Next Article in Journal
Whole-Chromosome Karyotyping of Fetal Nucleated Red Blood Cells Using the Ion Proton Sequencing Platform
Previous Article in Journal
Novel Insights into MEG3/miR664a-3p/ADH4 Axis and Its Possible Role in Hepatocellular Carcinoma from an in Silico Perspective
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 13
Issue 12
10.3390/genes13122256
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification and Expression Profile of the HD-Zip Transcription Factor Family Associated with Seed Germination and Abiotic Stress Response in Miscanthus sinensis

by
Jiayi Chen
1,†,
Qifan Ran
2,†,
Zhongfu Yang
1,
Ying Zhou
1,
Zhihang Yuan
1,
Huiqi Lai
1,
Jing Wang
1,
Gang Nie
1,* and
Yongqun Zhu
3,*
1
Department of Forage Breeding and Cultivation, College of Grassland Science and Technology, Sichuan Agricultural University, Chengdu 611130, China
2
Institute of Grassland Sciences, Chongqing Academy of Animal Science, Chongqing 402460, China
3
Institute of Agricultural Resources and Environment, Sichuan Academy of Agricultural Sciences, Chengdu 610066, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Genes 2022, 13(12), 2256; https://doi.org/10.3390/genes13122256
Submission received: 10 November 2022 / Revised: 21 November 2022 / Accepted: 27 November 2022 / Published: 30 November 2022
(This article belongs to the Section Plant Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
Miscanthus sinensis is an ornamental grass, non-food bioenergy crop, and forage with high feeding value. It can adapt to many kinds of soil conditions due to its high level of resistance to various abiotic stresses. However, a low level of seed germination restricts the utilization and application of M. sinensis. It is reported that the Homeodomain-leucine zipper (HD-Zip) gene family participates in plant growth and development and ability to cope with outside environment stresses, which may potentially regulate seed germination and stress resistance in M. sinensis. In this study, a complete overview of M. sinensis HD-Zip genes was conducted, including gene structure, conserved motifs, chromosomal distribution, and gene duplication patterns. A total of 169 members were identified, and the HD-Zip proteins were divided into four subgroups. Inter-chromosomal evolutionary analysis revealed that four pairs of tandem duplicate genes and 72 segmental duplications were detected, suggesting the possible role of gene replication events in the amplification of the M. sinensis HD-Zip gene family. There was an uneven distribution of HD-Zip genes on 19 chromosomes of M. sinensis. Also, evolutionary analysis showed that M. sinensis HD-Zip gene family members had more collinearity with monocotyledons and less with dicotyledons. The gene structure analysis showed that there were 93.5% of proteins with motif 1 and motif 4, while motif 10 was only found in group IV. Based on the cis-elements analysis, it appeared that most of the genes were related to plant growth and development, various hormones, and abiotic stress. Furthermore, qRT-PCR analysis showed that Misin06G303300.1 was significantly expressed in seed germination and Misin05G030000.1 and Misin06G303300.1 were highly expressed under chromium, salt, and drought stress. Results in this study will provide a basis for further exploring the potential role of HD-Zip genes in stress responses and genetic improvement of M. sinensis seed germination.

1. Introduction

As a perennial rhizomatous grass of the C4 family, Miscanthus sinensis is cultivated as a forage, ornamental, and energy crop due to its high biomass, photosynthesis potential, and water use efficiency [1,2,3,4]. It is reported that M. sinensis can adapt to multiple kinds of soil conditions due to its strong resistance to various abiotic stresses, such as heavy metal, salt, and drought stress [5,6,7]. However, it is widely believed that seed dormancy hindered free germination, which widely existed in M. sinensis [8,9,10], and seed germination characteristics contribute to the establishment of M. sinensis [11,12]. The seeds of M. sinensis are small, heterogeneous [13,14], and under natural conditions, the germination rate of seeds is only about 65% [12]. Therefore, it is imperative to explore the genes related to M. sinensis seed germination for further genetic improvement. There is increasing evidence indicating that Homeodomain-leucine zipper (HD-Zip) proteins have played a critical role in plant growth and development [15,16]. The HD-Zip gene family is a transcription factor family only found in plants, and it comprises two conserved functional domains: a homologous domain (HD) that specifically binds to DNA, and a leucine zipper (LZ) motif mediating the formation of protein dimers [17]. Additionally, HD-Zip proteins can be divided into four subfamilies: HD-Zip I (two domains, including a highly conserved HD and a less conserved LZ), HD-Zip II (HD domains, LZ domains, CPSCEs, and N-terminal consensus sequences exhibit high conservation), HD-Zip III (all members share the conserved domain “MEKHLA”, conservative amino acids account for more than half, and a START domain and SAD domain are contained), and HD-Zip IV (in comparison to HD-Zip III, the LZ domain has a loop in the middle and MEKHLA has disappeared) [18].
In recent years, plants from a wide range of species have been analyzed and identified to the function with HD-Zip genes. In Arabidopsis thaliana, ATHB2 acted as a light-induced factor, which negatively regulated and maintained the expression of transcription factors to prevent seed germination, as a gene of HD-Zip class I [19]. Furthermore, several HD-Zip proteins are involved in regulating the expression of NCED9, which can mediate the synthesis of ABA and gibberellin-dependent seed germination [20]. With the development of cotton roots, HD-Zip class I gene GhHB1 transcripts accumulated at the early stage and decreased dramatically as they developed [21]. As an Arabidopsis thaliana HD-Zip subgroup I gene, ATHB23 was controlled by GA and other activators such as PHABULOSA and participated in the establishment of polarity during leaf development [22]. As an HD-Zip IV gene in transgenic maize, the ectopic expression of OCL1 led to polyphenotypic aberrations, among which the most significant phenotypic aberrations were significantly delayed flowering [23]. VvHB58, a gene of the transgenic Grape HD-Zip I subfamily, was involved in regulating fruit size and seed yield [24]. ZeHB10, ZeHB11, and ZeHB12, three genes belonging to the HD-Zip III family in Zinnia, were reported to participate in the synthesis of lignin and the formation of lateral tissues [25].
Furthermore, HD-Zip genes also participated in regulating stress-responsive mechanisms under abiotic conditions. In rice, Oshox22 positively regulated salt and drought tolerance via ABA-dependent signal pathways as an HD-Zip I gene [26]. In Arabidopsis thaliana, ATHB17 knockout lines showed ABA-insensitivity and drought-sensitivity, indicating that ATHB17 responded to ABA and water-stress as an HD-Zip IV gene [27]. Moreover, TDF #170-1 and 170-1k, two proteins of the HD-Zip I subfamily in Citrus sinensis root, were significantly upregulated under manganese (Mn) toxicity, showing that they participated in Mn stress tolerance [28]. SlHZ24 was a gene belonging to the tomato HD-Zip class I, and it was reported that the overexpression of SlHZ24 could promote acetylsalicylic acid biosynthesis and resulted in enhanced oxidative stress tolerance [29]. Furthermore, Transgenic Arabidopsis improved osmotic tolerance when supplemented with GaHDG11, a gene of cotton HD-Zip class IV [30]. The silence of OsHB4, an Oryza sativa HD-Zip I transcription factor, was reported to reduce Cd accumulation in leaves and grains [31].
There is no research conducted on the HD-Zip gene family in M. sinensis despite the fact that HD-Zip genes have been proven to play a significant role in plant growth and development and abiotic stress. In this study, genome-wide identification, duplication, gene structure, and phylogenetic characteristics of M. sinensis HD-Zip genes were comprehensively analyzed based on the genome sequence of M. sinensis. In addition, the expression level of HD-Zip genes in seed germination and their responses to various abiotic stresses were also investigated. The results will be helpful to investigate the molecular mechanisms and the functional characteristics of M. sinensis HD-Zip genes associated with seed germination and abiotic stress.

2. Materials and Methods

2.1. Identification of HD-Zip Genes in M. Sinensis

The M. sinensis genome resource was downloaded from the PHYTOZOME database (https://phytozome-next.jgi.doe.gov/info/Msinensis_v7_1, accessed on 1 June 2022) [32]. To identify the M. sinensis HD-Zip members, the leucine zipper (LZ) domain (PF02183) and the Hidden Markov Model (HMM) file of the conserved HD domain of homeobox (PF00046) were obtained from the Pfam database (http://pfam.sanger.ac.uk/, accessed on 1 June 2022). Firstly, HMMER 3.0 was used to conduct a local BLSATP search against the proteome sequences of M. sinensis using the HMM profile as a query. An e-value cut-off of 1×E at minus 5 was used for the query. After removing all redundant sequences, all candidate protein sequences were confirmed by SMART (http://smart.emblheidelberg.de/smart/save_user_preferences.pl, accessed on 2 June 2022) tools and NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 2 June 2022) for further verification.
Further, the CD length, molecular weight (MW), and theoretical isoelectric points (pI) of HD-Zip proteins were analyzed by ProtParam (https://web.expasy.org/protparam/, accessed on 2 June 2022), and the subcellular localization of the HD-Zip proteins was predicted by the online ProtComp 9.0 (http://linux1.softberry.com/berry.phtml, accessed on 3 June 2022) [33]. In order to predict the membrane-bound HD-Zip proteins, the TMHMM server v.2.0 (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 3 June 2022) was used [34]. An analysis of the cis-acting regulatory elements was performed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 3 June 2022) [35].

2.2. Phylogenetic Analysis and Classification of HD-Zip Genes

The HD-Zip protein sequences of Arabidopsis thaliana were obtained from the TAIR 11 [36] (https://www.arabidopsis.org/, accessed on 4 June 2022). Amino acid sequences of M. sinensis HD-Zip proteins at full-length were used for multiple alignments conducted by muscle and automatedly aligned in Trimal. The phylogenetic tree was constructed by IQ-TREE [37]. Model_finder was used to find the best tree composition model for VT + F + R7 [38], and ultrafast bootstrap was performed using 1000 replicates with the partial deletion model for gaps/missing data [39]. Phylogenetic analysis of the transcription factors of M. sinensis and A. thaliana shows that the HD-Zip genes in M. sinensis can be further subdivided into different subfamilies.

2.3. Gene Structure and Motif Composition of HD-Zip Genes

Through the online Gene Structure Display Server 2.0 tools, the exon-intron structures of HD-Zip genes were displayed graphically to gain insight into their structures. In order to identify the conserved motif in HD-Zip proteins of M. sinensis, the program Multiple Em for Motif Elicitation (MEME) was used with default settings [40].

2.4. Chromosomal Distribution, Gene Duplication, and Synteny Analysis of HD-Zip Genes

The chromosomal distribution information of the HD-Zip genes was obtained from the M. sinensis genome annotation. Additionally, MG2C was used to draw the chromosomal positions map of the HD-Zip genes and specific nomenclatures were assigned based on the order of chromosomes. An analysis of segmental and tandem duplications was carried out by a Multiple collinear scanning toolkits (MCScanX) with default parameters [41]. TBtools platform was used to create the corresponding plot using Dual Synteny Plot for MCscanx [42]. To discuss the synteny relationship of orthologous HD-Zip genes between M. sinensis and ten other plants (Arabidopsis thaliana, Medicago truncatula, Glycine max, Trifolium repens, Sorghum bicolor, Bracypodium distachyon, Panicum hallii, Saccharun spontaneum, Zea mays, and Setaria italica), the synteny analysis maps were generated with TBtools Dual Synteny Plotter (https://github.com/CJ-Chen/TBtools, accessed on 4 June 2022).

2.5. Plant Growth and Treatments

To detect the expression of M. sinensis HD-Zip genes during seed germination, mature seeds of M. sinensis ‘M20100819’ were placed into Petri dishes and cultured in incubators (day/night temperature of 30/20 °C, light and dark cycle of 16/8 h) [8,10]. Distilled water was poured every 24 h. Whole plant samples were taken at 0 h, 2 h, 1 d, 3 d, and 5 d respectively, three biological replicates were taken at each time point.
Furthermore, the expression levels of HD-Zip genes in M. sinensis root under stress conditions were determined. The seedlings were cultured in plastic pots with a matrix of quartz sand and watered with Hoagland’s nutrient solution every day. The growth conditions of plant materials were 28/25 °C, with a light and dark cycle of 12/12 h, respectively, in the growth chamber [43]. Three different stress treatments were carried out for six days when plants had 5–6 leaves, including salt stress (300 mmol/L NaCl), drought stress (20% PEG), and chromium stress (200 mg/L K2Cr2O7). Three biological repetitions for each treatment were used to collect root samples on days 0, 1, 3, and 6. As soon as the samples were collected, they were frozen in liquid nitrogen and then stored at −80 °C.

2.6. RNA Extraction, cDNA Synthesis, and qRT-PCR Gene Expression Analysis

In order to perform qRT-PCR analysis, total RNA was extracted with the help of an RNA extraction kit (Zymo Research, Beijing, China). Elimination of genomic DNA contamination was performed by DNase I (Zymo Research, Beijing, China). The concentration of RNA was determined with a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and the integrity of RNA was monitored with agarose gel electrophoresis. With the 5X All-In-One RT MasterMix (Abm, Richmond, VA, USA), the first-strand cDNA was synthesized according to the manufacturer’s instructions.
The qRT-PCR primers (Table S1) were designed by Primer 3.0 software. The expression analysis of the selected genes was conducted by qRT-PCR with SYBR Premix ExTaq II (TaKaRa). Unigene33024 was used as the reference gene in this qRT–PCR experiments [43]. The data were calculated by the 2−(∆∆Ct) method [44].

3. Results

3.1. Identification of HD-Zip Genes in M. sinensis

A BLASTP search was conducted against the M. sinensis genome database with the HMM profile being used as a query to retrieve consensus protein sequences, and a total of 169 M. sinensis HD-Zip genes were identified. As can be found from Table S2, among these 169 HD-Zip proteins, the smallest protein was Misin05G360200.1, which contains 96 amino acids, and the largest was Misin17G240000.1 (which contains 1856 amino acids). Also, HD-Zip genes ranged from 288 bp (Misin05G360200.1) to 5568 bp (Misin17G240000.1) in length. The pI ranged from 4.38 (Misin03G157300.1) to 10.74 (Misin15G189700.1), with an average of 6.95. In addition, subcellular localization predicted that most of the M. sinensis HD-Zip proteins (~99.41%) were in the nucleus, and all proteins except protein Misin16G051700.1 were transmembrane proteins. Based on the cis-elements analysis of the M. sinensis HD-Zip genes, it appeared that most of the genes were associated with plant growth and development, various hormones, and abiotic stress (Table S3).

3.2. Phylogenetic Analysis and Classification of HD-Zip Genes

To explore the classification of HD-Zip protein in M. sinensis, the NJ rootless phylogenetic tree was constructed using HD-Zip protein sequences from 169 M. sinensis members and 58 A. thaliana members. The results showed that the M. sinensis HD-Zip gene family was divided into four classes: HD-Zip I–IV. Among the 169 HD-Zip proteins, 27 members were identified from the HD-Zip I subgroup, 32 members were from the HD-Zip II subgroup, 35 members belonged to the HD-Zip III subfamily, and 75 members belonged to the HD-Zip IV subfamily (Figure 1). The HD-Zip I–III subgroups contained a similar number of M. sinensis HD-Zip proteins, whereas the HD-Zip IV subgroup contained the largest amount of M. sinensis HD-Zip proteins.

3.3. Gene Structure and Motif Composition of the HD-Zip Gene Family

In order to further reveal the structural similarity and diversification of M. sinensis HD-Zip proteins, the intron and exon structure of M. sinensis HD-Zip genes was determined based on ten conserved motifs with the help of a MEME tool (Figure 2). Various lengths of conserved motifs were found, ranging from 19 to 42 amino acids, which showed a highly diverse distribution. Among 169 genes, there were 27 genes with only one intron, whereas the highest number of introns (a total of eighteen) were present in Misin17G240000.1, Misin02G197400.1, Misin02G509800.1, and Misin01G516200.1. In addition, the number of exons was a minimum of two and a maximum of eight. Of the proteins, 93.5% had motif 1 and motif 4. Notably, motif 10 was only found in group Ⅳ, which showed that this group may be related to specific functions. The details of the sequence logos of each motif can be found in Figure S1.

3.4. Chromosomal Distribution and Gene Duplication of the HD-Zip Genes

A total of 169 M. sinensis HD-Zip genes showed an unequal distribution on 19 M. sinensis chromosomes (Figure 3). The largest number of M. sinensis HD-Zip genes was detected on chromosome 2 (24, ~14.20%), while chromosome 10 contained the least (1, ~0.01%).
A genome duplication event analysis was carried out for M. sinensis HD-Zip genes as well. Tandem and segmental gene duplication could produce a great quantity of gene families. In this study, four pairs of M. sinensis HD-Zip genes linked by red lines were examined as tandem repeats (Figure 4), including a pair of tandem repeats on chromosomes 4, 5, 18, and 19, respectively. Also, a total of 72 segmental duplication gene pairs linked by blue lines were identified in the M. sinensis HD-Zip gene family (Table S4). According to these findings, some HD-Zip genes may have evolved through replication events, which were the main driving forces for the evolution of the HD-Zip gene family.

3.5. Evolutionary Analysis of HD-Zip Genes in Several Different Species

To deduce the evolutionary relationship of HD-Zip genes between M. sinensis and related species, comparative syntenic maps were conducted for four dicotyledonous plants (Arabidopsis thaliana, Medicago truncatula, Trifolium repens, and Glycine max) and six species of monocotyledons (Bracypodium distachyon, Saccharun spontaneum, Panicum hallii, Sorghum bicolor, Zea mays, and Setaria italica). M. sinensis and these ten species showed 28, 43, 4, 95, 187, 242, 195, 202, 268, and 191 homologous pairs, respectively (Table S5). As can be found in Figure 5, there was a large amount of collinearity between HD-Zip gene family members of M. sinensis and other monocotyledons, which showed that there were differences in the evolutionary branches of dicotyledons and monocotyledons in the process of systematic evolution.

3.6. Expression Analysis of HD-Zip Genes during Different Seed Germination Stages of M. sinensis

A qRT-PCR analysis of HD-Zip members was performed to determine their expression during M. sinensis seed germination. The expression patterns of 12 randomly selected HD-Zip genes during M. sinensis seed germination were analyzed (Figure 6). The expression patterns varied in different genes during seed germination, while some genes had similar expression patterns, suggesting that these genes have similar functions. The relative expression levels of Misin02G460500.1 and Misin01G096800.1 showed a downward trend, while those of Misin04G257500.1 and Misin05G030000.1 were the opposite. Genes whose relative expression level increased first and then decreased were Misin17G242100.1, Misin01G234600.1, Misin13G046000.1, Misin17G242100.1, Misin01G234600.1, Misin13G046000.1, Misin19G174900.1, Misin06G303300.1, Misin01G516200.1, and Misin02G271800.1.
Additionally, the expression level of Misin06G303300.1 was greatly higher than the other 11 genes in the process of seed germination, indicating that Misin06G303300.1 played a crucial role in seed germination, but the specific function of this gene remains to be studied (Figure S2).

3.7. Expression Patterns of HD-Zip Genes in Response to Abiotic Stress

In order to explore the roles of HD-Zip genes involved in responding to osmotic stress, the expression profiles of HD-Zip genes in M. sinensis root under three stresses were examined. According to the results, the expression levels of these 12 genes under the three stresses were significantly different. The data were presented with clusters using log2 fold-change values (Figure 7). It can be found that the majority of the selected genes were highly expressed in drought stress, and few genes were induced or inhibited by chromium stress and salt stress. Under drought stress, Misin05G030000.1 and Misin19G174900.1 were significantly upregulated in M. sinensis root. Furthermore, Misin19G174900.1 was significantly expressed under salt stress and Misin02G460500.1 was significantly upregulated under chromium stress (log2 based fold change ≥ 6).

4. Discussion

As a promising crop for non-food bioenergy, M. sinensis is widely planted all over the world and can withstand various abiotic stresses. The HD-Zip gene family is one of the most important transcription factors of higher plants, which plays a crucial role in the growth and development of plants, as well as stress responses [18,45]. A recent publication of chromosome scale assembly of the M. sinensis genome provided a precious genetic resource for the improvement of Miscanthus breeding and the mining of a stress-resistant molecular regulatory network.
Previous studies showed that there is no significant correlation between genome size and HD-Zip gene number [46]. Indeed, in this study, there were 169 HD-Zip genes in M. sinensis (1.88 Gb) [32], 32 members in barley (5.1 Gb) [47,48], 47 members in foxtail millet (423 Mb) [49,50], and 47 members in Arabidopsis thaliana (125 Mb) [18,51]. The results were consistent with the previous conclusions. On the other hand, as a dominant factor in species evolution, genome replication events were common in angiosperm evolution, usually resulting in gene family expansion [52]. There were 72 segmental duplication gene pairs and 4 tandem repeats in the M. sinensis HD-Zip gene family. It was inferred that gene replication promoted the evolution of the HD-Zip gene family in M. sinensis. Additionally, M. sinensis was a genetic diploid with an allotetraploid history, and the heterotetraploid was thought to originate from 2.5 Mya due to the outbreak of transposon activity [32]. The variation of gene structure was considered to be one of the representative traces of gene family evolution [53]. Different subfamilies had different motif structures and composition, but members in the same branch were similar in domain, as well as in number and length of introns, which indicated that there was a close relationship between the diversity of gene structure and the evolution of the M. sinensis HD-Zip gene family. Moreover, M. sinensis presented similar intron counts and motif compositions within each HD-Zip subgroup, which further supported phylogenetic grouping.
Genes in the same branch usually have a similar function in a multispecies phylogenetic tree [54]. The potential roles of these HD-Zip proteins could be predicted by clustering their sequences in the phylogenetic tree with those from Arabidopsis. During embryogenesis, REV, PHV, and PHB were responsible for two major processes: establishing bilateral symmetry at the apex and establishing the shoot apical meristem (SAM) [55]. In this study, these three HD-Zip genes (REV, PHV, and PHB) were in the same subfamily as Misin06G303300.1, indicating that Misin06G303300.1 may have similar functions to them. Also, it was found that Misin06G303300.1 was highly expressed in seed germination, and had the highest expression level in early germination, suggesting that this gene was related to the development of mature seed embryos, and then affected seed germination. ATML1 and PDF2 are two HD-Zip IV members, the silent expression of which can inhibit epidermal gene expression and seed germination [56]. Misin19G174900.1, a gene located in the same subgroup as ATML1 and PDF2, had its relative expression level increased at 2 h before decreasing gradually, showing that the germination rate at 2 h may be higher than that of other time points. In Arabidopsis, ATHB2, a gene of HD-Zip II subgroup, involved in seed germination acted as a light-induced factor and belonged to the same subfamily as Misin02G271800.1, Misin01G096800.1, and Misin01G234600.1. The result provided us with an opportunity to explore potential roles of these genes in seed germination.
In addition, transcriptional factors typically modulate gene expression in response to stress in plants when the environment stimulates them [57]. In our study, HD-Zip genes were actively involved in drought stress, salt stress, and chromium stress, which was consistent with previous studies [58,59,60]. ATHB13 was a gene belonging to the HD-Zip I subfamily. A study reported that transgenic Arabidopsis overexpressing JUNGBRUNNEN1 lowered levels of cellular H2O2 and increased tolerance to drought stress [61]. Transgenic Arabidopsis plants containing ATHB13, which acted as an upstream regulator of JUB1, were more drought-resistant [62]. During drought conditions, Misin02G460500.1, an HD-Zip I gene, was significantly upregulated. Thus, Misin02G460500.1 might have a similar function to ATHB13 regarding drought resistance. Members of the HD-Zip III subgroup have been shown to be related to heavy metal, salt, and drought stress. Previous studies have shown that transgenic lines overexpressing miRNA166 inhibited Cd accumulation in rice grains [31]. Also, plants overexpressing miRNA166 showed development changes, such as leaf curl and xylem diameter reduction, so as to improve its drought resistance [63]. There were differences in the expression level between MtHDZ5, MtHDZ13, and MtHDZ22, three HD-Zip genes in Medicago truncatula, under stress conditions of 180 mM and 200 mM NaCl, indicating their response to salt stress [64]. In our study, Misin05G030000.1 and Misin06G303300.1, two members of HD-Zip class III, were highly expressed under chromium, salt, and drought stress. It can be speculated that Misin05G030000.1 and Misin06G303300.1 have similar functions with the above genes in stress resistance.

5. Conclusions

In the current study, the HD-Zip gene family in M. sinensis was first comprehensively and systematically analyzed. One hundred and sixty-nine identified M. sinensis HD-Zip members were divided into four subgroups, and their distribution was uneven on 19 chromosomes. In addition, subcellular localization predicted that most of the proteins (~99.41%) were in the nucleus, and all proteins except protein Misin16G051700.1 were transmembrane proteins. According to the cis-elements analysis, it appeared that most of the genes were related to plant growth and development, various hormones, and abiotic stress. Indeed, there were 72 segmental duplication gene pairs and 4 tandem repeats. Gene expression analysis results demonstrated that Misin06G303300.1 could critically participate in the seed germination of M. sinensis. In addition, Misin05G030000.1 and Misin06G303300.1 played a potential role responding to chromium, salt, and drought stress. This study will provide a basis of information for future studies of HD-Zip genes in stress-responsive traits, as well as possible genetic improvements for M. sinensis seed germination.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes13122256/s1, Table S1: Primer information for selected M. sinensis HD-Zip genes in qRT-PCR validation and reference genes in this study. Table S2: The basic information of all identified M. sinensis HD-Zip genes. Table S3: Analysis of cis-acting element of HD-Zip genes in M. sinensis. Table S4: Tandem and segmental gene duplication of HD-Zip genes in M. sinensis. Table S5: Homologous pairs between M. sinensis and other 10 species. Figure S1: The details of the sequence logos of each motif. Figure S2: Expression profiles of 12 genes at the same time point, including 0 h, 2 h, 1 d, 3 d, 5 d, in response to seed germination. Data were normalized to reference gene Misin17G054400.1.

Author Contributions

Conceptualization, J.C.; Q.R., G.N. and Z.Y. (Yongqun Zhu); validation, Z.Y. (Zhihang Yuan), H.L. and J.W.; formal analysis, J.C., Q.R. and Z.Y. (Zhongfu Yang); investigation, Y.Z. (Ying Zhou) and Z.Y. (Zhihang Yuan); resources, G.N. and Z.Y. (Yongqun Zhu); writing—original draft preparation, J.C. and Q.R.; writing—review and editing, all authors; funding acquisition, G.N., Z.Y. (Yongqun Zhu) and Q.R. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the earmarked fund for CARS (CARS-34), Agricultural Science and Technology Achievement Transformation Project in Sichuan Province (2021NZZJ0004) and Sichuan Forage Grass Innovation Team (SCCXTD-2020-16).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Publicly available data sets were analyzed in this study. These data can be found at: https://phytozome-next.jgi.doe.gov/info/Msinensis_v7_1, accessed on 1 June 2022.

Acknowledgments

We gratefully acknowledge Therese Mitros from the University of California Berkeley for providing Miscanthus sinensis genomic resources.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Clifton-Brown, J.; Lewandowski, I. Screening Miscanthus genotypes in field trials to optimise biomass yield and quality in Southern Germany. Eur. J. Agron. 2002, 16, 97–110. [Google Scholar] [CrossRef]
  2. Nie, G.; Liao, Z.; Zhong, M.; Zhou, J.; Cai, J.; Liu, A.; Wang, X.; Zhang, X. MicroRNA-Mediated Responses to Chromium Stress Provide Insight Into Tolerance Characteristics of Miscanthus sinensis. Front. Plant Sci. 2021, 12, 666117. [Google Scholar] [CrossRef]
  3. Heaton, E.A.; Long, S.P.; Voigt, T.B.; Jones, M.B.; Clifton-Brown, J. Miscanthus for Renewable Energy Generation: European Union Experience and Projections for Illinois. Mitig. Adapt. Strat. Glob. Chang. 2004, 9, 433–451. [Google Scholar] [CrossRef]
  4. Nie, G.; Huang, L.; Ma, X.; Ji, Z.; Zhang, Y.; Tang, L.; Zhang, X. Enriching Genomic Resources and Transcriptional Profile Analysis of Miscanthus sinensis under Drought Stress Based on RNA Sequencing. Int. J. Genom. 2017, 2017, 9184731. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Nsanganwimana, F.; Waterlot, C.; Louvel, B.; Pourrut, B.; Douay, F. Metal, nutrient and biomass accumulation during the growing cycle of Miscanthus established on metal-contaminated soils. J. Soil Sci. Plant Nutr. 2016, 179, 257–269. [Google Scholar] [CrossRef]
  6. Ye, B.; Saito, A.; Minamisawa, K. Effect of Inoculation with Anaerobic Nitrogen-Fixing Consortium on Salt Tolerance of Miscanthus sinensis. Soil Sci. Plant Nutr. 2005, 51, 243–249. [Google Scholar] [CrossRef] [Green Version]
  7. Ezaki, B.; Nagao, E.; Yamamoto, Y.; Nakashima, S.; Enomoto, T. Wild plants, Andropogon virginicus L. and Miscanthus sinensis Anders, are tolerant to multiple stresses including aluminum, heavy metals and oxidative stresses. Plant Cell Rep. 2008, 27, 951–961. [Google Scholar] [CrossRef]
  8. Aso, T. Studies on the Germination of Seeds of Miscanthus sinensis ANDERSS. Science Reports of the Yokohama National University, Section II. Biol. Geology 1976, 23, 27–34. [Google Scholar]
  9. Bewley, J.D. Seed Germination and Dormancy. Plant Cell 1997, 9, 1055–1066. [Google Scholar] [CrossRef] [Green Version]
  10. Christian, E.J.; Goggi, A.S.; Moore, K.J. Temperature and Light Requirements for Miscanthus sinensis Laboratory Germination Test. Crop Sci. 2014, 54, 791–795. [Google Scholar] [CrossRef] [Green Version]
  11. Awty-Carroll, D.; Clifton-Brown, J.; Robson, P. Using k-NN to analyse images of diverse germination phenotypes and detect single seed germination in Miscanthus sinensis. Plant Methods 2018, 14, 5. [Google Scholar] [CrossRef] [PubMed]
  12. Nie, G.; Sun, M.; Huang, L.; Ma, X.; Zhang, X. Effect of moist pre-chill and dry pre-heat treatment on the germination of Miscanthus sinensis seed from southwest China. Grassl. Sci. 2017, 63, 93–100. [Google Scholar] [CrossRef]
  13. Clifton-Brown, J.; McCalmont, J.; Hastings, A. Development of Miscanthus as a bioenergy crop: Biofuels and bioenergy. In Biofuels and Bioenergy; Elsevier: Amsterdam, The Netherlands, 2017. [Google Scholar]
  14. Clifton-Brown, J.; Schwarz, K.-U.; Hastings, A. History of the development of Miscanthus as a bioenergy crop: From small beginnings to potential realisation. In Biology and Environment: Proceedings of the Royal Irish Academy; Royal Irish Academy: Dublin, Ireland, 2015; Volume 115B, p. 45. [Google Scholar] [CrossRef]
  15. Yang, Y.; Al-Baidhani, H.H.J.; Harris, J.; Riboni, M.; Li, Y.; Mazonka, I.; Bazanova, N.; Chirkova, L.; Hussain, S.S.; Hrmova, M.; et al. DREB/CBF expression in wheat and barley using the stress-inducible promoters of HD-Zip I genes: Impact on plant development, stress tolerance and yield. Plant Biotechnol. J. 2020, 18, 829–844. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Harris, J.C.; Hrmova, M.; Lopato, S.; Langridge, P. Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli. New Phytol. 2011, 190, 823–837. [Google Scholar] [CrossRef]
  17. Elhiti, M.; Stasolla, C. Structure and function of homodomain-leucine zipper (HD-Zip) proteins. Plant Signal. Behav. 2009, 4, 86–88. [Google Scholar] [CrossRef] [Green Version]
  18. Ariel, F.D.; Manavella, P.A.; Dezar, C.A.; Chan, R.L. The true story of the HD-Zip family. Trends Plant Sci. 2007, 12, 419–426. [Google Scholar] [CrossRef]
  19. Tognacca, R.S.; Carabelli, M.; Morelli, G.; Ruberti, I.; Botto, J.F. Author Correction: ATHB2 is a negative regulator of germination in Arabidopsis thaliana seeds. Sci. Rep. 2021, 11, 12879. [Google Scholar] [CrossRef]
  20. Seo, M.; Kanno, Y.; Frey, A.; North, H.M.; Marion-Poll, A. Dissection of Arabidopsis NCED9 promoter regulatory regions reveals a role for ABA synthesized in embryos in the regulation of GA-dependent seed germination. Plant Sci. 2016, 246, 91–97. [Google Scholar] [CrossRef]
  21. Ni, Y.; Wang, X.; Li, D.; Wu, Y.; Xu, W.; Li, X. Novel cotton homeobox gene and its expression profiling in root development and in response to stresses and phytohormones. Acta Biochim. Biophys. Sin. 2010, 40, 78–84. [Google Scholar] [CrossRef] [Green Version]
  22. Kim, Y.-K.; Son, O.; Kim, M.-R.; Nam, K.-H.; Kim, G.-T.; Lee, M.-S.; Choi, S.-Y.; Cheon, C.-I. ATHB23, an Arabidopsis class I homeodomain-leucine zipper gene, is expressed in the adaxial region of young leaves. Plant Cell Rep. 2007, 26, 1179–1185. [Google Scholar] [CrossRef]
  23. Depège-Fargeix, N.; Javelle, M.; Chambrier, P.; Frangne, N.; Gerentes, D.; Perez, P.; Rogowsky, P.M.; Vernoud, V. Functional characterization of the HD-ZIP IV transcription factor OCL1 from maize. J. Exp. Bot. 2011, 62, 293–305. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Li, Y.; Zhang, S.; Dong, R.; Wang, L.; Yao, J.; van Nocker, S.; Wang, X. The grapevine homeobox gene VvHB58 influences seed and fruit development through multiple hormonal signaling pathways. BMC Plant Biol. 2019, 19, 523. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Ohashi-Ito, K.; Kubo, M.; Demura, T.; Fukuda, H. Class III Homeodomain Leucine-Zipper Proteins Regulate Xylem Cell Differentiation. Plant Cell Physiol. 2005, 46, 1646–1656. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Zhang, S.; Haider, I.; Kohlen, W.; Jiang, L.; Bouwmeester, H.; Meijer, A.H.; Schluepmann, H.; Liu, C.-M.; Ouwerkerk, P.B.F. Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Mol. Biol. 2012, 80, 571–585. [Google Scholar] [CrossRef] [PubMed]
  27. Park, M.Y.; Kim, S.-A.; Lee, S.-J.; Kim, S.Y. ATHB17 is a positive regulator of abscisic acid response during early seedling growth. Mol. Cells 2013, 35, 125–133. [Google Scholar] [CrossRef] [Green Version]
  28. Zhou, C.-P.; Li, C.-P.; Liang, W.-W.; Guo, P.; Yang, L.-T.; Chen, L.-S. Identification of manganese-toxicity-responsive genes in roots of two citrus species differing in manganese tolerance using cDNA-AFLP. Trees 2017, 31, 813–831. [Google Scholar] [CrossRef]
  29. Hu, T.; Ye, J.; Tao, P.; Li, H.; Zhang, J.; Zhang, Y.; Ye, Z. The tomato HD-Zip I transcription factor SlHZ24 modulates ascorbate accumulation through positive regulation of thed-mannose/l-galactose pathway. Plant J. 2016, 85, 16–29. [Google Scholar] [CrossRef] [Green Version]
  30. Chen, E.; Zhang, X.; Yang, Z.; Wang, X.; Yang, Z.; Zhang, C.; Wu, Z.; Kong, D.; Liu, Z.; Zhao, G.; et al. Genome-wide analysis of the HD-ZIP IV transcription factor family in Gossypium arboreum and GaHDG11 involved in osmotic tolerance in transgenic Arabidopsis. Genet. Mol. Res. 2017, 292, 593–609. [Google Scholar] [CrossRef]
  31. Ding, Y.F.; Gong, S.H.; Wang, Y.; Wang, F.J.; Bao, H.X.G.D.L.; Sun, J.W.; Cai, C.; Yi, K.K.; Chen, Z.X.; Zhu, C. MicroRNA166 Modulates Cadmium Tolerance and Accumulation in Rice. Plant Physiol. 2018, 177, 1691–1703. [Google Scholar] [CrossRef] [Green Version]
  32. Mitros, T.; Session, A.M.; James, B.T.; Wu, G.A.; Belaffif, M.B.; Clark, L.V.; Shu, S.; Dong, H.; Barling, A.; Holmes, J.R.; et al. Genome biology of the paleotetraploid perennial biomass crop Miscanthus. Nat. Commun. 2020, 11, 5442. [Google Scholar] [CrossRef]
  33. Wilkins, M.R.; Gasteiger, E.; Bairoch, A.; Sanchez, J.C.; Williams, K.L.; Appel, R.D.; Hochstrasser, D.F. Protein Identification and Analysis Tools in the ExPASy Server. In 2-D Proteome Analysis Protocols. Methods in Molecular Biology; Humana Press: Totowa, NJ, USA, 1999; Volume 112, pp. 531–552. [Google Scholar] [CrossRef]
  34. Zhuo, X.; Zheng, T.; Zhang, Z.; Zhang, Y.; Jiang, L.; Ahmad, S.; Sun, L.; Wang, J.; Cheng, T.; Zhang, Q. Genome-Wide Analysis of the NAC Transcription Factor Gene Family Reveals Differential Expression Patterns and Cold-Stress Responses in the Woody Plant Prunus mume. Genes 2018, 9, 494. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  36. Rhee, S.Y.; Beavis, W.; Berardini, T.Z.; Chen, G.; Dixon, D.; Doyle, A.; Garcia-Hernandez, M.; Huala, E.; Lander, G.; Montoya, M.; et al. The Arabidopsis Information Resource (TAIR): A model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res. 2003, 31, 224–228. [Google Scholar] [CrossRef] [Green Version]
  37. Nguyen, L.-T.; Schmidt, H.A.; Von Haeseler, A.; Minh, B.Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [Google Scholar] [CrossRef]
  38. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.F.; Von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2013, 14, 587–589. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Hoang, D.T.; Chernomor, O.; Haeseler, A.v.; Minh, B.Q.; Vinh, L.S. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol. Biol. Evol. 2017, 35, 518–522. [Google Scholar] [CrossRef]
  40. Bailey, T.L.; Johnson, J.; Grant, C.E.; Noble, W.S. The MEME Suite. Nucleic Acids Res. 2015, 43, W39–W49. [Google Scholar] [CrossRef] [Green Version]
  41. Wang, Y.; Tang, H.; DeBarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-H.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [Green Version]
  42. Chen, C.J.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.H.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  43. Zhong, M.; Yang, X.; Hu, Y.; Huang, L.; Peng, Y.; Li, Z.; Liu, Q.; Wang, X.; Zhang, X.; Nie, G. Identification of candidate reference genes for quantitative RT-PCR in Miscanthus sinensis subjected to various abiotic stresses. Mol. Biol. Rep. 2020, 47, 2913–2927. [Google Scholar] [CrossRef]
  44. Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 2008, 3, 1101–1108. [Google Scholar] [CrossRef] [PubMed]
  45. Henriksson, E.; Olsson, A.S.; Johannesson, H.; Hanson, J.; Engström, P.; Söderman, E. Homeodomain Leucine Zipper Class I Genes in Arabidopsis. Expression Patterns and Phylogenetic Relationships. Plant Physiol. 2005, 139, 509–518. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Wei, M.; Liu, A.; Zhang, Y.; Zhou, Y.; Li, D.; Dossa, K.; Zhou, R.; Zhang, X.; You, J. Genome-wide characterization and expression analysis of the HD-Zip gene family in response to drought and salinity stresses in sesame. BMC Genom. 2019, 20, 748. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Mayer, K.F.X.; Waugh, R.; Langridge, P.; Close, T.J.; Wise, R.P.; Graner, A.; Matsumoto, T.; Sato, K.; Schulman, A.; Muehlbauer, G.J.; et al. A physical, genetic and functional sequence assembly of the barley genome. Nature 2012, 491, 711–716. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Li, Y.; Xiong, H.; Cuo, D.; Wu, X.; Duan, R. Genome-wide characterization and expression profiling of the relation of the HD-Zip gene family to abiotic stress in barley (Hordeum vulgare L.). Plant Physiol. Biochem. 2019, 141, 250–258. [Google Scholar] [CrossRef] [PubMed]
  49. Chai, W.; Si, W.; Ji, W.; Qin, Q.; Zhao, M.; Jiang, H. Genome-Wide Investigation and Expression Profiling of HD-Zip Transcription Factors in Foxtail Millet (Setaria italica L.). BioMed Res. Int. 2018, 2018, 8457614. [Google Scholar] [CrossRef] [Green Version]
  50. Zhang, G.; Liu, X.; Quan, Z.; Cheng, S.; Xu, X.; Pan, S.; Xie, M.; Zeng, P.; Yue, Z.; Wang, W.; et al. Genome sequence of foxtail millet (Setaria italica) provides insights into grass evolution and biofuel potential. Nat. Biotechnol. 2012, 30, 549–554. [Google Scholar] [CrossRef] [Green Version]
  51. The Arabidopsis Genome Initiative. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 2000, 408, 796–815. [Google Scholar] [CrossRef] [Green Version]
  52. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4, 10. [Google Scholar] [CrossRef]
  53. Tian, R.; Yang, Y.; Chen, M. Genome-wide survey of the amino acid transporter gene family in wheat (Triticum aestivum L.): Identification, expression analysis and response to abiotic stress. Int. J. Biol. Macromol. 2020, 162, 1372–1387. [Google Scholar] [CrossRef]
  54. Liu, M.; Ma, Z.; Sun, W.; Huang, L.; Wu, Q.; Tang, Z.; Bu, T.; Li, C.; Chen, H. Genome-wide analysis of the NAC transcription factor family in Tartary buckwheat (Fagopyrum tataricum). BMC Genom. 2019, 20, 113. [Google Scholar] [CrossRef] [Green Version]
  55. Prigge, M.J.; Otsuga, D.; Alonso, J.M.; Ecker, J.R.; Drews, G.N.; Clark, S.E. Class III Homeodomain-Leucine Zipper Gene Family Members Have Overlapping, Antagonistic, and Distinct Roles in Arabidopsis Development. Plant Cell 2005, 17, 61–76. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Rombolá-Caldentey, B.; Rueda-Romero, P.; Iglesias-Fernández, R.; Carbonero, P.; Oñate-Sánchez, L. Arabidopsis DELLA and Two HD-ZIP Transcription Factors Regulate GA Signaling in the Epidermis through the L1 Box cis-Element. Plant Cell 2014, 26, 2905–2919. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Yamaguchi-Shinozaki, K.; Shinozaki, K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu. Rev. Plant Biol. 2006, 57, 781–803. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  58. Zhang, Z.; Zhu, R.; Ji, X.; Li, H.J.; Lv, H.; Zhang, H.Y. Genome-Wide Characterization and Expression Analysis of the HD-ZIP Gene Family in Response to Salt Stress in Pepper. Int. J. Genom. 2021, 2021, 8105124. [Google Scholar] [CrossRef]
  59. Zhao, S.; Gao, H.; Jia, X.; Wang, H.; Ke, M.; Ma, F. The HD-Zip I transcription factor MdHB-7 regulates drought tolerance in transgenic apple (Malus domestica). Environ. Exp. Bot. 2020, 180, 104246. [Google Scholar] [CrossRef]
  60. Hong, Y.; Liu, Y.; Zhang, Y.; Jia, L.; Yang, X.; Zhang, X.; Liu, J.; Luan, Y. Genome-wide characterization of homeobox-leucine zipper gene family in tomato (Solanum lycopersicum) and functional analysis of SlHDZ34 (III sub-family member) under salinity stress. Environ. Exp. Bot. 2021, 192, 104652. [Google Scholar] [CrossRef]
  61. Ebrahimian-Motlagh, S.; Ribone, P.A.; Thirumalaikumar, V.P.; Allu, A.D.; Chan, R.L.; Mueller-Roeber, B.; Balazadeh, S. JUNGBRUNNEN1 Confers Drought Tolerance Downstream of the HD-Zip I Transcription Factor AtHB13. Front. Plant Sci. 2017, 8, 2118. [Google Scholar] [CrossRef] [Green Version]
  62. Cabello, J.V.; Chan, R.L. The homologous homeodomain-leucine zipper transcription factors HaHB1 and AtHB13 confer tolerance to drought and salinity stresses via the induction of proteins that stabilize membranes. Plant Biotechnol. J. 2012, 10, 815–825. [Google Scholar] [CrossRef]
  63. Zhang, J.; Zhang, H.; Srivastava, A.K.; Pan, Y.; Bai, J.; Fang, J.; Shi, H.; Zhu, J.-K.; Zhang, J.; Zhang, H.; et al. Knockdown of Rice MicroRNA166 Confers Drought Resistance by Causing Leaf Rolling and Altering Stem Xylem Development. Plant Physiol. 2018, 176, 2082–2094. [Google Scholar] [CrossRef] [Green Version]
  64. Li, Z.; Gao, Z.; Li, R.; Xu, Y.; Kong, Y.; Zhou, G.; Meng, C.; Hu, R. Genome-wide identification and expression profiling of HD-ZIP gene family in Medicago truncatula. Genomics 2020, 112, 3624–3635. [Google Scholar] [CrossRef] [PubMed]
Genes 13 02256 g001 550
Figure 1. Unrooted phylogenetic tree illustrating links between the 169 HD-Zip proteins in Miscanthus sinensis and 47 HD-Zip members in Arabidopsis thaliana. The phylogenetic tree was constructed by IQ-TREE.
Figure 1. Unrooted phylogenetic tree illustrating links between the 169 HD-Zip proteins in Miscanthus sinensis and 47 HD-Zip members in Arabidopsis thaliana. The phylogenetic tree was constructed by IQ-TREE.
Genes 13 02256 g001
Genes 13 02256 g002 550
Figure 2. A phylogenetic analysis of M. sinensis HD-Zip genes, as well as the pattern of motifs and gene structure. By using MEME, the HD-Zip proteins’ motifs composition was identified, and gray lines indicate non-conserved sequences. An overview of the sequence information for 10 motifs can be found in Figure S1. With the online tool GSDS, the exon-intron structure of M. sinensis HD-Zip genes was analyzed. The lengths of exons and introns were proportionately displayed.
Figure 2. A phylogenetic analysis of M. sinensis HD-Zip genes, as well as the pattern of motifs and gene structure. By using MEME, the HD-Zip proteins’ motifs composition was identified, and gray lines indicate non-conserved sequences. An overview of the sequence information for 10 motifs can be found in Figure S1. With the online tool GSDS, the exon-intron structure of M. sinensis HD-Zip genes was analyzed. The lengths of exons and introns were proportionately displayed.
Genes 13 02256 g002
Genes 13 02256 g003 550
Figure 3. Distribution of 169 HD-Zip genes on 19 M. sinensis chromosomes, represented by vertical bars, with chromosomes numbered at the top. On the left, the scale represents chromosome length in megabytes (Mb).
Figure 3. Distribution of 169 HD-Zip genes on 19 M. sinensis chromosomes, represented by vertical bars, with chromosomes numbered at the top. On the left, the scale represents chromosome length in megabytes (Mb).
Genes 13 02256 g003
Genes 13 02256 g004 550
Figure 4. Diagrams depicting the interchromosomal relationship of M. sinensis HD-Zip genes. All synteny blocks in the genome were represented by gray lines, while segmental duplicates were shown by blue lines, and tandem duplicates were shown by red lines.
Figure 4. Diagrams depicting the interchromosomal relationship of M. sinensis HD-Zip genes. All synteny blocks in the genome were represented by gray lines, while segmental duplicates were shown by blue lines, and tandem duplicates were shown by red lines.
Genes 13 02256 g004
Genes 13 02256 g005 550
Figure 5. HD-Zip gene synteny analysis between M. sinensis and ten species. Collinear blocks from M. sinensis and other plant genomes were indicated by a gray background line, whereas syntenic HD-Zip gene pairs were indicated by a red line.
Figure 5. HD-Zip gene synteny analysis between M. sinensis and ten species. Collinear blocks from M. sinensis and other plant genomes were indicated by a gray background line, whereas syntenic HD-Zip gene pairs were indicated by a red line.
Genes 13 02256 g005
Genes 13 02256 g006 550
Figure 6. (a) Expression patterns of 12 HD-Zip genes from M. sinensis related to seed germination chosen at random. The gene Unigene33024 has been used to standardize all the data. Significant differences (p < 0.05) between the treatments were denoted by lowercase letter(s) above all the bars. (b) Seed morphological changes during seed germination.
Figure 6. (a) Expression patterns of 12 HD-Zip genes from M. sinensis related to seed germination chosen at random. The gene Unigene33024 has been used to standardize all the data. Significant differences (p < 0.05) between the treatments were denoted by lowercase letter(s) above all the bars. (b) Seed morphological changes during seed germination.
Genes 13 02256 g006
Genes 13 02256 g007 550
Figure 7. Expression patterns of 12 M. sinensis HD-Zip genes in different abiotic stresses: (a) a heatmap of 12 M. sinensis HD-Zip genes under drought stress; (b) a heatmap of 12 M. sinensis HD-Zip genes under salt stress; (c) a heatmap of 12 M. sinensis HD-Zip genes under chromium stress. Expression changes of M. sinensis HD-Zip genes under abiotic stresses were shown by log2 change folds, and the levels of expression were illustrated by color gradations.
Figure 7. Expression patterns of 12 M. sinensis HD-Zip genes in different abiotic stresses: (a) a heatmap of 12 M. sinensis HD-Zip genes under drought stress; (b) a heatmap of 12 M. sinensis HD-Zip genes under salt stress; (c) a heatmap of 12 M. sinensis HD-Zip genes under chromium stress. Expression changes of M. sinensis HD-Zip genes under abiotic stresses were shown by log2 change folds, and the levels of expression were illustrated by color gradations.
Genes 13 02256 g007
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Chen, J.; Ran, Q.; Yang, Z.; Zhou, Y.; Yuan, Z.; Lai, H.; Wang, J.; Nie, G.; Zhu, Y. Genome-Wide Identification and Expression Profile of the HD-Zip Transcription Factor Family Associated with Seed Germination and Abiotic Stress Response in Miscanthus sinensis. Genes 2022, 13, 2256. https://doi.org/10.3390/genes13122256

AMA Style

Chen J, Ran Q, Yang Z, Zhou Y, Yuan Z, Lai H, Wang J, Nie G, Zhu Y. Genome-Wide Identification and Expression Profile of the HD-Zip Transcription Factor Family Associated with Seed Germination and Abiotic Stress Response in Miscanthus sinensis. Genes. 2022; 13(12):2256. https://doi.org/10.3390/genes13122256

Chicago/Turabian Style

Chen, Jiayi, Qifan Ran, Zhongfu Yang, Ying Zhou, Zhihang Yuan, Huiqi Lai, Jing Wang, Gang Nie, and Yongqun Zhu. 2022. "Genome-Wide Identification and Expression Profile of the HD-Zip Transcription Factor Family Associated with Seed Germination and Abiotic Stress Response in Miscanthus sinensis" Genes 13, no. 12: 2256. https://doi.org/10.3390/genes13122256

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop