, N.; Yadav, P.; Kulkarni, P.; Kumar, A.; Shasany, A.K. Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (−)-menthol biosynthesis in Mentha species. Physiol. Plant. 2017, 160, 128–141. [Google Scholar] [CrossRef]
  20. Yang, C.; Ye, Z. Trichomes as models for studying plant cell differentiation. Cell. Mol. Life Sci. 2013, 70, 1937–1948. [Google Scholar] [CrossRef]
  21. Dubos, C.; Stracke, R.; Grotewold, E.; Weisshaar, B.; Martin, C.; Lepiniec, L. MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010, 15, 573–581. [Google Scholar] [CrossRef] [PubMed]
  22. Matus, J.T.; Aquea, F.; Arce-Johnson, P. Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes. BMC Plant Biol. 2008, 8, 83. [Google Scholar] [CrossRef] [PubMed]
  23. Ito, M.; Iwase, M.; Kodama, H.; Lavisse, P.; Komamine, A.; Nishihama, R.; Machida, Y.; Watanabe, A. A Novel cis-Acting Element in Promoters of Plant B-Type Cyclin Genes Activates M Phase–Specific Transcription. Plant Cell 1998, 10, 331–341. [Google Scholar] [CrossRef] [PubMed]
  24. Ambawat, S.; Sharma, P.; Yadav, N.R.; Yadav, R.C. MYB transcription factor genes as regulators for plant responses: An overview. Physiol. Mol. Biol. Plants 2013, 19, 307–321. [Google Scholar] [CrossRef]
  25. Chezem, W.R.; Clay, N.K. Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs. Phytochemistry 2016, 131, 26–43. [Google Scholar] [CrossRef]
  26. An, X.-H.; Tian, Y.; Chen, K.-Q.; Liu, X.-J.; Liu, D.-D.; Xie, X.-B.; Cheng, C.-G.; Cong, P.-H.; Hao, Y.-J. MdMYB9 and MdMYB11 are Involved in the Regulation of the JA-Induced Biosynthesis of Anthocyanin and Proanthocyanidin in Apples. Plant Cell Physiol. 2015, 56, 650–662. [Google Scholar] [CrossRef]
  27. Chezem, W.R.; Memon, A.; Li, F.-S.; Weng, J.-K.; Clay, N.K. SG2-Type R2R3-MYB Transcription Factor MYB15 Controls Defense-Induced Lignification and Basal Immunity in Arabidopsis. Plant Cell 2017, 29, 1907–1926. [Google Scholar] [CrossRef]
  28. Oppenheimer, D.G.; Herman, P.L.; Sivakumaran, S.; Esch, J.; Marks, M.D. A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules. Cell 1991, 67, 483–493. [Google Scholar] [CrossRef]
  29. Schliep, M.; Ebert, B.; Simon-Rosin, U.; Zoeller, D.; Fisahn, J. Quantitative expression analysis of selected transcription factors in pavement, basal and trichome cells of mature leaves from Arabidopsis thaliana. Protoplasma 2010, 241, 29–36. [Google Scholar] [CrossRef]
  30. Yu, C.-Y.; Sharma, O.; Nguyen, P.H.T.; Hartono, C.D.; Kanehara, K. A pair of DUF538 domain-containing proteins modulates plant growth and trichome development through the transcriptional regulation of GLABRA1 in Arabidopsis thaliana. Plant J. 2021, 108, 992–1004. [Google Scholar] [CrossRef]
  31. Sun, W.; Gao, Z.; Wang, J.; Huang, Y.; Chen, Y.; Li, J.; Lv, M.; Wang, J.; Luo, M.; Zuo, K. Cotton fiber elongation requires the transcription factor GhMYB212 to regulate sucrose transportation into expanding fibers. New Phytol. 2019, 222, 864–881. [Google Scholar] [CrossRef]
  32. Yan, T.; Li, L.; Xie, L.; Chen, M.; Shen, Q.; Pan, Q.; Fu, X.; Shi, P.; Tang, Y.; Huang, H.; et al. A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua. New Phytol. 2018, 218, 567–578. [Google Scholar] [CrossRef]
  33. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  34. Zhou, X.; Liu, J.; Bao, S.; Yang, Y.; Zhuang, Y. Molecular Cloning and Characterization of a Wild Eggplant Solanum aculeatissimum NBS-LRR Gene, Involved in Plant Resistance to Meloidogyne incognita. Int. J. Mol. Sci. 2018, 19, 583. [Google Scholar] [CrossRef] [PubMed]
  35. Mahanty, A.; Purohit, G.K.; Mohanty, S.; Nayak, N.R.; Mohanty, B.P. Suitable reference gene for quantitative real-time PCR analysis of gene expression in gonadal tissues of minnow Puntius sophore under high-temperature stress. BMC Genom. 2017, 18, 617. [Google Scholar] [CrossRef] [PubMed]
  36. Newman, L.J.; Perazza, D.E.; Juda, L.; Campbell, M.M. Involvement of the R2R3-MYB, AtMYB61, in the ectopic lignification and dark-photomorphogenic components of the det3 mutant phenotype. Plant J. 2004, 37, 239–250. [Google Scholar] [CrossRef]
  37. Kang, L.; Teng, Y.; Cen, Q.; Fang, Y.; Tian, Q.; Zhang, X.; Wang, H.; Zhang, X.; Xue, D. Genome-Wide Identification of R2R3-MYB Transcription Factor and Expression Analysis under Abiotic Stress in Rice. Plants 2022, 11, 1928. [Google Scholar] [CrossRef]
  38. Ma, D.; Reichelt, M.; Yoshida, K.; Gershenzon, J.; Constabel, C.P. Two R2R3-MYB proteins are broad repressors of flavonoid and phenylpropanoid metabolism in poplar. Plant J. 2018, 96, 949–965. [Google Scholar] [CrossRef]
  39. Gonzalez, A.; Zhao, M.; Leavitt, J.M.; Lloyd, A.M. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. 2008, 53, 814–827. [Google Scholar] [CrossRef]
  40. Lepiniec, L.; Debeaujon, I.; Routaboul, J.-M.; Baudry, A.; Pourcel, L.; Nesi, N.; Caboche, M. GENETICS AND BIOCHEMISTRY OF SEED FLAVONOIDS. Annu. Rev. Plant Biol. 2006, 57, 405–430. [Google Scholar] [CrossRef]
  41. Reyes, J.L.; Chua, N.-H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J. 2007, 49, 592–606. [Google Scholar] [CrossRef] [PubMed]
  42. Jung, C.; Seo, J.S.; Han, S.W.; Koo, Y.J.; Kim, C.H.; Song, S.I.; Nahm, B.H.; Do Choi, Y.; Cheong, J.-J. Overexpression of AtMYB44 Enhances Stomatal Closure to Confer Abiotic Stress Tolerance in Transgenic Arabidopsis. Plant Physiol. 2008, 146, 623. [Google Scholar] [CrossRef] [PubMed]
  43. Yan, A.; Pan, J.; An, L.; Gan, Y.; Feng, H. The responses of trichome mutants to enhanced ultraviolet-B radiation in Arabidopsis thaliana. J. Photochem. Photobiol. B Biol. 2012, 113, 29–35. [Google Scholar] [CrossRef] [PubMed]
  44. Andrade, M.C.; da Silva, A.A.; Neiva, I.P.; Oliveira, I.R.C.; De Castro, E.M.; Francis, D.M.; Maluf, W.R. Inheritance of type IV glandular trichome density and its association with whitefly resistance from Solanum galapagense accession LA1401. Euphytica 2017, 213, 52. [Google Scholar] [CrossRef]
  45. Posluszny, U.; Fisher, J.B. Thorn and hook ontogeny in Artabotrys hexapetalus (Annonaceae). Am. J. Bot. 2000, 87, 1561–1570. [Google Scholar] [CrossRef]
  46. Kang, Y.H.; Kirik, V.; Hulskamp, M.; Nam, K.H.; Hagely, K.; Lee, M.M.; Schiefelbein, J. The MYB23 Gene Provides a Positive Feedback Loop for Cell Fate Specification in the Arabidopsis Root Epidermis. Plant Cell 2009, 21, 1080–1094. [Google Scholar] [CrossRef]
  47. Kirik, V.; Lee, M.M.; Wester, K.; Herrmann, U.; Zheng, Z.; Oppenheimer, D.; Schiefelbein, J.; Hulskamp, M. Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation. Development 2005, 132, 1477–1485. [Google Scholar] [CrossRef]
  48. Zhong, R.; Lee, C.; Zhou, J.; McCarthy, R.L.; Ye, Z.-H. A Battery of Transcription Factors Involved in the Regulation of Secondary Cell Wall Biosynthesis in Arabidopsis. Plant Cell 2008, 20, 2763–2782. [Google Scholar] [CrossRef]
  49. Jakoby, M.J.; Falkenhan, D.; Mader, M.T.; Brininstool, G.; Wischnitzki, E.; Platz, N.; Hudson, A.; Hülskamp, M.; Larkin, J.; Schnittger, A. Transcriptional Profiling of Mature Arabidopsis Trichomes Reveals That NOECK Encodes the MIXTA-Like Transcriptional Regulator MYB106. Plant Physiol. 2008, 148, 1583–1602. [Google Scholar] [CrossRef]
  50. Telfer, A.; Bollman, K.M.; Poethig, R.S. Phase change and the regulation of trichome distribution in Arabidopsis thaliana. Development 1997, 124, 645–654. [Google Scholar] [CrossRef]
  51. Gan, Y.; Kumimoto, R.; Liu, C.; Ratcliffe, O.; Yu, H.; Broun, P. GLABROUS INFLORESCENCE STEMS Modulates the Regulation by Gibberellins of Epidermal Differentiation and Shoot Maturation in Arabidopsis. Plant Cell 2006, 18, 1383–1395. [Google Scholar] [CrossRef] [PubMed]
  52. Li, L.; Zhao, Y.; McCaig, B.C.; Wingerd, B.A.; Wang, J.; Whalon, M.E.; Pichersky, E.; Howe, G.A. The Tomato Homolog of CORONATINE-INSENSITIVE1 Is Required for the Maternal Control of Seed Maturation, Jasmonate-Signaled Defense Responses, and Glandular Trichome Development. Plant Cell 2004, 16, 126–143. [Google Scholar] [CrossRef] [PubMed]
  53. Qi, T.; Song, S.; Ren, Q.; Wu, D.; Huang, H.; Chen, Y.; Fan, M.; Peng, W.; Ren, C.; Xie, D. The Jasmonate-ZIM-Domain Proteins Interact with the WD-Repeat/bHLH/MYB Complexes to Regulate Jasmonate-Mediated Anthocyanin Accumulation and Trichome Initiation in Arabidopsis thaliana. Plant Cell 2011, 23, 1795–1814. [Google Scholar] [CrossRef] [PubMed]
  54. Bowling, S.A.; Clarke, J.D.; Liu, Y.; Klessig, D.F.; Dong, X. The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 1997, 9, 1573–1584. [Google Scholar] [CrossRef]
  55. Stracke, R.; Werber, M.; Weisshaar, B. The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 2001, 4, 447–456. [Google Scholar] [CrossRef]
  56. Wang, S.; Chen, J.-G. Regulation of cell fate determination by single-repeat R3 MYB transcription factors in Arabidopsis. Front. Plant Sci. 2014, 5, 133. [Google Scholar] [CrossRef]
  57. Zulfiqar, S.; Farooq, M.A.; Zhao, T.; Wang, P.; Tabusam, J.; Wang, Y.; Xuan, S.; Zhao, J.; Chen, X.; Shen, S.; et al. Virus-Induced Gene Silencing (VIGS): A Powerful Tool for Crop Improvement and Its Advancement towards Epigenetics. Int. J. Mol. Sci. 2023, 24, 5608. [Google Scholar] [CrossRef]
  58. Li, S.F.; Milliken, O.N.; Pham, H.; Seyit, R.; Napoli, R.; Preston, J.; Koltunow, A.M.; Parish, R.W. The Arabidopsis MYB5 Transcription Factor Regulates Mucilage Synthesis, Seed Coat Development, and Trichome Morphogenesis. Plant Cell 2009, 21, 72–89. [Google Scholar] [CrossRef]
  59. Castro-Rodríguez, V.; García-Gutiérrez, A.; Cañas, R.A.; Pascual, M.B.; Avila, C.; Cánovas, F.M. Redundancy and metabolic function of the glutamine synthetase gene family in poplar. BMC Plant Biol. 2015, 15, 20. [Google Scholar] [CrossRef]
  60. Abe, H.; Urao, T.; Ito, T.; Seki, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as Transcriptional Activators in Abscisic Acid Signaling. Plant Cell 2003, 15, 63–78. [Google Scholar] [CrossRef]
  61. Li, T.; Zhang, S.; Li, Y.; Zhang, L.; Song, W.; Chen, C. Overexpression of AtMYB2 Promotes Tolerance to Salt Stress and Accumulations of Tanshinones and Phenolic Acid in Salvia miltiorrhiza. Int. J. Mol. Sci. 2024, 25, 4111. [Google Scholar] [CrossRef]
  62. Nesi, N.; Jond, C.; Debeaujon, I.; Caboche, M.; Lepiniec, L.c. The Arabidopsis TT2 Gene Encodes an R2R3 MYB Domain Protein That Acts as a Key Determinant for Proanthocyanidin Accumulation in Developing Seed. Plant Cell 2001, 13, 2099–2114. [Google Scholar] [CrossRef]
  63. Baudry, A.; Heim, M.A.; Dubreucq, B.; Caboche, M.; Weisshaar, B.; Lepiniec, L. TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J. 2004, 39, 366–380. [Google Scholar] [CrossRef]
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Figure 1. Chromosome localization of the R2R3-MYB genes in S. aculeatissimum. The left scale indicates the chromosome length (Mb). Different colors represent the distribution of genes: red indicates regions with higher gene density, and blue indicates regions with lower gene density.
Figure 1. Chromosome localization of the R2R3-MYB genes in S. aculeatissimum. The left scale indicates the chromosome length (Mb). Different colors represent the distribution of genes: red indicates regions with higher gene density, and blue indicates regions with lower gene density.
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Figure 2. Phylogenetic analysis of SaMYB members. Background color of gene numbers represents 10 main groups. Differently colored boxes of the outer ring represent 36 subgroups. The red star represents R2R3-MYB transcription factors of A. thaliana.
Figure 2. Phylogenetic analysis of SaMYB members. Background color of gene numbers represents 10 main groups. Differently colored boxes of the outer ring represent 36 subgroups. The red star represents R2R3-MYB transcription factors of A. thaliana.
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Figure 3. Conserved structure and cis-acting elements prediction analysis of SaMYB genes. (a) The neighbor joining tree with 1000 bootstrap replicates of all SaMYB members, (b) motif compositions, (c) conserved domains, and (d) cis-acting elements.
Figure 3. Conserved structure and cis-acting elements prediction analysis of SaMYB genes. (a) The neighbor joining tree with 1000 bootstrap replicates of all SaMYB members, (b) motif compositions, (c) conserved domains, and (d) cis-acting elements.
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Figure 4. Synteny analysis of SaMYB genes. The gray lines indicate all syntenic pairs in S. aculeatissimum, and red lines indicate syntenic pairs in SaMYB members.
Figure 4. Synteny analysis of SaMYB genes. The gray lines indicate all syntenic pairs in S. aculeatissimum, and red lines indicate syntenic pairs in SaMYB members.
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Figure 5. The expression patterns of SaMYB genes. (A) The expression patterns of SaMYB genes in leaves of wild-type (WT) and sparse-trichome (SC), 3 replicates per sample. The expression patterns were analyzed based on RNA-seq. The hierarchical clustering heatmap was plotted according to the FPKM values. The red color indicated high expression levels, and the blue color indicated low levels. (B) Positive regulation of SaMYB gene expression patterns in leaves at different growth stages, 3 replicates per sample. YC1 is tender leaves, YC2 is growing leaves, and YC3 is mature leaves. Different letters indicate significant differences between groups (p < 0.05).
Figure 5. The expression patterns of SaMYB genes. (A) The expression patterns of SaMYB genes in leaves of wild-type (WT) and sparse-trichome (SC), 3 replicates per sample. The expression patterns were analyzed based on RNA-seq. The hierarchical clustering heatmap was plotted according to the FPKM values. The red color indicated high expression levels, and the blue color indicated low levels. (B) Positive regulation of SaMYB gene expression patterns in leaves at different growth stages, 3 replicates per sample. YC1 is tender leaves, YC2 is growing leaves, and YC3 is mature leaves. Different letters indicate significant differences between groups (p < 0.05).
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Figure 6. SaMYB59 silencing decreases trichome density in S. aculeatissimum. (A) PCR detection of different treatment plants. M: 5000-bp marker, 1–3: plants inoculated with empty TRV2, 5–7: H2O control, 9–12: plants inoculated with TRV2-SaMYB1; 13–15: plants inoculated with TRV2-SaMYB36, and 17–19: plants inoculated with TRV2-SaMYB59. (B) qRT-PCR analysis of target gene mRNA transcript levels in silenced plants and empty TRV2 and H2O control plants. Different letters indicate significant differences between groups (p < 0.05). (C) Scanning electron microscope observation of leaf trichomes.
Figure 6. SaMYB59 silencing decreases trichome density in S. aculeatissimum. (A) PCR detection of different treatment plants. M: 5000-bp marker, 1–3: plants inoculated with empty TRV2, 5–7: H2O control, 9–12: plants inoculated with TRV2-SaMYB1; 13–15: plants inoculated with TRV2-SaMYB36, and 17–19: plants inoculated with TRV2-SaMYB59. (B) qRT-PCR analysis of target gene mRNA transcript levels in silenced plants and empty TRV2 and H2O control plants. Different letters indicate significant differences between groups (p < 0.05). (C) Scanning electron microscope observation of leaf trichomes.
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Yang, F.; Yang, Y.; Li, W.; Cai, Q.; Miao, M.; Sun, Z.; Li, L. The Regulatory Role of R2R3-MYB Family Genes in Trichome Formation in Solanum aculeatissimum. Agronomy 2025, 15, 2637. https://doi.org/10.3390/agronomy15112637

AMA Style

Yang F, Yang Y, Li W, Cai Q, Miao M, Sun Z, Li L. The Regulatory Role of R2R3-MYB Family Genes in Trichome Formation in Solanum aculeatissimum. Agronomy. 2025; 15(11):2637. https://doi.org/10.3390/agronomy15112637

Chicago/Turabian Style

Yang, Fan, Yanbo Yang, Wanqi Li, Qihang Cai, Man Miao, Zhenghai Sun, and Liping Li. 2025. "The Regulatory Role of R2R3-MYB Family Genes in Trichome Formation in Solanum aculeatissimum" Agronomy 15, no. 11: 2637. https://doi.org/10.3390/agronomy15112637

APA Style

Yang, F., Yang, Y., Li, W., Cai, Q., Miao, M., Sun, Z., & Li, L. (2025). The Regulatory Role of R2R3-MYB Family Genes in Trichome Formation in Solanum aculeatissimum. Agronomy, 15(11), 2637. https://doi.org/10.3390/agronomy15112637

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