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. 2023 Jan 6;16(1):e20295. doi: 10.1002/tpg2.20295

Genome‐wide identification of HD‐ZIP gene family and screening of genes related to prickle development in Zanthoxylum armatum

Xiaoxi Zhang 1, Zexiong Chen 2, Caini Wang 1, Xian Zhou 1, Ning Tang 2, Weiwei Zhang 1,3,✉, Feng Xu 1,✉, Zhiwu Yang 4, Chengrong Luo 4, Yongling Liao 1, Jiabao Ye 1
PMCID: PMC12806939  PMID: 36606521

Abstract

Zanthoxylum armatum is an important cash crop for medicinal and food purposes in Asia. However, its stems and leaves are covered with a large number of prickles, which cause many problems in the production process. The homeodomain leucine zipper (HD‐ZIP) gene family is a class of transcription factors unique to plants that play an important role in biological processes such as morphogenesis, signal transduction, and secondary metabolite synthesis. However, little is known about HD‐ZIP gene information that may be involved in prickle development of Z. armatum. Here, we identified 76 ZaHDZ genes from the Z. armatum genome and classified them into four subfamilies (I–IV) based on phylogenetic analysis, a classification further supported by gene structure and conserved motif analysis. Seventy‐six ZaHDZ genes were unevenly distributed on chromosomes. Evolutionary analysis revealed that the expansion of ZaHDZ genes mainly were due to whole‐genome duplication (WGD) or segmental duplication, and they experienced strong purifying selection pressure in the process of evolution. A total of 47 cis‐elements were identified in the promoter region of ZaHDZ genes. Quantitative real‐time polymerase chain reaction analysis was performed on subfamily IV ZaHDZ gene expression levels in five tissues and under four hormone treatments. Finally, ZaHDZ16 was predicted to be the candidate gene most likely to be involved in prickle development of Z. armatum. These results contribute to a better understanding of the characteristics of HD‐ZIP gene family and lay a foundation for further study on the function of genes related to prickle development of Z. armatum.

Core Ideas

  • Seventy‐six HD‐ZIP genes were identified from the Zanthoxylum armatum genome.

  • Some IV subfamily ZaHDZ genes responded to different hormone treatments.

  • ZaHDZ16 is most likely to be involved in prickle development of Zanthoxylum armatum.

Abbreviations

6‐BA

6‐benzylaminopurine

ABA

abscisic acid

GA

gibberellin

GA3

gibberellic acid‐3

HD

homeodomain

HD‐ZIP

homeodomain leucine zipper

Ka

nonsynonymous substitution rate

Ks

synonymous substitution rate

LZ

leucine zipper

MeJA

methyl jasmonate

qRT‐PCR

quantitative real‐time polymerase chain reaction

SA

salicylic acid

WGD

whole‐genome duplication

1. INTRODUCTION

Zanthoxylum armatum (genus Zanthoxylum, family Rutaceae) is widely distributed in China, India, Thailand, Japan, South Korea, and other Asian countries. Its mature peel is often used as a traditional medicine and food flavoring and has high economic value (Phuyal et al., 2019). Zanthoxylum armatum is the main cultivated variety in southwest China (Xiang et al., 2016). It is widely loved by people because of its strong fragrance and long hemp flavor. In addition, Z. armatum has the characteristics of rapid growth, barren resistance, and drought resistance. It plays an important role in water and soil conservation and in returning farmland to forests in Southwest China. At present, its cultivation area has been further expanded. However, the stem and leaf surface of Z. armatum have many hard prickles, which complicates field management and fruit harvesting. Therefore, the mechanism of Z. armatum prickle formation needs to be elucidated to cultivate varieties with no or few prickles and reduce the production cost.

The plant epidermis is the first line of defense against biotic and abiotic stresses. It is usually distributed with accessory structures such as trichomes and spines (called epiderms) to assist in exercising defense functions (Zhou, Simonneau et al., 2021). Trichomes are highly specialized epidermal protrusions, often located in the leaves, stems, flowers, petioles, sepals, tendrils, seed coats, and other parts, and play an important role in defending against alien organisms such as insects or large herbivores (biological stress) and coping with abiotic stress (Liu et al., 2016; Zhu et al., 2018). Trichomes originate from protocortical cells and have different manifestations in different plants (Han et al., 2022). Prickles and trichomes have certain correlations and similarities (Swarnkar et al., 2021; Zhou, Simonneau et al., 2021), and some studies suggest that spines originate from multiple cell divisions in the epidermis, develop from modified glandular trichome, and then lignify to form rigid spiny appendages, which are the result of the lignification of epidermal hairs (KelloggAllicia et al., 2011; Khadgi & Weber, 2020; Qian et al., 2021). Swarnkar et al. (2021) found that prickle formation in Rosa hybrida L. is controlled by the canonical MBW transcriptional complex. The origin and developmental regulation mode of prickles and trichomes are considered to be similar, and prickles have been proposed to have evolved from trichomes.

In Arabidopsis thaliana (L.) Heynh., the regulation mechanism of the formation of single‐cell trichomes (nonglandular trichome) has been widely studied. Transcription factors play an important role in the development and regulation of trichomes mainly including MYB, bHLH, WD40, HD‐ZIP, WRKY, and other transcription factor families. The R2‐R3 MYB transcription factor GL1 (GLABRA1), bHLH transcription factor GL3 (GLABRA3), and its homologue EGL3 (ENHANCER OF GLABRA3) and WD40 repeat protein TTG1 (TRANSPARENT TESTA GLABRA 1) form MYB‐bHLH‐WDR (MBW) complex to activate the expression of downstream gene GL2(GLABRA2) and then initiate the differentiation of trichomes (Pesch & Hülskamp, 2009; Wang & Chen, 2008; Zhao et al., 2008).

Homeodomain leucine zipper (HD‐ZIP) is a kind of transcription factor unique to plants. Its typical structural feature is composed of a highly conserved homeodomain (HD) composed of 60–61 amino acids at the C‐terminus and a leucine zipper (LZ) immediately behind it. The HD can specifically bind DNA, and LZ acts as a dimerization motif (Ariel et al., 2007). The HD‐ZIP gene is further divided into four subfamilies according to the structure, physiological function, and additional conserved domains of the gene (Mukherjee et al., 2009). The structure of HD‐ZIP I and II subfamily members is similar, and both have a highly conserved HD and a relatively less conserved LZ. The main difference is that there is a CPSCE conserved motif composed of Cys, Pro, Ser, Cys, and Glu at the C‐terminal of type II protein; HD‐ZIP type III and IV have additional START (steroidogenic acute regulatory protein‐related lipid transfer domain) and SAD (START associated domain) domains. In addition, the C‐terminus of HD‐ZIP III subfamily members contains a specific MEKHLA domain, which is not available to other subfamily members (Chen et al., 2014). Different structures endow HD‐ZIP genes with abundant functions, thus enabling them to participate in diverse biological processes. Class I HD‐ZIP genes play an important role in abiotic stress, sugar signaling, blue light signaling, leaf development, flower induction, and de‐etiolation (Ariel et al., 2007; Gong et al., 2019; Henriksson et al., 2005; Liu et al., 2013; Shao et al., 2018; Sharif et al., 2021). Class II HD‐ZIP genes are mainly involved in the shade avoidance response or light signal, auxin induction, and plant development (Ariel et al., 2007; Ciarbelli et al., 2008). Class III HD‐ZIP genes are closely related to biological processes such as polarity maintenance of lateral organs, meristem regulation, root formation, vascular development, and embryogenesis of plants (Ariel et al., 2007; Prigge et al., 2005; Sharif et al., 2021). Class IV HD‐ZIP genes are associated with plant epidermal cell differentiation; trichome formation; development of root hair, stomata, and cuticle (Ariel et al., 2007; Nakamura et al., 2006; Sharif et al., 2021); abiotic stress response (Zhang et al., 2019); and biosynthesis of anthocyanins (Chen & Wang, 2019) and flavonols (Wang et al., 2020).

The Gl2 gene was the first Arabidopsis HD‐ZIP gene identified to be associated with epidermal cell fate determination (Chen & Wang, 2019; Di Cristina et al., 1996; Wang et al., 2007). This gene is highly expressed in mature trichomes and is necessary for the initiation and maintenance of trichomes during cell differentiation (Marks et al., 2009); gl2 mutants exhibit abnormal trichome expansion and ectopic root hair differentiation (Di Cristina et, al., 1996; Hülskamp et al., 1994; Rerie et al., 1994). Type IV genes hdg11 and hdg12 play a role in trichome formation in Arabidopsis. The hdg11 mutant shows the excessive branching phenotype of the trichome, whereas the hdg12 mutant has more normal trichomes than the wild type. Moreover, hdg12 can enhance the excessive branching phenotype of the hdg11 mutant (Nakamura et al., 2006). Four type IV HD‐ZIP genes were implicated in tobacco (Nicotiana tabacum L.) glandular trichome induction (Zhang et al., 2019). Type IV HD‐ZIP gene CmGL controls multicellular trichome initiation in melon (Cucumis melo L.) (Zhu et al., 2018). In tomato (Solanum lycopersicum L.), two HD‐ZIP type IV genes related to trichome formation have been reported; Wo (Woolly) is involved in the formation and embryonic development of tomato type I trichomes (Yang et al., 2011). CD2 (CUTIN DEFINIENT2) is preferentially expressed in the epidermal cells of tomato stems and plays an important role in regulating trichome density, cuticle synthesis and anthocyanin accumulation (Nadakuduti et al., 2012). The HD‐ZIP IV subfamily gene AaHD8 can directly promote the expression of AaHD1 in the initiation of glandular hair of annual mugwort (Artemisia annua L.) and is an important positive regulator in the process of trichome formation. In addition, AaHD8 interacts with the MYB family gene aamixta1, which can enhance the development of the stratum corneum and the transcriptional activity of glandular trichome initiation genes (Yan et al., 2017, 2018). HD‐ZIP class I gene CsGL1 and class IV gene CsGL3 related to trichome formation were found in cucumber (Cucumis sativus L.) (Cui et al., 2016; Li et al., 2015). The tree cotton (Gossypium arboreum L.) HD‐ZIP type IV gene GAHOX1 is a functional homolog of Arabidopsis GL2 and is involved in the development of cotton fiber (Guan et al., 2008). The HD‐ZIP IV gene OCL4 can inhibit the development of maize (Zea mays L.) and Arabidopsis trichomes and is necessary for the formation of maize trichomes (Vernoud et al., 2009). At present, the whole genome identification of HD‐ZIP gene has been identified in many plant species such as Arabidopsis (48) (Ciarbelli et al., 2008; Henriksson et al., 2005; Nakamura et al., 2006; Prigge et al., 2005), rice (Oryza sativa L.) (48) (Jain et al., 2008), poplar (Populus trichocarpa Torr. & A. Gray) (63) (Hu et al., 2012), pineapple [Ananas comosus (L.) Merr. var. comosus] (33) (Zhou, Liu et al., 2021), apple [Malus domestica (Suckow) Borkh.] (48) (Liu et al., 2022), barrel clover (Medicago truncatula Gaertn.) (52) (Li et al., 2020), and Dendrobium officinale K. Kimura & Migo (37) (Yang et al., 2022). To our knowledge, HD‐ZIP family genes related to Z. armatum development have not been reported so far. The formation of prickle in Z. armatum is a complex process and may be regulated by a variety of transcription factors. Previous studies have found that the MYB family genes ZaMYB86 and ZaMYB5 work together to regulate prickle formation in Z. armatum. Through the phenotype of transgenic Arabidopsis, ZaMYB86 is found to be a negative regulator of trichome development (Liu et al., 2022a) and whether it participates in prickle formation through the typical ‘MBW‐GL2’ pathway is unknown. Therefore, this study identified and classified the HD‐ZIP family genes of Z. armatum based on genomic data and screened the genes that may be involved in Z. armatum prickle development through expression analysis, laying a theoretical foundation for subsequent functional studies and molecular mechanism analysis of Z. armatum prickle formation.

Core Ideas

  • Seventy‐six HD‐ZIP genes were identified from the Zanthoxylum armatum genome.

  • Some IV subfamily ZaHDZ genes responded to different hormone treatments.

  • ZaHDZ16 is most likely to be involved in prickle development of Zanthoxylum armatum.

2. MATERIALS AND METHODS

2.1. Plant materials and treatments

Zanthoxylum armatum was planted in the Agricultural Science and Technology Innovation Park (30°37′ N, 112°07′ E) at Yangtze University. It was grown naturally in an open‐air environment to carry out unified water and fertilizer management. The Z. armatum leaves were treated with methyl jasmonate (MeJA) (1 mM, 10 mM), salicylic acid (SA) (1 mM, 10 mM), gibberellic acid‐3 (GA3) (5 mM, 10 mM), and 6‐benzylaminopurine (6‐BA, a synthetic cytokinin) (5 mM, 10 mM). Each treatment was sprayed with three plants, and each plant was sprayed with 500 ml (all solutions contained 0.05% [v/v] Tween‐80). A 0.05% (v/v) Tween‐80 water was added as a control (the concentrations of the different hormones were determined on the basis of pre‐experiments). After 24 h spray treatment, the leaves of each treatment group were collected. After the leaves were frozen in liquid nitrogen, they were placed in a −80 °C refrigerator for later use. All control groups and processing groups had three parallel replicates.

The roots, mature leaves, young stems (with prickles removed), fruits, and unlignified prickles of 3‐yr‐old Z. armatum were collected simultaneously in June 2022 and quickly frozen in liquid nitrogen and then stored in −80 °C refrigerator for tissue expression analysis.

2.2. Identification of HD‐ZIP gene in Zanthoxylum armatum

The genome data of Z. armatum were downloaded from figshare (https://doi.org/10.6084/m9.figshare.14400884.v1) (Wang et al., 2021). Hidden Markov model profile of the HD (PF00046) and the LZ domain (PF02183) were downloaded from the PFAM database (http://pfam.xfam.org), and Hmmer 3.1 was used to search for HD‐ZIP gene family members in the protein sequence of Z. armatum (E‐value ≤ 1 × 10−5). The HD‐ZIP gene family members of Z. armatum were predicted on PlantTFDB (http://planttfdb.gao‐lab.org/). The HD‐ZIP protein sequence of Arabidopsis was downloaded and used as the query sequence. BLAST was used to identify the HD‐ZIP gene in the protein sequence of Z. armatum (E‐value ≤ 1 × 10−5) and comprehensively obtain the HD‐ZIP candidate protein sequence of Z. armatum. The obtained HD‐ZIP candidate protein sequences were then identified using Pfam, CD‐Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART (https://smart.embl.de/), and the final data were obtained after manual removal of prediction errors and redundant sequences.

2.3. Multiple sequence alignment and phylogenetic tree analysis of HD‐ZIP gene in Zanthoxylum armatum

The HD‐ZIP protein sequences of sweet orange [Citrus ×sinensis (L.) Osbeck] were downloaded from PlantTFDB, and the HD‐ZIP protein sequences of Z. armatum, Arabidopsis (AtHDZ), and sweet orange (CsHDZ) were compared in multiple sequences by the MAFFT (Katoh & Standley, 2013) algorithm in the PhyloSuite (Zhang et al., 2020) software. The phylogenetic tree was constructed using neighbor joining in MEGA 11 software (parameter setting: bootstrap, 1,000; substitution model, p‐distance; missing data treatment, pairwise deletion). The phylogenetic tree was embellished using the iTOL (Letunic & Bork, 2021) online software (https://itol.embl.de/). The HD‐ZIP sequence information for the three species is presented in Supplemental Table S1.

2.4. Analysis of ZaHDZ protein characteristics

The physicochemical properties of the Z. armatum HD‐ZIP protein were analyzed by the ProtParam tool on the ExPASy online website (https://web.expasy.org/protparam/). The subcellular localization and signal peptides of the Z. armatum HD‐ZIP protein were predicted by Wolf Psort (https://wolfpsort.hgc.jp/) and SignaIP‐5.0 (http://www.cbs.dtu.dk/services/SignalP/) online software, respectively.

2.5. Chromosomal localization and synteny analysis of ZaHDZ gene

Information on gene density of chromosomes and location of ZaHDZ genes on chromosomes was extracted from GFF3 files of Z. armatum by TBtools software (Chen et al., 2020). The synteny information of Z. armatum was obtained by MCScanX (Tang et al., 2008; Wang et al., 2012). The genome sequence and annotation files of Arabidopsis and sweet orange were obtained from NCBI (https://www.ncbi.nlm.nih.gov/). The collinearity between Z. armatum and Arabidopsis and sweet orange genomes was analyzed using MCScanX software. Visualization is conducted through TBtools software.

2.6. Analysis of ZaHDZ gene structure, conserved motifs, and cis‐acting elements

The Batch CD‐Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) tool in NCBI was used to predict the conserved structural domains of the ZaHDZ protein. The conserved motifs of ZaHDZ proteins were analyzed by MEME (https://meme‐suite.org/meme/tools/meme) online software, the number of motifs filtered was set to 20, and default values were used for other parameters. The ZaHDZ gene structure and protein conserved motifs were visualized using TBtools software.

The sequence 2,000 bp upstream of the start codon in the Z. armatum genome was extracted as a promoter sequence and submitted to the PlantCARE (Lescot et al., 2002) database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict cis‐acting elements. The predicted results were visualized using Excel 2019 software and then visualized by TBtools software.

2.7. RNA extraction and qRT‐PCR analysis

The extraction of total RNA from plant material was performed according to the instructions of the plant RNA extraction kit (TaKaRa MiniBEST Plant RNA Extraction Kit) from TaKaRa Corporation (Beijing, China). First strand cDNA synthesis was performed using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme). Specific qPCR primers were designed using Primer Premier 5 and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific primers used for qPCR and the primers for the housekeeping genes (Liu et al., 2022b) are shown in Supplemental Table S2. The quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis was performed using a real‐time quantitative PCR instrument (LineGene 9600Plues). Reactions were performed according to the ChamQ Universal SYBR qPCR Master Mix (Vazyme) instructions: reaction system, 2 × ChamQ Universal SYBR qPCR Master Mix, 10 μl; primer forward, 0.4 μl; primer reverse, 0.4 μl; cDNA, 2 μl; ddH2O to 20 μl. Reaction procedures include the following: constant temperature section, 95 °C, 30 s; cycling section, 95 °C, 10 s, 60 °C, 30 s; and melting section, 95 °C, 15 s, 60 °C, 60 s, 95 °C, 15 s. Three biological replicates were used for each sample, and the relative expression levels of genes were calculated by the 2−ΔΔCT method. The statistical significance of differences between the means of each sample was tested by one‐way ANOVA with Tukey's post hoc HSD test using SPSS 25 software.

3. RESULTS

3.1. Identification and phylogenetic analysis of HD‐ZIP gene in Zanthoxylum armatum

In this study, a total of 76 HD‐ZIP genes were identified from the Z. armatum genome—1.58 times more than those in Arabidopsis (48) and slightly more than those in sweet orange (64), which belong to the same Rutaceae family. These genes were named ZaHDZ01 through ZaHDZ076 in order according to their position on the chromosome.

To investigate the phylogenetic relationships of HD‐ZIP genes between Z. armatum and other species, we constructed a phylogenetic tree containing HD‐ZIP genes from Z. armatum (76), Arabidopsis (48), and sweet orange (64) by the neighbor‐joining method (Figure 1). Consistent with the expected results, these genes were divided into four subfamilies (HD‐ZIP I through IV), with the most members in class I (68) and the least in class III (21). The results of the evolutionary tree of 76 HD‐ZIP genes in Z. armatum showed that there were 23, 24, 9, and 20 members of subfamilies ZaHDZ I to IV, respectively. The HD‐ZIP genes of different species were scattered in almost all evolutionary branches, indicating that the HD‐ZIP genes had completed differentiation before the separation of the species. In general, genes with close phylogenetic relationships may have similar structural or biological functions. For example, in subfamily IV, ZaHDZ16 has relatively high homology with AtHDZ27 (ANTHOCYANINLESS2, ANL2); ZaHDZ32 and ZaHDZ39 with AtHDZ11 (GL2); and ZaHDZ46, ZaHDZ47, and ZaHDZ64 with AtHDZ02 (HDG12) and AtHDZ10 (HDG11), whereas all four genes in Arabidopsis are involved in trichome formation or epidermal cell development and differentiation (Di Cristina et al., 1996; Kubo & Hayashi, 2011; Marks et al., 2009; Nakamura et al., 2006). It is therefore speculated that these six ZaHDZ genes may have similar functions.

FIGURE 1.

FIGURE 1

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Phylogenetic classification of homeodomain leucine zipper (HD‐ZIP) genes in Zanthoxylum armatum, Arabidopsis thaliana, and Citrus sinensis. Phylogenetic tree was constructed by neighbor‐joining method in Mega 11 software, with 1,000 bootstrap value. Red squares, circles, and stars represent HD‐ZIP genes of A. thaliana, Z. armatum, and C. sinensis, respectively. In the figure, the green band represents the type I HD‐ZIP genes, the orange band represents the type II HD‐ZIP genes, the purple band represents the type III HD‐ZIP genes, and the blue band represents the type IV HD‐ZIP genes

3.2. Analysis of the physicochemical properties of ZaHDZ protein

Analysis of the physicochemical properties of ZaHDZ proteins (Supplemental Table S3) showed that the encoded proteins ranged from 151 to 867 amino acids in length with an average length of 457. Members of subfamilies I and II had shorter proteins with an average of 267 amino acids, whereas those members of subfamilies III and IV had an average of 765 amino acids. The molecular masses ranged from 17.13 to 95.44 kD with an average of 50.84 kD. The theoretical isoelectric point ranged from 4.55 to 9.11, with most of the proteins in subfamily II having an isoelectric point >7, indicating that the composition of these proteins contains more basic amino acids. The average index of hydrophilicity of all proteins was <0, and they were hydrophilic proteins. The results of subcellular localization prediction showed that most members of ZaHDZ proteins were localized to the nucleus, four to the chloroplast (ZaHDZ03, ZaHDZ14, ZaHDZ52, and ZaHDZ55), and two to the cytoplasm (ZaHDZ40, ZaHDZ67), indicating that ZaHDZ genes may play different roles in different biological processes in Z. armatum. In addition, no signal peptide was predicted in the ZaHDZ protein.

3.3. Chromosomal localization and synteny analysis of ZaHDZ gene

A total of 66 of the 76 ZaHDZ genes were localized to 27 chromosomes (HD‐ZIP genes were not distributed on the remaining six chromosomes) (Figure 2), and 10 were localized to unanchored scaffolds. The HD‐ZIP genes of Z. armatum were unevenly distributed in chromosomes, with the number of distributed genes ranging from one to six. Chromosome 32 had the highest number of distributed genes with six, whereas chromosomes 4, 8, 13, 19, 20, 28, and 30 all had only one HD‐ZIP gene distributed. Overall, the number of genes distributed at the proximal or distal end of the chromosome was much higher than the number of genes at the central position.

FIGURE 2.

FIGURE 2

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Distribution of ZaHDZ genes on chromosomes. A total of 66 ZaHDZ genes were distributed on 27 chromosomes, and the remaining 10 were located on unanchored scaffold. Different colors represent homeodomain leucine zipper (HD‐ZIP) gene members of different subfamilies, and green, orange, purple, and blue represent HD‐ZIP genes of subfamilies I–IV, respectively

To understand the evolutionary mechanism of the HD‐ZIP gene family of Z. armatum, we investigated its replication events by MCScanX software (Supplemental Table S4; Figure 3). The results showed that the expansion of ZaHDZ gene mainly originated from whole‐genome duplication (WGD) events or segmental duplication, accounting for 60.6% (40), followed by dispersed duplication, accounting for 36.4% (24). Only two HD‐Zip genes were proximal duplication types, accounting for 3.0%, and no tandem duplication events were found. In addition, a total of 34 paralogous homologous HD‐ZIP gene pairs were identified in the Z. armatum genome (Supplemental Table S5), with 14, 12, 4, and 4 paralogous homologous gene pairs of types I–IV, respectively. We also calculated the ratio of nonsynonymous (Ka) to synonymous (Ks) substitutions (Ka/Ks) for these 34 pairs of paralogous homologs. In brief, Ka/Ks < 1, = 1, and > 1 represent purifying selection, neutral selection, and positive selection, respectively. The results showed that the Ka/Ks ratios of all gene pairs ranged from 0.071 (ZaHDZ34/ZaHDZ35) to 0.519 (ZaHDZ48/ZaHDZ49), with a mean value of 0.233, indicating that the ZaHDZ genes may have experienced strong selection pressure for purification during the evolutionary process. The higher Ka/Ks ratio of ZaHDZ48/ZaHDZ49 compared with other homologous gene pairs suggests that they may have undergone a relatively slow evolution after replication.

FIGURE 3.

FIGURE 3

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Collinearity analysis of ZaHDZ genes. The gray line in the circle represents all collinear blocks in the genome of Zanthoxylum armatum, and the blue line represents collinear ZaHDZ gene pairs. The heat map in the inner square represents gene density, and the outer blue square represents chromosomes

The synteny of HD‐ZIP genes of Z. armatum, Arabidopsis, and sweet orange was obtained by MCScanX (Figure 4), and the results showed that there were 54 and 73 HD‐ZIP orthologous gene pairs between Z. armatum and Arabidopsis and sweet orange genomes, respectively, with 29 HD‐ZIP orthologous genes in the Arabidopsis genome and 25 HD‐ZIP orthologous genes in the sweet orange genome. This suggests that ZaHDZ has a closer homologous evolutionary relationship with CsHDZ.

FIGURE 4.

FIGURE 4

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Synteny analysis of homeodomain leucine zipper (HD‐ZIP) genes in Zanthoxylum armatum, Arabidopsis thaliana, and Citrus sinensis. The gray line in the figure represents all collinear blocks in the genomes of Z. armatum, A. thaliana, and C. sinensis, and the blue line signify the syntenic HD‐ZIP gene pairs

3.4. Analysis of ZaHDZ gene structure and conserved motifs

To gain insights into the structure of ZaHDZ protein and to further confirm the classification accuracy of each subfamily, we determined the conserved motif structure and gene structure of the ZaHDZ protein (Figure 5) and identified a total of 20 conserved motifs. Among them, Motif1 and Motif2 are present in all ZaHDZ genes and represent the LZ and HD structural domains, respectively. Motif14 is specifically present in class III genes and corresponds to the MEKHLA structural domain. Motif18 corresponds to the CPSCE motif and is present in most type II genes and almost all type III and IV genes. Compared with subfamilies III and IV, the members of subfamilies I and II have a smaller number of motif compositions and simpler gene structures. The HD‐ZIP members of the same subfamily have similar conserved motif composition, consistent with the phylogenetic tree classification results, and the similar structure implies that they may have similar biological functions.

FIGURE 5.

FIGURE 5

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Phylogenetic tree, conserved motifs, and genetic structures of 76 ZaHDZ genes. (a) Phylogenetic tree of ZaHDZ genes. Phylogenetic tree was constructed by neighbor‐joining method in Mega 11 software, with 1,000 bootstrap value. The color bands of different colors represent HD‐ZIP genes of different subfamilies. (b) The conserved motifs of ZaHDZ genes. Different colors signify different motif types. (c) The characteristic conserved domains of homeodomain leucine zipper (HD‐ZIP) genes in different subfamilies. (d) Structure of ZaHDZ genes. Length of motifs and coding sequences is proportionally represented

Gene structure analysis showed that the ZaHDZ‐encoding gene contained between two and 20 exons (Figure 5d). Types I and II HD‐ZIP genes contained a smaller number of exons, between two and six with an average of three. Types III and IV had a larger number of exons, between five and 20 with an average of 18 and 10 exons for types III and IV genes, respectively. Gene members of the same subfamily have similar exon numbers or exon–intron structures, and the significant differences between different subfamilies further support the phylogenetic classification results.

3.5. Analysis of the cis‐acting element of the ZaHDZ promoters

Cis‐acting elements in gene promoters usually regulate gene expression levels and functions. To investigate the regulatory and expression properties of ZaHDZ, cis‐acting elements were predicted at 2,000 bp upstream of the promoter region. A total of 47 cis‐acting elements were predicted (Supplemental Table S6), which are mainly related to functions such as light response, hormone response, environmental response, and development. We focused on 10 types of components (Figure 6), mainly including hormone response components (gibberellin [GA], SA, abscisic acid, and MeJA) and stress‐related response components (anaerobic, drought, defense, and stress, and low temperature). Among the phytohormone‐related elements, the largest number of MeJA‐responsive elements (168) was dispersed among 48 ZaHDZ genes; the largest number of stress‐response‐related elements were anaerobic‐inducible elements (181), which were widely distributed among 64 ZaHDZ genes. Among the 10 components of interest, ZaHDZ36 contains the largest number (27) and the most abundant type (9) of components, significantly more than the other genes, suggesting that this gene may be involved in the regulation of multiple biological processes. However, the ZaHDZ76 gene does not contain any of the 10 elements. In addition, some cis‐elements between adjacent homologous gene pairs have significant differences such as ZaHDZ76 and ZaHDZ74 and ZaHDZ70 and ZaHDZ73. Moreover, these differences may reflect their functional differences during evolution. Overall, the diverse cis‐acting elements of ZaHDZ genes confer the ability to participate in a wide range of biological responses.

FIGURE 6.

FIGURE 6

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Ten cis‐elements in the promoter regions of ZaHDZ genes. Different colors represent different identification elements. See Supplemental Table S6 for more information

3.6. Expression profiles of type IV ZaHDZ genes in different tissues

To understand the expression of subfamily IV ZaHDZ genes in different tissues, we analyzed the expression levels of 20 type IV genes in the roots, stems, leaves, prickles, and fruits and generated an expression heat map of subfamily IV ZaHDZ genes by using TBtools software (Figure 7). The results showed that the expression of subfamily IV members was generally low in the roots, and most genes had high expression levels in the leaves, suggesting that the genes of this subfamily may exercise biological functions mainly in the growth and development of leaves. In addition, we found that ZaHDZ04, ZaHDZ05, ZaHDZ32, ZaHDZ39, and ZaHDZ50 were highly expressed in the fruits, presumably involved in the regulation of fruit development. The expression of ZaHDZ16, ZaHDZ20, ZaHDZ44, ZaHDZ45, and ZaHDZ53 was higher in the prickles. In particular, the expression of ZaHDZ16 and ZaHDZ44 was significantly higher in the prickles than in other tissues, suggesting that they may play an important role in prickle formation. Notably, some genes with close phylogenetic relationships also have similar expression patterns, such as ZaHDZ04/ZaHDZ05, ZaHDZ27/ZaHDZ51, and ZaHDZ32/ZaHDZ39, suggesting that they may have the same or similar functions.

FIGURE 7.

FIGURE 7

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Expression profile of ZaHDZ genes of subfamily IV in different tissues. Expression values were normalized using SPSS 25 software and heat maps were generated by TBtools software. Expression profiles of 20 type IV ZaHDZ genes in roots, stems, leaves, prickles, and fruits of Zanthoxylum armatum. R, root; S, stem; L, leaf; P, prickle; F, fruit. In the figure, red represents the high expression of this gene in the corresponding tissues, and blue represents the low expression. The color code representing the expression level is displayed on the right

3.7. Expression profiles of ZaHDZ genes under phytohormones treatment

In this study, the expression profiles of five candidate ZaHDZ genes potentially involved in Z. armatum prickle development were evaluated under treatment with four plant hormones (MeJA, GA3, SA, 6‐BA) (Figure 8). Overall, all four hormones caused changes in the expression of candidate genes, but the expression levels of individual candidate genes showed variability under different hormones or different concentrations of the same hormone treatment. Among the five candidate genes, almost all of them responded to SA and MeJA to different degrees. For example, ZaHDZ20 expression was significantly elevated under 1 mM SA treatment, but no significant difference was observed at 10 mM SA treatment. The expression level of ZaHDZ53 was significantly increased under 10 mM MeJA treatment but decreased in 5 mM 6‐BA and GA3 treatment. By contrast, ZaHDZ44 and ZaHDZ45 did not respond significantly to different concentrations of 6‐BA and GA3 treatments, which may be related to the differences in the hormone‐responsive elements in the promoter sequences of different genes. In addition, ZaHDZ16, which may be closely related to Z. armatum prickle development, responded to SA, MeJA, and 6‐BA, whereas ZaHDZ44 responded to SA and MeJA only.

FIGURE 8.

FIGURE 8

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Relative expression levels of five candidate ZaHDZ genes under different hormone treatment conditions analyzed by quantitative real‐time polymerase chain reaction. The y axis shows the relative levels of gene expression. The x axis represents the treatment with different concentrations of hormones. (A–D) Gene expression profiles under salicylic acid (SA), methyl jasmonate (MeJA), cytokinin (CK), and gibberellic acid‐3 (GA3) treatments, respectively. Data represent the mean ± SD of three independent biological replicates. Asterisks represent significant differences (p < .05, one‐way ANOVA with Tukey's post hoc HSD test)

4. DISCUSSION

As a medicinal and food species, Z. armatum has a high economic value and a promising development prospect. However, the numerous prickles on Z. armatum plants complicate field management and fruit harvesting, which greatly increases the production cost. Therefore, it is an urgent problem to analyze the mechanism of Z. armatum prickle formation and to cultivate prickle‐free varieties.

As a kind of transcription factors unique to plants, HD‐ZIP family genes play an important role in several aspects of plant growth and development and have been identified in various plant species. Numerous studies have shown that type IV HD‐ZIP genes play important roles in plant epidermal cell fate determination; trichome initiation; root hair formation; cuticle development; abiotic stress response; and biosynthesis of anthocyanins, flavonols, mucilage, and lipids (Ariel et al., 2007; Chen & Wang, 2019; Nakamura et al., 2006; Sharif et al., 2021; Shen et al., 2006; Wang et al., 2020; Zhang et al., 2019). Among them, HD‐ZIP genes associated with trichome formation have been reported in several species, such as GL2, HDG11, and HDG12 in Arabidopsis (Marks et al., 2009; Nakamura et al., 2006); CmGL in melon (Zhu et al., 2018), Wo (Woolly) and CD2 (CUTIN DEFICIENT2) in tomato (Nadakuduti et al., 2012; Yang et al., 2011); AaHD1 and AaHD8 in annual mugwort (Yan et al., 2017, 2018); and GaHOX1 in cotton (Guan et al., 2008). However, the characteristics and functions of the HD‐ZIP gene of Z. armatum are still unknown, and no HD‐ZIP gene related to Z. armatum prickle development has been reported.

In this study, a total of 76 ZaHDZ genes were identified from the Z. armatum genome, which is more than most of the reported plants such as Dendrobium officinale (37) (Yang et al., 2022), Arabidopsis (48), poplar (63) (Hu et al., 2012), and sweet orange (64), but less than soybean [Glycine max (L.) Merr.] (88) HD‐ZIP family genes, which may be related to the two rounds of WGD events that the soybean genome underwent (Chen et al., 2014). Gene duplication is one of the main evolutionary mechanisms for generating new genes that help organisms to cope with variable environments (Bowers et al., 2003). Previous studies have shown that Z. armatum experienced an independent WGD event ∼26.6 million yr ago (Wang et al., 2021). A total of 40 (59%) ZaHDZ genes affected by WGD or segmental duplication were identified in this study, and no tandem duplication events were found, suggesting that WGD or segmental duplication made an important contribution to the formation of the ZaHDZ gene family. Furthermore, the proportion of ZaHDZ genes affected by segmental duplication (60%) was close to that of maize (62%) (Zhao et al., 2011) and rice (54%) (Jain et al., 2008) HD‐ZIP genes and lower than that of Arabidopsis (75%) (Ciarbelli et al., 2008; Henriksson et al., 2005; Nakamura et al., 2006; Prigge et al., 2005), suggesting that segmental duplication may play a dominant role in the expansion of the HD‐ZIP gene family in plant species. The Ka/Ks ratio is considered an important indicator for assessing sequence evolution based on selection pressure and duplication type (Hurst, 2002). In this study, the mean value of Ka/Ks for 34 paralogous gene pairs was 0.233, implying that ZaHDZ genes may have experienced strong purifying selection pressure during evolution. In poplar (Hu et al., 2012), soybean (Chen et al., 2014), and barrel clover (Li et al., 2020) HD‐ ZIP genes, similar findings were obtained.

Duplicated genes usually have a high degree of sequence similarity and theory, suggesting that there are three alternative outcomes of duplicated genes during evolution: nonfunctionalization, neofunctionalization, and subfunctionalization (Lynch & Conery, 2000). In general, the different evolutionary fates of duplicated genes can be implied by differences in their expression profiles under different tissues and abiotic stresses or hormonal treatments (Li et al., 2020). In the present study, we found that paralogous genes usually showed the same or similar expression patterns in different tissues of ZaHDZ04/ZaHDZ05 and ZaHDZ32/ZaHDZ39 (Figure 7). Similar examples were found under hormone treatment conditions. For example, ZaHDZ44/ZaHDZ45 (Figure 8), ZaHDZ32/ZaHDZ39, and ZaHDZ46/ZaHDZ47 (Supplemental Figure S1) all had similar expression profiles under four hormone treatment conditions, suggesting that paralogous gene pairs may play the same role or exhibit functional redundancy. Different paralogous gene pairs exhibit more or less differential expression between them, implying a diversity of their functions.

Similar to Arabidopsis, ZaHDZ genes were divided into four subfamilies based on phylogenetic analysis, and ZaHDZ genes of the same subfamily have higher structural and functional similarity. Among the four subfamilies, subfamilies III and IV had similar and the largest number of members compared with subfamilies I (23) and II (24). Subfamily III had the smallest number (9), consistent with the distribution characteristics of rice (Jain et al., 2008), maize (Zhao et al., 2011), and poplar (Hu et al., 2012). The conserved motif structure and gene structure of ZaHDZ protein also well support the results of ZaHDZ gene classification. The number of conserved motifs and exons in the HD‐ZIP genes of subfamilies I and II of Z. armatum were significantly smaller than those of the gene members of subfamilies III and IV, having a simpler structure. For example, the MEKHLA structural domain in the HD‐ZIP gene structure of subfamily III is not found in members of other subfamilies, which is consistent with the reported HD‐ZIP gene characteristics of other plant species, indicating that this gene family is highly conserved during the evolutionary process.

The type IV HD‐ZIP gene plays an important regulatory role in plant epidermal trichome development. Numerous studies have shown that GL2 (AtHDZ11) expression promotes the initiation of Arabidopsis trichomes, negatively regulates the formation of root hairs in roots, and is required for the inhibition of root hairs. In addition, GL2 is required in the synthesis pathways of flavonols, anthocyanins, mucilage, and other substances. The MBW complex consists of different genes interacting with each other and participating in the above biological processes by regulating the expression of the same GL2 gene (Chen & Wang, 2019). We compared Arabidopsis GL2 as the query sequence to all protein data of Z. armatum and obtained three genes with the highest identity, namely, ZaHDZ39 (65.17%), ZaHDZ32 (64.16%), and ZaHDZ16 (40.07%). The sequence similarity implies that they may have similar functions to GL2. Combining the tissue expression patterns of these three genes, ZaHDZ16 is most likely to be associated with Z. armatum prickle development with significantly high expression in prickles, high expression in fruits, and almost no expression in roots. By analogy with the function of GL2, we hypothesized that ZaHDZ16 can promote Z. armatum prickle formation and participate in the synthesis of secondary metabolites such as flavonols in fruits, whereas its low expression in roots facilitates root hair formation. In addition, the results of phylogenetic analysis show that ZaHDZ16 has high homology with AtHDZ25 and AtHDZ27 (Figure 1). Previous studies have shown that AtHDZ27 (ANTHOCYANINLESS2, ANL2) is involved in the accumulation of anthocyanins in subepidermal cells and root development in Arabidopsis (Kubo et al., 1999), regulates the mechanical properties of the cell wall (Mabuchi et al., 2016), and is essential for the maintenance of the epidermal cell lineage (Kubo & Hayashi, 2011). Blast results show that ZaHDZ16 has 69.98% identity with AtHDZ27. The ability of ANL2 to be involved in regulating the mechanical properties of the cell wall provides a new perspective for understanding the development of plant epidermal hairs. It has been suggested that plant cell growth is due to relaxation of the cell wall in response to mechanical forces generated by expansion pressure. The growth of cells is anisotropic, and the principal direction of growth is usually associated with the direction of lower cell wall stiffness (Majda et al., 2022). Plant cells trapped in the cell wall are unable to migrate and therefore morphogenesis depends on the directionality of cell division, the precise positioning of the division plane, and the anisotropy of cell growth, which are mediated by regulated mechanical inhomogeneity of the cell wall (Gorelova et al., 2021). Morphogenesis of organisms relies on the integration of biochemical and mechanical signals (Hamant & Traas, 2010), which is regulated by genes to complete the process of growth or differentiation. We speculate that the formation of Z. armatum prickles involves an orderly and precise division of cells in a certain direction, and this process may be regulated by genes such as ZaHDZ16. For ZaHDZ39 and ZaHDZ32, they were significantly highly expressed in fruits and leaves; there was no significant difference in the expression in roots and prickles. In this period, the Z. armatum fruit is in the stage of rapid growth and development, which is accumulating a large number of secondary metabolites. The high expression of ZaHDZ39 and ZaHDZ32 genes implies that they may play an important role in the biosynthetic pathways of flavonols, anthocyanins, and mucilage. Some researchers have pointed out that the coupling of secondary metabolite accumulation and epidermal growth in plants is a general phenomenon that is conserved throughout the species (Swarnkar et al., 2021). Although ZaHDZ39 and ZaHDZ32 did not show specific high expression in the prickles, we cannot deny their possible role in prickle formation. In addition, we aligned ZaHDZ39, ZaHDZ32, and ZaHDZ16 with other reported type IV HD‐ZIP genes associated with trichome formation (Supplemental Table S7). Interestingly, ZaHDZ16 had the highest similarity to the tomato CD2 gene (82.2%), followed by the annual mugwort AaHD8 gene (78.5%), whereas ZaHDZ39 and ZaHDZ32 had the highest similarity to the cotton GaHOX1 gene (81.45 and 82.21%, respectively), suggesting that they may have similar biological functions. In a previous study, we used transcriptome data of Z. armatum ‘Rongchangwuci’ (prickle‐free type) and Z. armatum ‘Zhuye’ (prickly type) leaves for differential expression analysis and identified 38 HD‐ZIP genes that may be involved in Z. armatum prickle development (Liu et al., 2022a). Further analysis revealed that ZaHDZ16, ZaHDZ32, and ZaHDZ39 were all differentially expressed in two different types of Z. armatum leaves. Specifically, the expression of all three genes was significantly higher in leaves of the Zhuye (prickly type) than in the Rongchangwuci (prickle‐free type), with ZaHDZ16 and ZaHDZ32 being significantly different in leaves at three stages (10, 60, and 120 d after sprouting, respectively), whereas ZaHDZ39 was significant only at the first two stages. In addition, their expression did not differ significantly in leaves of the same type at different stages. This conclusion increases the possibility that ZaHDZ16, ZaHDZ32, and ZaHDZ39 are involved in Z. armatum prickle development. However, the functions of these genes need to be further verified by experiments.

Plant hormones play an indispensable role in the growth and development of plants. Recent studies have shown that many plant hormones are involved in trichome development such as JA, abscisic acid, GAs, growth hormone, cytokinin, and SA (Fambrini & Pugliesi, 2019; Li et al., 2021; Traw & Bergelson, 2003). In Arabidopsis, JA and GAs can induce trichome formation, and both have strong synergistic effects in trichome formation. However, SA treatment decreases their trichome density and number (Hua et al., 2021). In other plants, JA promotes the development of glandular trichome on tobacco (Zhang et al., 2019), annual mugwort leaves (Yan et al., 2017), and trichome elongation in tomato (Hua et al., 2021). Growth hormone is involved in the formation of tomato trichomes and fiber elongation in cotton (Zhang et al., 2017, 2015). There is growing evidence that plant hormones play an important role in the formation and regulation of plant trichomes. In the present study, we used four hormones to treat Z. armatum and found that different ZaHDZ genes responded to different hormones or hormone concentrations to different degrees (Figure 8). For example, ZaHDZ16 gene expression was significantly increased at 1 mM SA treatment, significantly decreased at 10 mM SA treatment, and significantly decreased at 1 and 10 mM MeJA treatment with no significant response at GA3 treatment. However, the effect of these hormones on Z. armatum prickle formation needs further study.

5. CONCLUSION

In this study, we identified 76 ZaHDZ genes in the Z. armatum genome and classified them into four subfamilies (I–IV) based on phylogenetic analysis, conserved motif composition, and gene structure analysis. The ZaHDZ gene was also analyzed in terms of chromosomal localization, synteny, and cis‐elements. Subsequently, for subfamily IV ZaHDZ genes, we evaluated their expression profiles in different tissues and under different hormone treatments. Finally, combining the available knowledge and evidence, we predicted an HD‐ZIP gene (ZaHDZ16) that is most likely involved in the developmental relevance of Z. armatum prickles. This study laid the theoretical foundation for subsequent functional studies of the HD‐ZIP gene.

AUTHOR CONTRIBUTIONS

Xiaoxi Zhang: Software; Visualization; writing – Original Draft. Zexiong Chen: Methodology; Software. Caini Wang: Methodolog. Xian Zhou: Software. Ning Tang: Methodology. Weiwei Zhang: Data curation; Project administration; Resources; Supervision; writing – Review & Editing. Feng Xu: Project administration; Visualization; writing – Review & Editing. Zhiwu Yang: Methodology, Chengrong Luo: Methodology. Yongling Liao: Software. Jiabao Ye: Visualization.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Supporting information

Supplemental Figure S1. Relative expression levels of ZaHDZ32, ZaHDZ39, ZaHDZ46, and ZaHDZ47 under different hormone treatments were analyzed by qRT‐PCR. The Y‐axis shows the relative levels of gene expression. x‐axis represents the treatment with different concentrations of hormones. Figure A, B, C, and D represent the gene expression profiles under SA, MeJA, 6‐BA, and GA3 treatments, respectively. Data represent the mean ± SD of three independent biological replicates. Asterisks represent significant differences (p < 0.05, one‐way ANOVA with Tukey's post hoc HSD test).

TPG2-16-e20295-s002.jpg (4.7MB, jpg)

Supplemental Table S1. The protein sequences of HD‐ZIP genes in Zanthoxylum armatum, Arabidopsis thaliana and Citrus sinensis.

TPG2-16-e20295-s003.xlsx (47.3KB, xlsx)

Supplemental Table S2. Primer information used in qRT‐PCR experiment.

TPG2-16-e20295-s005.xlsx (10.3KB, xlsx)

Supplemental Table S3. Basic information on the HD‐ZIP genes of Zanthoxylum armatum.

TPG2-16-e20295-s007.xlsx (15.5KB, xlsx)

Supplemental Table S4. Duplication Type information of ZaHDZ genes.

TPG2-16-e20295-s004.xlsx (10.9KB, xlsx)

Supplemental Table S5. The information of gene replication type, Ka, Ks, and Ka/Ks ratio values of ZaHDZ genes.

TPG2-16-e20295-s001.xlsx (13.2KB, xlsx)

Supplemental Table S6. Information on all cis‐acting elements detected in the promoter regions of ZaHDZ genes.

TPG2-16-e20295-s006.xlsx (332KB, xlsx)

Supplemental Table S7. Sequence alignment of ZaHDZ39, ZaHDZ32, ZaHDZ16 and trichome formation‐related type IV HD‐ZIP genes.

TPG2-16-e20295-s008.xlsx (13.1KB, xlsx)

ACKNOWLEDGMENTS

This work was supported by National Key R&D Program of China (2019YFD1001200), Projects for Innovative Research Groups of Chongqing Universities (CXQT21028), Scientific Technological Research Program of Chongqing Municipal Education Commission (KJZD‐K201901303), and Breakthrough Breeding of New Forest Varieties and Innovation of Breeding Mterials(2021YFYZ0032).

Zhang, X. , Chen, Z. , Wang, C. , Zhou, X. , Tang, N. , Zhang, W. , Xu, F. , Yang, Z. , Luo, C. , Liao, Y. , & Ye, J. (2023). Genome‐wide identification of HD‐ZIP gene family and screening of genes related to prickle development in Zanthoxylum armatum . The Plant Genome, 16, e20295. 10.1002/tpg2.20295

Assigned to Associate Editor Lin Li.

Contributor Information

Weiwei Zhang, Email: wwzhangchn@163.com.

Feng Xu, Email: xufeng@yangtzeu.edu.cn.

DATA AVAILABILITY STATEMENT

All the data were presented in the main manuscript and additional supporting files.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Figure S1. Relative expression levels of ZaHDZ32, ZaHDZ39, ZaHDZ46, and ZaHDZ47 under different hormone treatments were analyzed by qRT‐PCR. The Y‐axis shows the relative levels of gene expression. x‐axis represents the treatment with different concentrations of hormones. Figure A, B, C, and D represent the gene expression profiles under SA, MeJA, 6‐BA, and GA3 treatments, respectively. Data represent the mean ± SD of three independent biological replicates. Asterisks represent significant differences (p < 0.05, one‐way ANOVA with Tukey's post hoc HSD test).

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Supplemental Table S1. The protein sequences of HD‐ZIP genes in Zanthoxylum armatum, Arabidopsis thaliana and Citrus sinensis.

TPG2-16-e20295-s003.xlsx (47.3KB, xlsx)

Supplemental Table S2. Primer information used in qRT‐PCR experiment.

TPG2-16-e20295-s005.xlsx (10.3KB, xlsx)

Supplemental Table S3. Basic information on the HD‐ZIP genes of Zanthoxylum armatum.

TPG2-16-e20295-s007.xlsx (15.5KB, xlsx)

Supplemental Table S4. Duplication Type information of ZaHDZ genes.

TPG2-16-e20295-s004.xlsx (10.9KB, xlsx)

Supplemental Table S5. The information of gene replication type, Ka, Ks, and Ka/Ks ratio values of ZaHDZ genes.

TPG2-16-e20295-s001.xlsx (13.2KB, xlsx)

Supplemental Table S6. Information on all cis‐acting elements detected in the promoter regions of ZaHDZ genes.

TPG2-16-e20295-s006.xlsx (332KB, xlsx)

Supplemental Table S7. Sequence alignment of ZaHDZ39, ZaHDZ32, ZaHDZ16 and trichome formation‐related type IV HD‐ZIP genes.

TPG2-16-e20295-s008.xlsx (13.1KB, xlsx)

Data Availability Statement

All the data were presented in the main manuscript and additional supporting files.

Articles from The Plant Genome are provided here courtesy of Wiley Periodicals LLC on behalf of Crop Science Society of America

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