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Article

Genome-Wide Analysis of the HD-Zip Gene Family in Chinese Cabbage (Brassica rapa subsp. pekinensis) and the Expression Pattern at High Temperatures and in Carotenoids Regulation

by
Lian Yin
1,
Yudong Sun
1,
Xuehao Chen
2,
Jiexia Liu
2,
Kai Feng
2,
Dexu Luo
1,
Manyi Sun
3,
Linchuang Wang
1,
Wenzhao Xu
1,
Lu Liu
1 and
Jianfeng Zhao
1,*
1
Vegetable Research and Development Center, Huaiyin Institute of Agricultural Science in Xuhuai Area of Jiangsu Province, Huaian 223001, China
2
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
3
State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(5), 1324; https://doi.org/10.3390/agronomy13051324
Submission received: 29 March 2023 / Revised: 27 April 2023 / Accepted: 5 May 2023 / Published: 9 May 2023
(This article belongs to the Special Issue Advances in Crop Molecular Breeding and Genetics)
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Versions Notes

Abstract

HD-Zip, a special class of transcription factors in high plants, has a role in plant development and responding to external environmental stress. Heat stress has always been an important factor affecting plant growth, quality, and yield. Carotenoid content is also an important factor affecting the color of the inner leaf blades of Chinese cabbage. In this study, the genomes of three Brassicaceae plants were selected: Chinese cabbage (Brassica rapa subsp. pekinensis), Brassica oleracea, and Brassica napus. We identified 93, 96, and 184 HD-Zip genes in the B. rapa, B. oleracea, and B. napus, respectively. The HD-Zip gene family was classified into four subfamilies based on phylogeny: I, II, III, and IV;. The results of cis-acting element analysis suggested that HD-Zip family genes may participate in various biological processes, such as pigment synthesis, cell cycle regulation, defense stress response, etc. Conserved motifs prediction revealed that three motifs exist among the four HD-Zip gene families and that different motifs exhibit significant effects on the structural differences in HD-Zips. Synteny, Ks, and 4DTv results displayed that genome-wide triplication events act in HD-Zip gene family expansion. Transcriptome data showed that 18 genes responded (>1.5-fold change) to heat stress in Chinese cabbage, and 14 of 18 genes were from the HD-Zip I subfamily. Three genes had up-regulation, and eight genes had down-regulation in high-carotenoid-content Chinese cabbage. The BraA09g011460.3C expression level was up-regulated after heat stress treatment and significantly reduced in varieties with high carotenoid content, indicating its potential for heat stress tolerance and carotenoid content regulation. This study provided important gene resources for the subsequent breeding of Chinese cabbage.

1. Introduction

The HD-Zip family, a transcription factor gene family in plants, consists of the HD (homeodomain) and LZ (leucine zipper) structural domains. In addition to higher plants, the HD-Zip protein was investigated in Pteridophyta [1] and Bryophyta [2]. Until now, the HD-Zip genes have been widely and systematically studied in Arabidopsis thaliana (Arabidopsis), cassava (Manihot esculenta), and maize (Zea mays) [3,4,5]. Numerous reports have shown that the HD-Zip gene family is closely associated with plant growth and tolerance to environmental stress [6]. In view of the existing reports, HD-Zips are usually divided into four groups (I–IV) [7]. HD-Zip I members only consist of HD and LZ domains without other motifs. The expression of HD-Zip I gene members is particularly regulated by abiotic stress, such as light and temperature [8]. Group I genes can also promote the fruit coloration process by regulating the carotenoid content [9]. Group II members have been widely proven in their light quality change response and shade aversion response, and group III has been extensively studied in embryonic plant development [8,10]. Furthermore, Turchi et al. [11] pointed out the interaction of the transcription factors of groups II and III in the auxin regulation of plant development. Group IV proteins were involved in plant morphogenesis, mainly displayed in the plant differentiation of the epidermis and epidermal cells and stomatal development [12,13].
Chinese cabbage (Brassica rapa subsp. Pekinensis) is an herbaceous plant in Brassicaceae with a leafy head. It is a specialty and important vegetable in China with a large cultivation area [14]. Heat stress is one factor affecting production, distribution, and quality. Chinese cabbage prefers cold and cool, and high temperatures affect its growth and development [14]. Therefore, it is of great theoretical and practical importance to research mechanisms and adaptation under high temperatures. Zhang et al. [15] selected heat-tolerant and heat-sensitive Chinese cabbage and measured physiological indicators using transcriptome. Several genes (Prx50, Prx52, Prx54, SOD1, and SOD2) related to reactive oxygen species (ROS) scavenging were identified, which were significantly up-regulated in heat-tolerant varieties. In addition, Quan et al. [16] found that glycine betaine (GB) and β-aminobutyric acid (BABA) could improve photosynthetic performance and antioxidant enzyme activity under high temperatures to alleviate heat stress on Chinese cabbage. Based on the sequenced Chinese cabbage genome, Huang et al. [17] identified 30 heat shock factors (Hsfs) that function in several organs of Chinese cabbage and also hypothesized that Hsfs might be essential in the developmental regulation of the underground parts of Chinese cabbage.
Transcription factors control the plant’s abiotic stress response and regulate the expression of the many downstream target genes of various metabolic processes [18]. Up to the present, the function of HD-Zips under heat stress in other species has been studied. In lily (Lilium longiflorum), the LlHB16 gene can positively regulate heat resistance by linking the heat response pathway and ABA signaling [19]. Li et al. [20] identified 43 HD-Zip genes on 12 chromosomes of potato (Solanum tuberosum L.) and found that the StHOX2 gene was significantly up-regulated in the root. To investigate how HD-Zips regulate the heat stress tolerance of the radish (Raphanus sativus L.), RsHDZ17 was isolated from HD-Zip group I and overexpressed in Arabidopsis. It was found that the gene enhanced the heat stress tolerance of radishes by improving photosynthesis and enhancing the scavenging activity of reactive oxygen [21]. Wang et al. [22] detected the transcription levels of group I genes in heat-tolerant and heat-sensitive perennial ryegrass lines and demonstrated that LpHOX21 was positively associated with heat tolerance. Taken together, HD-Zip genes are essential in the plant lifecycle, including growth, development, reproduction, differentiation, and morphogenesis [23]. However, the effects of the HD-Zip gene family in B. rapa are still being unraveled.
In this study, we identified the HD-Zip gene family in B. rapa, and phylogenetic tree construction, motif and cis-element prediction, and collinearity and Ka/Ks analysis were carried out. Simultaneously, the transcription patterns of HD-Zip genes under heat stress and Chinese cabbage varieties with different carotenoid content were analyzed. The results identified several candidate genes related to heat stress and carotenoid accumulation. We provided a foundation for research on HD-Zip genes in the mechanism of carotenoid accumulation and heat stress response in Chinese cabbage leaf blades.

2. Materials and Methods

2.1. Plant Materials, Heat Stress Treatment, and Carotenoid Content Measurement

The Chinese cabbage seeds of the hybrid one-generation ‘Gailiang Qingza 3’ were purchased from Qingdao International Seedling Co. (Qingdao, China), and the high-generation inbred line ‘54’ were conserved in the Huaian Key Laboratory for Facility Vegetables (33°53′ N, 119°04′ E). Under heat stress treatment, the seeds were sowed in 96-hole cell trays, transplanted into 32 cm diameter pots at 30 days, and the 40-old-day seedlings were cultivated in a light incubator (40 °C for 16 h in the daytime, 30 °C for 8 h at night, with a relative humidity of 60%). The leaf blades of the Chinese cabbages were taken at 0, 4, 8, and 10 day, separately, and the samples were immediately frozen in liquid nitrogen. Under normal conditions, the seeds were sowed in 96-hole cell trays on 8 August 2022 and transplanted into the field on 2 September. The head of the outer (the fifth part) and inner leaves of the 90-day-old line of the ‘54’ cabbage and ‘Gailiang Qingza 3’ were collected to determine the carotenoid content. Carotenoid content was measured according to previous methods [24]. Thus, 50 mg of the sample was ground into a powder by ball mill was extracted with 0.5 mL of a hexane/acetone/ethanol (1:1:1, v/v/v) mixture containing 0.1% BHT (butylated hydroxytoluene), vortexed for 20 min at room temperature, centrifuged at 12,000 r/min for 5 min, and the supernatant was extracted and then repeated twice. The combined supernatant was redissolved with 100 uL of a methanol/methyl tert-butyl ether mixture, and the content of carotenoids was determined by LC-MS/MS after filtration.

2.2. GenomeWide Identification of HD-Zip Genes

All protein sequences of B. rapa, B. oleracea, and B. napus were downloaded from the Brassicaceae Database (http://www.brassicadb.cn/, accessed on 8 October 2022) for HD-Zip gene identification. Candidate HD-Zip genes were searched against known HD-Zip protein sequences using the BLASTP program. Hidden Markov Model (HMM) profiles of the homeodomain (PF00046) and the leucine zipper domain (PF02183) were downloaded from the PFAM database [25] for search by HMMER3.0 [26]. All candidate sequences were further examined using the NCBI-CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 8 October 2022) to confirm the presence of the HD and LZ domains.

2.3. Multiple Sequence Alignment and Phylogenetic Analysis

All HD-Zip protein sequences in B. rapa, Arabidopsis, B. oleracea, and B. napus were aligned with ClustalW2 [27]. The tree was constructed by using IQTREE software [28] (version 1.6) with the max likelihood (ML) method, and 1000 ultrafast bootstraps were estimated. The model was selected using the ‘MF’ function.

2.4. Gene Structure, Motif, and Cis-Regulatory Elements Analysis

Gene structural information for HD-Zip genes was extracted from whole genome data and displayed using Tbtools software [29]. The MEME website (http://meme-suite.org/tools/meme, accessed on 10 October 2022) was used to identify the motif sequence. The upstream 2000 bp regions of the HD-Zip genes were extracted, and Plant CARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 9 October 2022) was used to identify the cis-regulatory elements of the promoter region of the HD-Zip genes.

2.5. Synteny Analysis of HD-Zip Genes

For analyzing HD-Zip gene duplication events, BLASTP was used to make an all-vs-all BLAST search (top five matches and e-value of 1 × 10−5) with all protein sequences as input data. The BLAST output and the whole genome annotation file were imported to MCScanX [30] for homologous pairs and syntenic regions identification. Via duplicate gene classifier [30], HD-Zip genes were classified into five duplication types [30].

2.6. Calculating the Ka, Ks, and 4DTv of HD-Zip Paralogs

The Ka, Ks, and Ka/Ks ratios of HD-Zip paralog gene pairs were calculated using ParaAT (v2.0) [31] and Kaks_calculator (v2.0) [32]. The ParaAT2.0 software was used to compare the coding and nucleotide sequences of the HD-Zip genes in Brassicaceae, and Ka, Ks, and Ka/Ks values were calculated via KaKs_calculator2.0 software [32] with the model set to the MYN model [33]. Additionally, 4DTv (4-fold synonymous third-codon transversion) was used to estimate the genetic distances of synteny gene pairs. We calculated 4DTv values of HD-Zip paralog pairs using an in-house Python script.

2.7. Expression Pattern Analysis of HD-Zip Genes

Transcriptome data of the highly inbred line ‘268’ with heat stress treatment were obtained from Yue et al. [34]. The transcriptome data of ‘QZ’ and ‘54’ were sequenced on the Illumina NovSeq6000 platform with 150 bp pair-end sequences (Illumina, California, USA). The raw data were trimmed using Trimmomatic (v0.39) [35]. Then, the high-quality reads were mapped to the reference genome (Chiifu_V3.0) [36] using HISAT2 (v2.2.1) [37]. StringTie (v2.1.7) [38] was used to quantify the read count and calculate the Fragments Per Kilobase of the exon model per million mapped fragments (FPKM). The expression heat map of the HD-Zip family was analyzed with TBtool software.
Extraction of total RNA and cDNA was performed using an RNA simple total RNA Kit (Tiangen, Beijing, China) and the Prime Script RT Reagent Kit (TaKaRa, Dalian, China). The expression analysis of HD-Zip genes was detected by quantitative real-time PCR analysis (qRT-PCR) using the SYBR GREEN method with BrActin1 as the internal reference gene [34]. The primers were designed by Primer Premier 6.0 (Supplemental Table S1). The formulation of the qRT-PCR reaction system (20 μL) included 10 μL SYBR Premix Ex Taq, 1 μL cDNA, 1 μL forward/reverse primers, and 7 μL ddH2O. Two-step qRT-PCR amplification conditions were set: 95 °C for 5 min, 55 cycles at 95 °C for 3 s, 60 °C for 10 s, and 72 °C for 30 s, followed by 72 °C for 3 min. Three replicates were set in each reaction. The relative transcription levels of genes were calculated with 2−ΔΔCt methods.

3. Results

3.1. Whole-Genome Identification of HD-Zip Genes in Brassicaceae Plants

HD-Zip genes in the B. rapa, B. oleracea, and B. napus genomes were identified by BLAST search and hmmsearch functions based on Hmmer 3.0 software. The HD-Zip protein contains a homeodomain (HD) and a leucine zipper (LZ) domain. A total of 93 HD-Zip genes were identified from the whole genome of B. rapa (AA, 2n = 20) (Table 1). Meanwhile, 96 and 184 genes were identified from B. oleracea (CC, 2n = 18) and B. napus (AACC, 2n = 38), respectively. The number of HD-Zip genes in B. napus was 1.98- and 1.92-fold higher than that in B. rapa and B. oleracea, respectively. The relative molecular weights of the HD-Zip family genes of Chinese cabbage ranged from a minimum of 17.93 kD (BraA02g014240) to a maximum of 99.25 kD (BraA09g015080), with theoretical pI of 4.54 to 10.24 (Supplemental Table S2).

3.2. Phylogenetic Analysis of the HD-Zip Genes

The phylogenetic tree was constructed using the full-length HD-Zip proteins of B. rapa and Arabidopsis (Figure 1a). The result showed that a total of 141 HD-Zip proteins from the two species were phylogenetically categorized into four subgroups and further named I, II, III, and IV based on the classification of HD-Zip genes in Arabidopsis (Figure 1b). From the four subfamilies, 39 (I), 18 (II), 10 (III), and 26 (IV) HD-Zip genes were obtained in the B. rapa genome, respectively, and subfamily I has a higher percentage of HD-Zip genes than classes II, III, and IV. Meanwhile, 41 (I), 20 (II), 10 (III), and 25 (IV) HD-Zip genes were identified from the B. oleracea genome (Figure S1), and 74 (I), 38 (II), 19 (III), and 53 (IV) HD-Zip genes identified from the B. napus genome (Figure S2). These results suggested that subfamily I contained the greatest number of HD-Zip genes in the three Brassicaceae species. Many AtHD-Zip genes contained at least two homology HD-Zip genes in the three Brassicaceae species, suggesting that genome-wide duplication has led to HD-Zip family expansion [39].

3.3. Conserved Motif Analysis and Gene Structural Analysis of HD-Zip Genes

Gene structures and conserved motifs of the 93 HD-Zips in B. rapa were predicted, and a total of 20 motifs were discovered using the MEME tool. The genes in the same subfamily possessed the same motif structure, such as class II and III (Figure 2a). It is noteworthy that three motifs (motifs 1, 2, and 3) appeared in all HD-Zip genes. Most subfamily I genes contained only three motifs, except for four genes (BraA02g005880.3C, BraA01g024200.3C, BraA03g006690.3C, and BraA09g015080.3C). Compared with HD-Zip I, motif 19 appeared at the C terminal of the HD-Zip II genes. HD-Zip III and IV genes had many more types of motifs than HD-Zip I and II. All HD-Zip III genes contained fourteen motifs, and fifteen types of motifs appeared in nearly all of HD-Zip IV. In addition, motifs 1, 2, and 3 also appeared in all genes of B. napus (Figure S3a). However, only motif 1 and motif 2 appeared in most HD-Zip genes of B. oleracea, and motif 3 was not in HD-Zip IV (Figure S4a). Consistently, the number of introns and exons of the HD-Zip III and IV genes was much more than in HD-Zip I and II in the three Brassicaceae species (Figure 2b, Figures S3b and S4b). These results suggested that the number of motifs and exons may result in the divergence of four subfamilies.

3.4. Cis-Acting Elements Analysis in the Putative Promoter of HD-Zip Genes

In general, cis-acting elements in the promoter region can influence gene function and response for several environment adaptations [40]. In this study, the upstream 2000 bp sequence of the HD-Zip genes in B. rapa was extracted for cis-acting element analysis, and 18 types of cis-acting elements appeared in three species (Figure 3, Figures S5 and S6). It is obvious that most of the elements in HD-Zips were related to the light-responsive element, anaerobic induction, and MeJA-responsiveness, suggesting that the expression of HD-Zip genes may be regulated by various factors. Previous studies reported that HD-Zip genes could regulate auxin perception or auxin response and further regulate plant development [11,41]. For instance, 45.16% (42/93) of genes contained an auxin-responsive element, suggesting that these genes may be important in the auxin-response pathway and plant growth (Figure 3). In addition, 43.01% (40/93) genes contained “defense and stress responsiveness” elements, 48.39% (45/93) genes contained “low-temperature responsiveness” elements, and 48.39% (45/93) genes contained “MYB binding site involved in drought inducibility” elements, suggesting that HD-Zip genes may respond to various environmental factors.

3.5. Chromosome Location and Gene Family Expansion Analysis of HD-Zip Genes in Brassicaceae Plants

Based on the HD-Zip chromosome location information of Chinese cabbage, 93 genes were unevenly distributed on 10 chromosomes (Figure 4a, Supplemental Table S2). Seven to fourteen HD-Zip genes were distributed on each chromosome, and chromosome A09 contained the most HD-Zip genes. HD-Zip I, II, and IV genes were distributed on all 10 chromosomes, and group 3 subfamily members were not discovered on chromosome 3 or 7. In B. oleracea, seven to fifteen genes were unevenly distributed on each of nine chromosomes (Supplemental Table S3), and chromosome 3 contained the most HD-Zip genes (Figure 4b). In B. napus, 184 genes were discovered on 19 chromosomes (Figure 4c), with a gene number ranging from six to sixteen on each chromosome (Supplemental Table S4).
The results of collinearity analysis within the genome showed that there were 76.34% (71/96) of HD-Zip genes in B. rapa and 75.53% (71/94) in B. oleracea that have paralogs (Figure 5a,b, Supplemental Table S5). In B. napus, 75.53% of genes in the A genome and 81.11% of genes in the C genome have paralogs (Figure 5c). Duplication gene classification analysis showed that 77.42% (72/93) of genes were classified into segmental/whole genome duplication (WGD), and 11.83% (11/93) of genes were dispersed and duplicated (Figure 5d). In B. oleracea and B. napus, more than 75% of genes were classified into segmental/whole genome duplication (WGD). These results suggest that HD-Zip gene family expansion was mainly caused by the WGD in the three Brassica species.

3.6. Estimating Dates and Driving Forces for the Evolution of the HD-Zip Gene Family

Many HD-Zip genes were involved in collinear regions, indicating that many genes underwent duplication events. In order to reveal which duplication event drove the evolution of the HD-Zip gene family, we calculated 4DTv and Ks values to evaluate the date of duplication events and further calculated Ka/Ks ratios to determine which selective pressures drove the evolution of HD-Zip genes in the three Brassica species. We found obvious 4DTv peaks within the genome at 0.10 to 0.13 (Figure 6a), and Ks peaked at 0.27 to 0.35 (Figure 6b), which is consistent with the ancient whole-genome triplication (WGT) event (Ks = 0.34) that occurred approximately 16 million years ago [42]. Two peaks were observed between the A and C genomes in B. napus; one was consistent with the peak of a WGT event, and another ranged from 0.07 to 0.12, consistent with the divergence of the A and C genomes [42]. These results suggested that HD-Zip gene family expansion was mainly driven by WGT events. The results of the Ka/Ks ratios of HD-Zip paralogs showed that most HD-Zip gene pairs were concentrated between 0.1 and 0.3 (Figure 6c), suggesting that HD-Zip genes were mainly under purifying selection in the three species. It was notable that the ratio of five orthologs between the A and C genomes in B. napus was more than one, indicating that these gene pairs were under positive selection.

3.7. Expression Patterns of HD-Zip Genes in Different Chinese Cabbage Varieties

Comparative transcriptome analysis was widely used to identify the potential role of gene function and provide a foundation for further functional analysis [43]. Previous studies found that HD-Zip genes participate in various abiotic stresses, such as heat, drought, and salt stress [44,45,46]. To reveal the HD-Zip genes related to the heat stress response in Chinese cabbage, transcriptome sequencing analysis was performed using a heat-resistant inbred line ‘268’, and the FPKM values were counted to represent the expression level (Figure 7). A total of 59 HD-Zip genes were expressed (FPKM > 1) in at least one sample, and 18 genes had more than a 1.5-fold mean FPKM difference between the three heat treatments (HT-4 day, 8 day, 10 day) and the CK (Control treatment) group. Thirteen of the 18 genes were highly expressed under heat treatment, 11 of which were from subfamily I, indicating that HD-Zip I genes have a positive function under heat stress. BraA07g002160.3C showed a 7.57-fold up-regulation under heat treatment and had the highest expression at 10 days of heat treatment (HT-10). BraA05g001160.3C had higher expression levels at three periods under heat treatments than the CK. In addition, BraA09g022670.3C was up-regulated under heat stress treatment (HT-8 day, 10 day). The expression level of BraA07g022080.3C, belonging to the HD-Zip IV subfamily, was 12.66-fold up-regulated upon heat stress treatment. We further selected eight different expression genes for qRT-PCR analysis in two B. rapa varieties (‘QZ’ and ‘54’). Six genes were up-regulated when ‘QZ’ was under heat stress treatment, and five genes showed up-regulation in ‘54’. BraA05g040600.3C showed significant up-regulation for heat stress treatment (from 2 day to 10 day) in ‘QZ’, but no up-regulation was observed in ‘54’, suggesting that BraA05g040600.3C has different expression patterns between the two varieties. The findings suggest that these genes may be important for the heat tolerance of Chinese cabbage.
A previous study found that the HD-Zip gene can regulate the carotenoid content of fruits and generate different fruit colors. To reveal the potential role of HD-Zip genes in the regulation of leaf carotenoid content, the leaf blades of two Chinese cabbage varieties (yellow leaf: ‘54’; white leaf: ‘Gailiang Qiuza 3’, QZ) were selected (Figure 8a). These have significantly (p value = 0.0026) different carotenoid contents (Figure 8b), and transcriptome data analysis was performed. The results showed that fifteen HD-Zip genes have more than a 2-fold change between ‘QZ’ and ‘54’ (FPKM value > 1 in at least one sample) (Figure 8c). Three genes in ‘54’ showed higher expression levels than in QZ, and 12 genes showed lower expression levels. Interestingly, all three up-regulated genes in ‘54’ belong to the HD-Zip I subfamily. BraA09g015080.3C has a high expression level (FPKM > 20) in ‘54’, but it was not expressed in QZ. BraA02g019420.3C and BraA04g000960.3C showed 5.57-fold and 2.46-fold up-regulation in ’54’, respectively. The qRT-PCR analysis was performed, and a high correlation between the transcriptome and qRT-PCR results suggests the accuracy of the transcriptome. These results suggest three HD-Zip genes may have positive potential in the carotenoid content regulation of Chinese cabbage.
Interestingly, BraA09g011460.3C, belonging to the HD-zip I subfamily, had up-regulated expression after eight days of heat treatment, and it also had high expression after heat treatment in ‘QZ’ and ‘54’. These results suggest BraA09g011460.3C has potential involvement in the heat stress tolerance of B. rapa. In addition, it had lower expression levels in ‘54’ (mean FPKM = 0.57) than in ‘QZ’ (mean FPKM = 3.31) and showed a significant (p-value = 9.82E-4) negative correlation with the total carotenoid content. The phylogenetic and BLAST search results showed that BraA09g011460.3C was orthologous with AT2G18550.1 (ATHB21), which can promote the expression of AtNCED3 [47]. NCED encode 9-CIS-EPOXICAROTENOIDDIOXIGENASE has been proven to lead to carotenoid cleavage [48] and the accumulation of abscisic acid (ABA), which is involved in acquired thermotolerance [49]. Therefore, we speculate that BraA09g011460.3C may improve heat stress tolerance and decrease the carotenoid content of B. rapa.

4. Discussion

4.1. Whole-Genome Identification and Phylogenetic Analysis of HD-Zip Genes in Chinese Cabbage

The HD-Zip genes encoding the HD and LZ domains are important in plant development and abiotic stress tolerance [50]. Thus far, HD-Zip transcription factors have been studied in plants such as Arabidopsis [3], Physic nut [51], peach [52], and cucumber [53]. Chinese cabbage, a member of Brassica, is an important vegetable with rich nutrients and high yield. In this study, we performed relatively rigorous criteria to identify candidate members of the HD-Zip genes in B. rapa and two other Brassica species. According to the criteria, 93 HD-Zip genes were identified in B. rapa, 96 in B. oleracea, and 184 in B. napus, and the expansion of HD-Zip genes in B. napus can be explained by B. napus (AACC, 2n  =  38) originating from the natural hybridization of B. rapa (AA, 2n  =  20) and B. oleracea (CC, 2n  =  18) [54].
Based on the phylogenetic results, a total of 93 HD-Zip genes of B. rapa were classified into four subfamilies (HD-Zip I, II, III, and IV). The percentage of HD-Zip I genes reached 41.9%, 42.7%, and 40.2% in B. rapa, B. oleracea, and B. napus, which was higher than the three other subfamilies. This percentage was slightly higher than Arabidopsis (35.41%), suggesting that more HD-Zip I genes were retained after the WGT event. HD-Zip I genes can participate in various abiotic stress response tolerances, such as salt tolerance in apples [55] and physic nuts [51], thermotolerance in lilies [19], and drought tolerance in rice [56]. Therefore, we speculate that a high percentage of HD-Zip genes may improve the abiotic stress tolerance and environmental adaptation of the three Brassica species.

4.2. The Evolution History of the HD-Zip Gene Family

The expansion of gene families was mainly driven by gene duplication, like and dispersed, and tandem or whole genome duplication [56]. In the evolution process of the Chinese cabbage genome, a WGT event occurred approximately 16 million years ago, which resulted in the explosion of genes in Brassica species. The gene duplication type classification and Ka/Ks value peaks suggest that the HD-Zip gene family was primarily expanded by a WGT event, and many duplication genes were retained. No gene was classified into tandem duplication, which was different from the NBS gene family, wherein 43.3% of genes were formed by tandem duplication, suggesting a different expansion pattern between the HD-Zip and NBS gene families [57]. The ratios of Ka/Ks were mostly less than 1, indicating that HD-Zip genes within this species are mainly subject to purifying selection. Interestingly, five orthologous gene pairs between the A and C genomes in B. napus showed positive selection, which can facilitate the prevalence of advantageous traits for a particular species’ evolution [58,59].

4.3. The Potential Roles of Chinese Cabbage HD-Zip Transcription Factors

Growing evidence suggests that HD-Zip genes are important in a plant’s abiotic stress tolerance. Typically, gene expression differences in different samples can be explored, and functional genes of plants can be mined based on transcriptome sequencing analysis [60,61]. In a previous study, MdHB7 (belonging to the HD-Zip I subfamily) could improve tolerance to salinity in apples [55]. Overexpression of Zmhdz10 in maize could enhance a plant’s tolerance to drought and salt stress and increase susceptivity to ABA [62]. In addition, overexpression of RsHDZ17 could improve heat tolerance in radishes [63]. LlHB16, the HD-Zip I gene in lilies, could regulate the basal heat-response pathway and ABA signal to promote thermotolerance [19]. In Eucalyptus grandis, EgHD-Zip37 also showed a response to temperature changes [45]. The 14 HD-Zip genes in B. rapa exhibited different expression levels during heat stress treatment. Of these, 11 belonged to the HD-Zip I gene family, suggesting their importance in Chinese cabbage thermotolerance. Interestingly, the large number of HD-Zip genes containing stress-response elements in promoter regions also indicates their importance in other stresses.
The yellow inner leaf of Chinese cabbage is an important agronomic trait, which is primarily caused by a high carotenoid content, such as lutein, β-carotene, or lycopene [63]. A lot of carotene synthesis genes were up-regulated in the yellow leafy head cultivar [64]. In watermelon, differences in carotenoid content resulted in differences in the flesh color among watermelon varieties, and a large number of HD-Zip genes showed different expression levels, suggesting their potential role in carotenoid regulation [65]. In addition, different transcript levels of carotenoid biosynthesis genes and HD-Zip genes were identified between citruses, with fruits having color variations [66]. In our study, 11 HD-Zip genes showed different expression patterns between yellow- and white-inner leaf varieties, and all of the three up-regulated genes belonged to the HD-Zip I subfamily. CaATHB-12 belongs to the HD-Zip I subfamily, and its overexpression could increase the carotenoid content under normal conditions in Capsicum annuum [7]. These results suggest that HD-Zip genes may also participate in carotenoid regulation, and the three up-regulated HD-Zip I genes may play positive roles in yellow-inner-leaf Chinese cabbage. A previous study found that the AtHB21 gene could regulate the NCED gene, which could decrease the carotenoid content [46,47,67] and promote ABA accumulation to improve heat stress tolerance [19]. BraA09g011460.3C was homologous to AtHB21 and was highly expressed after heat treatment and lowly expressed in high-carotenoid varieties, suggesting a regulatory effect on heat tolerance and the carotenoid content in B. rapa.

5. Conclusions

In our study, a total of 93, 96, and 184 HD-Zip genes were identified in the B. rapa, B. oleracea, and B. napus genomes, and they were further divided into four subfamilies (I to IV) based on gene structure. The results of the gene structure and evolutionary trajectories predicted that members in the same subclade possessed the same motif structure, and motifs 1, 2, and 3 were commonly present in all HD-Zip family genes. Notably, stress-response-related elements and hormone-response elements were discovered in the promoters of all three of these genes. Collinearity and Ks results showed that HD-Zip gene family expansion was driven by WGT events, and most HD-Zip genes were under purifying selection. Transcriptome analysis identified 14 genes showing different expressions between the CK and heat treatment groups, and 11 of the 14 genes were from the HD-Zip I subfamily. In addition, three up-regulated genes were identified in high-carotenoid-content Chinese cabbages. BraA09g011460.3C showed up-regulation after heat treatment and low expression in high-carotenoid-content varieties, suggesting its regulation in heat stress tolerance and the carotenoid content in B. rapa.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13051324/s1, Figure S1: A phylogenetic tree of HD-Zip proteins of B. oleracea and Arabidopsis. Purple branches indicate HD-Zip subfamily I, yellow branches indicate II, red branches indicate III and cyan branches indicate IV. Red squares indicate genes coming from Arabidopsis and blue squares indicate genes coming from B. napus. Figure S2: A phylogenetic tree of HD-Zip proteins of B. napus and Arabidopsis. Purple branches indicate HD-Zip subfamily I, yellow branches indicate II, red branches indicate III and cyan branches indicate IV. Red squares indicate genes coming from Arabidopsis and blue squares indicate genes coming from B. oleracea. Figure S3: The phylogenetic relationship, conserved motifs and gene structure of HD-Zip proteins in B. oleracea. (a) A phylogenetic tree of HD-Zip proteins was constructed using the Max likelihood (ML) method and 1000 ultrafast bootstraps. (b) The distribution of conserved motifs across B. oleracea HD-Zip proteins. A total of 20 motifs were predicted using MEME tool. (c) The gene structure of HD-Zip genes in B. oleracea, including intron and exon. The black lines indicate intron and green squares indicates coding sequence (CDS). Figure S4: The phylogenetic relationship, conserved motifs and gene structure of HD-Zip proteins in B. napus. (a) A phylogenetic tree of HD-Zip proteins was constructed using the Max likelihood (ML) method and 1000 ultrafast bootstraps. (b) The distribution of conserved motifs across B. napus HD-Zip proteins. A total of 20 motifs were predicted using MEME tool. (c) The gene structure of HD-Zip genes in B. napus, including intron and exon. The black lines indicate intron and yellow squares indicates coding sequence (CDS). Figure S5: The cis-acting elements predication on putative promoters of HD-Zip genes in B. oleracea. The number of cis-acting elements on putative promoters of HD-Zip genes. A total of eighteen cis-acting elements were investigated in our study, including: (1) Auxin-responsive element; (2) Cell cycle regulation; (3) Defense and stress responsiveness; (4) Gibberellin-responsive element; (5) Light responsive element; (6) Low-temperature responsiveness; (7) Phytochrome down-regulation expression; (8) Salicylic acid responsiveness; (9) Abscisic acid responsiveness; (10) Anaerobic induction; (11) Circadian control; (12) MeJA-responsiveness; (13) Zein metabolism regulation; (14) Meristem expression; (15) Cis-regulatory element involved in endosperm expression; (16) MYB binding site involved in drought-inducibility; (17) MYB binding site involved in flavonoid biosynthetic genes regulation; (18) MYB binding site involved in light responsiveness. Figure S6: The cis-acting elements predication on putative promoters of HD-Zip genes in B. napus. The number of cis-acting elements on putative promoters of HD-Zip genes. A total of eighteen cis-acting elements were investigated in our study, including: (1) Auxin-responsive element; (2) Cell cycle regulation; (3) Defense and stress responsiveness; (4) Gibberellin-responsive element; (5) Light responsive element; (6) Low-temperature responsiveness; (7) Phytochrome down-regulation expression; (8) Salicylic acid responsiveness; (9) Abscisic acid responsiveness; (10) Anaerobic induction; (11) Circadian control; (12) MeJA-responsiveness; (13) Zein metabolism regulation; (14) Meristem expression; (15) Cis-regulatory element involved in endosperm expression; (16) MYB binding site involved in drought-inducibility; (17) MYB binding site involved in flavonoid biosynthetic genes regulation; (18) MYB binding site involved in light responsiveness. Supplemental Table S1: Primers of HD-Zip genes in B. rapa. Supplemental Table S2: The information of HD-Zip genes in B. rapa. Supplemental Table S3: The information of HD-Zip genes in B. oleracea. Supplemental Table S4: The information of HD-Zip genes in B. napus. Supplemental Table S5: The duplication information of HD-Zip genes in three species.

Author Contributions

J.Z. and L.Y. initiated and designed the research; L.Y., L.W., W.X. and L.L. performed the experiments; L.Y., Y.S., X.C., D.L. and M.S. analyzed the data; L.Y. and J.Z. wrote the paper; J.Z. contributed to and edited the paper; J.Z., Y.S., X.C., J.L. and K.F. revised the paper; J.L. and K.F. polished the language. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Independent Innovation Fund Project of Agricultural Science and Technology in Jiangsu Province (CX(21)2020), the Seed Industry Revitalization Project of Jiangsu Province (JBGS(2021)073), and the Research and Development Fund of Huai’an Academy of Agricultural Sciences (HNY202131).

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 13 01324 g001
Figure 1. Phylogenetic tree and the classification of B. rapa, B. oleracea, and B. napus. (a) A phylogenetic tree of the HD-Zip proteins of B. rapa, B. oleracea, B. napus, and Arabidopsis. The tree was constructed by using IQTREE software (version 1.6) with the max likelihood (ML) method and 1000 ultrafast bootstraps. Purple represents the HD-Zip subfamily I, yellow represents II, red represents III, and cyan represents IV. (b) A phylogenetic tree of the HD-Zip proteins of B. rapa and Arabidopsis. Purple branches indicate the HD-Zip subfamily I, yellow branches indicate II, red branches indicate III, and cyan branches indicate IV. Red rectangles indicate genes coming from Arabidopsis, and blue squares indicate genes coming from B. rapa.
Figure 1. Phylogenetic tree and the classification of B. rapa, B. oleracea, and B. napus. (a) A phylogenetic tree of the HD-Zip proteins of B. rapa, B. oleracea, B. napus, and Arabidopsis. The tree was constructed by using IQTREE software (version 1.6) with the max likelihood (ML) method and 1000 ultrafast bootstraps. Purple represents the HD-Zip subfamily I, yellow represents II, red represents III, and cyan represents IV. (b) A phylogenetic tree of the HD-Zip proteins of B. rapa and Arabidopsis. Purple branches indicate the HD-Zip subfamily I, yellow branches indicate II, red branches indicate III, and cyan branches indicate IV. Red rectangles indicate genes coming from Arabidopsis, and blue squares indicate genes coming from B. rapa.
Agronomy 13 01324 g001
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Figure 2. The conserved motifs and gene structure of HD-Zip proteins in B. rapa. (a) A phylogenetic tree of HD-Zip proteins was constructed using the Max likelihood (ML) method. The distribution of conserved motifs across B. rapa HD-Zip proteins. (b) The gene structure of HD-Zip genes in B. rapa, including intron and exon genes. The black lines indicate introns, and the green squares indicate a coding sequence (CDS).
Figure 2. The conserved motifs and gene structure of HD-Zip proteins in B. rapa. (a) A phylogenetic tree of HD-Zip proteins was constructed using the Max likelihood (ML) method. The distribution of conserved motifs across B. rapa HD-Zip proteins. (b) The gene structure of HD-Zip genes in B. rapa, including intron and exon genes. The black lines indicate introns, and the green squares indicate a coding sequence (CDS).
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Figure 3. The cis-acting elements prediction on putative promoters of HD-Zip genes. The number of cis-acting elements on putative promoters of HD-Zip genes. A total of eighteen cis-acting elements were investigated in our study.
Figure 3. The cis-acting elements prediction on putative promoters of HD-Zip genes. The number of cis-acting elements on putative promoters of HD-Zip genes. A total of eighteen cis-acting elements were investigated in our study.
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Figure 4. Chromosome location of HD-Zip genes in B. rapa (a), B. oleracea (b), and B. napus (c). Different colors represent different types of HD-Zip genes. Red (subfamily III), cyan (subfamily IV), yellow (subfamily II), and purple (subfamily I). The x-axis represents the chromosome number, and the y-axis represents chromosome length.
Figure 4. Chromosome location of HD-Zip genes in B. rapa (a), B. oleracea (b), and B. napus (c). Different colors represent different types of HD-Zip genes. Red (subfamily III), cyan (subfamily IV), yellow (subfamily II), and purple (subfamily I). The x-axis represents the chromosome number, and the y-axis represents chromosome length.
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Figure 5. Synteny analysis of the HD-Zip genes in B. rapa (a), B. napus (b), and B. oleracea (c). The letter ‘a’ represents the chromosome of the ‘A’ genome in B. rapa and B. napus, and the letter ‘c’ represents the chromosome of the ‘C’ genome in B. oleracea and B. napus. Blue lines mean collinearity relationships among genes within a genome (within A genome or within C genome), and orange lines mean collinearity relationships among genes between A and C genomes. (d) Classification of duplication gene pairs in three Brassica species. The details of the classification are displayed in the method section.
Figure 5. Synteny analysis of the HD-Zip genes in B. rapa (a), B. napus (b), and B. oleracea (c). The letter ‘a’ represents the chromosome of the ‘A’ genome in B. rapa and B. napus, and the letter ‘c’ represents the chromosome of the ‘C’ genome in B. oleracea and B. napus. Blue lines mean collinearity relationships among genes within a genome (within A genome or within C genome), and orange lines mean collinearity relationships among genes between A and C genomes. (d) Classification of duplication gene pairs in three Brassica species. The details of the classification are displayed in the method section.
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Figure 6. The evolution analysis of the HD-Zip gene family. The distribution of 4DTv (a) and Ks (b) values of homologous gene pairs in three species. (c) The distribution Ka/Ks values of homology gene pairs. ‘A’ means homology gene pairs in the A genome, ‘C’ means homology gene pairs in the C genome, and ‘AC’ means homology gene pairs between the A and C genomes.
Figure 6. The evolution analysis of the HD-Zip gene family. The distribution of 4DTv (a) and Ks (b) values of homologous gene pairs in three species. (c) The distribution Ka/Ks values of homology gene pairs. ‘A’ means homology gene pairs in the A genome, ‘C’ means homology gene pairs in the C genome, and ‘AC’ means homology gene pairs between the A and C genomes.
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Figure 7. Expression pattern of HD-zip genes in B. rapa (‘268’, a heat-resistant inbred line) under heat stress treatment. (a) The heat maps were plotted by the R/Pheatmap package. A red color indicates high expression, blue indicates low expression, and gray indicates no data. ‘CK’ means normal treatment, and ‘HT’ means heat treatment (40 °C). The FPKM value was normalized by using the z-score method. (b) qRT-PCR analysis of the eight different expression genes. Two B. rapa varieties (‘QZ’ and ‘54’) under heat stress treatment (0 d, 2 d, 4 d, 6 d, 8 d, and 10 d) were used for qRT-PCR analysis. SPSS software was used to analyze the difference in expression level at the 0.05 level. The yellow inner-leaf variety is represented by ‘54’, and ‘QZ’ represents ‘Gailiang Qiuza 3’ (white inner-leaf variety). Different lower case letters indicate significant difference at 0.05 level.
Figure 7. Expression pattern of HD-zip genes in B. rapa (‘268’, a heat-resistant inbred line) under heat stress treatment. (a) The heat maps were plotted by the R/Pheatmap package. A red color indicates high expression, blue indicates low expression, and gray indicates no data. ‘CK’ means normal treatment, and ‘HT’ means heat treatment (40 °C). The FPKM value was normalized by using the z-score method. (b) qRT-PCR analysis of the eight different expression genes. Two B. rapa varieties (‘QZ’ and ‘54’) under heat stress treatment (0 d, 2 d, 4 d, 6 d, 8 d, and 10 d) were used for qRT-PCR analysis. SPSS software was used to analyze the difference in expression level at the 0.05 level. The yellow inner-leaf variety is represented by ‘54’, and ‘QZ’ represents ‘Gailiang Qiuza 3’ (white inner-leaf variety). Different lower case letters indicate significant difference at 0.05 level.
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Figure 8. Leaf color (a) and carotenoid content (b) in two Chinese cabbage varieties. The yellow inner-leaf variety is represented by ‘54’, and ‘QZ’ represents ‘Gailiang Qiuza 3’ (white inner-leaf variety). The three replicated are represented by ‘1, 2, and 3’. (c) Expression profiles of 15 genes in two Chinese cabbages by qRT-PCR analysis and RNA-seq data. Lines represent the value of FPKM, and bars represent the value of the relative expression level.
Figure 8. Leaf color (a) and carotenoid content (b) in two Chinese cabbage varieties. The yellow inner-leaf variety is represented by ‘54’, and ‘QZ’ represents ‘Gailiang Qiuza 3’ (white inner-leaf variety). The three replicated are represented by ‘1, 2, and 3’. (c) Expression profiles of 15 genes in two Chinese cabbages by qRT-PCR analysis and RNA-seq data. Lines represent the value of FPKM, and bars represent the value of the relative expression level.
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Table 1. Summary of the HD-zip gene family in three Brassica species.
Table 1. Summary of the HD-zip gene family in three Brassica species.
SpeciesGenome Size Chromosome Number (2n)Whole Gene NumberHD-Zip Gene Number
IIIIIIIV
Brassica rapa351.062046,25039181026
Brassica oleracea561.161859,06441201025
Brassica napus92438108,19074381953
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Yin, L.; Sun, Y.; Chen, X.; Liu, J.; Feng, K.; Luo, D.; Sun, M.; Wang, L.; Xu, W.; Liu, L.; et al. Genome-Wide Analysis of the HD-Zip Gene Family in Chinese Cabbage (Brassica rapa subsp. pekinensis) and the Expression Pattern at High Temperatures and in Carotenoids Regulation. Agronomy 2023, 13, 1324. https://doi.org/10.3390/agronomy13051324

AMA Style

Yin L, Sun Y, Chen X, Liu J, Feng K, Luo D, Sun M, Wang L, Xu W, Liu L, et al. Genome-Wide Analysis of the HD-Zip Gene Family in Chinese Cabbage (Brassica rapa subsp. pekinensis) and the Expression Pattern at High Temperatures and in Carotenoids Regulation. Agronomy. 2023; 13(5):1324. https://doi.org/10.3390/agronomy13051324

Chicago/Turabian Style

Yin, Lian, Yudong Sun, Xuehao Chen, Jiexia Liu, Kai Feng, Dexu Luo, Manyi Sun, Linchuang Wang, Wenzhao Xu, Lu Liu, and et al. 2023. "Genome-Wide Analysis of the HD-Zip Gene Family in Chinese Cabbage (Brassica rapa subsp. pekinensis) and the Expression Pattern at High Temperatures and in Carotenoids Regulation" Agronomy 13, no. 5: 1324. https://doi.org/10.3390/agronomy13051324

APA Style

Yin, L., Sun, Y., Chen, X., Liu, J., Feng, K., Luo, D., Sun, M., Wang, L., Xu, W., Liu, L., & Zhao, J. (2023). Genome-Wide Analysis of the HD-Zip Gene Family in Chinese Cabbage (Brassica rapa subsp. pekinensis) and the Expression Pattern at High Temperatures and in Carotenoids Regulation. Agronomy, 13(5), 1324. https://doi.org/10.3390/agronomy13051324

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