Systematic Analysis of Zinc Finger-Homeodomain Transcription Factors (ZF-HDs) in Barley (Hordeum vulgare L.)

Zinc finger-homeodomain transcription factors (ZF-HDs) are pivotal in regulating plant growth, development, and diverse stress responses. In this study, we found 8 ZF-HD genes in barley genome. Theses eight HvZF-HD genes were located on five chromosomes, and classified into ZHD and MIF subfamily. The collinearity, gene structure, conserved motif, and cis-elements of HvZF-HD genes were also analyzed. Real-time PCR results suggested that the expression of HvZF-HD4, HvZF-HD6, HvZF-HD7 and HvZF-HD8 were up-regulated after hormones (ABA, GA3 and MeJA) or PEG treatments, especially HvZF-HD6 was significantly induced. These results provide useful information of ZF-HD genes to future study aimed at barley breeding.


Introduction
Barley (Hordeum vulgare L.) is the fourth largest crop all over the world, which has important applications in food, feed, and brewing [1].As sessile organism, barley has to suffer various adverse conditions including drought stress, resulting in the loss of yield.Consequently, identification of stress-tolerance genes and breeding stress-tolerance varieties are the key strategies for enhancing the quality and yield of barley.
Zinc-finger homologous domain proteins (ZF-HDs), a kind of plant-specific transcription factors, belong to C2H2 type zinc finger proteins and play vital functions in vegetative growth, flowering, bearing, and stress resistance of plants [2][3][4].A standard ZF-HD protein comprises a zinc finger structure (ZF) at the N-terminus and a homologous domain (HD) at the C-terminus.The HD domain is a conserved DNA binding domain with about 60 amino acid residues, which participates in plants growth and development by regulating the expression level of downstream target genes [5].Most HD proteins include other domains that can be involved in interacting with other proteins [6].The ZF domain comprises two pairs of conserved cysteine and/or histidine residues, which coordinate with a lone zinc ion to configure a finger-like loop structure, thus enhancing the stability of the motif [7].Zinc finger domain is related to protein nucleic acid recognition and protein-protein interaction [8,9].Based on conserved motifs and phylogenetic relationships, ZF-HD gene family is essentially classified to two subfamilies: ZHD and MIF (mini zinc finger) subfamilies [10].The ZHD subfamily genes contain both HD and ZF domains [4], and MIF subfamily genes encode ZF-HD proteins with the ZF domains but without the HD domains [11].
ZF-HD genes have been investigated in many plant species, but ZF-HD family genes in barley have not been identified and analyzed.In this study, we found 8 ZF-HD genes in barley genome, and then the physical and chemical properties, chromosomal location, collinearity, exon-intron structure, conserved motif, cis-elements and gene expression pattern of HvZF-HD genes were analyzed.These results provide useful information of ZF-HD genes to future function stud in barley.

Identification of ZF-HD Genes in Barley
The barley protein sequences were downloaded from EnsemblPlant website (https: //plants.ensembl.org/index.html)(accessed on 20 January 2024).Hidden Markov Model (HMM) search was used to obtained the proteins with ZF-HD dimerization region (PF04770) in local barley protein sequences [18].All of the identified HvZF-HD proteins were verified by using SMART (https://smart.embl-heidelberg.de)(accessed on 20 January 2024) and InterPro (http://www.ebi.ac.uk/interpro/) (accessed on 20 January 2024) online services.The ExPASY tools (https://web.expasy.org/protparam/)(accessed on 20 January 2024) was used to predict the physical and chemical parameters of HvZF-HD proteins.

Multiple Sequence Alignment and Phylogenetic Tree Construction
Multiple sequence alignment was performed with ClustalW of MEGA 11 software [19].The neighbor-joining (NJ) tree was constructed by MEGA 11 with 1000 bootstraps [19].The ZF-HD protein sequences of Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa and, Zea mays were downloaded from the EnsemblPlant website.

Chromosome Location and Synteny Analysis
The chromosome location of HvZF-HD genes was mapped to chromosome according to barley genome annotation information by using the MapChart [22].The genome sequences and annotation files of Arabidopsis, G. max, H. vulgare, O. sativa, and Z. mays were obtained from EnsemblPlant database.The Multiple collinear scanning toolkits (MC-ScanX) [23,24] and TBtools [25] were used to analyze the collinear relationships between barley and other species.

Plant Materials and Treatments
The seeds of barley cultivar "MoreX" were germinated on moist filter paper and grown at 23 • C with a 16 h light/8 h dark photoperiod.For stress and hormone treatments, twenty seedlings grown in hydroponic culture for 5 days were treated with 100 µM GA3 (gibberellin A3), 100 µM ABA (abscisic acid), 100 µM MeJA (methyl jasmonate) and 10% PEG8000 (polyethylene glycol 8000, w/v) for 36 h, respectively.Then, the shoot tissues were collected, and stored at −80 • C.

RNA Isolation and Real-Time PCR Analysis
SteadyPure Universal RNA Extraction Kit (Accurate Biology) was used to isolate total RNA, and the first-strand cDNA was synthesized by using the Evo M-MLV kit with gDNA clean for qPCR II (Accurate Biology).The specific primers of HvZF-HD and HvActin genes were showed in Table S1 [26].Real-time PCR experiments were conducted using TransTaq-T DNA Polymerase (TransGen, Beijing, China) under the following cycling program: initial denaturation at 95 • C for 3 min, followed by 40 cycles of denaturation at 95 • C for 5 s and annealing/extension at 60 • C for 30 s. Real-time PCR experiments were performed in three independent biological replicates and three technical replications to determine the average Ct values.The relative expression levels of HvZF-HD genes were calculated by 2 −∆∆CT method [27].

Identification and Characteristic Analysis of ZF-HD Gene Family in Barley
A total of 8 ZF-HD gene were identified by searching for the proteins with ZF-HD dimerization region (PF04770) in local barley protein sequences (Table 1), and SMART and InrerPro online services were used to verified the reliability of these 8 ZF-HD genes.Based on chromosome locations of ZF-HD genes in barley genome, we named them from HvZF-HD1 to HvZF-HD8 (Table 1 and Figure 1).HvZF-HD1-HvZF-HD8 were distributed on 5 chromosomes, i.e., chromosome 1H, 2H, 3H, 4H, 4H, 5H, 5H, and 5H, respectively.
HvZF-HD genes encoded polypeptides ranging from 94 to 420 amino acids, with the predicted molecular weights varying from 9.9 to 44.3 kDa (Table 1).The isoelectric point (pI) value ranged from 6.9 to 9.5.The calculated grand average of hydrophilic index (GRAVY) ranged from −0.5 to −1.0, indicating that HvZF-HD proteins were hydrophilic proteins (Table 1).Subcellular localization analysis indicated that HvZF-HD proteins were located in the nucleus (Table 1), which confirmed once again that ZF-HD protein was transcription factors.HvZF-HD genes encoded polypeptides ranging from 94 to 420 amino acids, with the predicted molecular weights varying from 9.9 to 44.3 kDa (Table 1).The isoelectric point (pI) value ranged from 6.9 to 9.5.The calculated grand average of hydrophilic index (GRAVY) ranged from −0.5 to −1.0, indicating that HvZF-HD proteins were hydrophilic proteins (Table 1).Subcellular localization analysis indicated that HvZF-HD proteins were located in the nucleus (Table 1), which confirmed once again that ZF-HD protein was transcription factors.
We further analyzed synteny between HvZF-HD genes in barley with ZF-HD genes in other plants, i.e., dicotyledonous plants (Arabidopsis and soybean) and monocotyledonous plants (rice and maize) (Figure 3 and Table S3).The results suggested that 0, 1, 10, and 14 orthologous gene pairs were identified between HvZF-HDs with other ZF-HD genes in Arabidopsis, soybean, rice, and maize, respectively (Figure 3).Some HvZF-HD genes had at least two orthologous genes with other ZF-HD gene in rice and maize, such as HvZF-HD4, HvZF-HD6, HvZF-HD7, and HvZF-HD8.These genes might play vital roles in the evolution and expansion of the ZF-HD family.The result showed that the genetic relationship of ZF-HD gene family was closer with barley in rice and maize than that in Arabidopsis, which is consistent with the results of phylogenetic tree (Figure 2).In addition, HvZF-HD genes and orthologous genes in other species (soybean, rice and maize) were still highly conserved in a long evolutionary process, suggesting these ZF-HD genes might still maintain the similar functions.We further analyzed synteny between HvZF-HD genes in barley with ZF-HD genes in other plants, i.e., dicotyledonous plants (Arabidopsis and soybean) and monocotyledonous plants (rice and maize) (Figure 3 and Table S3).The results suggested that 0, 1, 10, and 14 orthologous gene pairs were identified between HvZF-HDs with other ZF-HD genes in Arabidopsis, soybean, rice, and maize, respectively (Figure 3).Some HvZF-HD genes had at least two orthologous genes with other ZF-HD gene in rice and maize, such as HvZF-HD4, HvZF-HD6, HvZF-HD7, and HvZF-HD8.These genes might play vital roles in the evolution and expansion of the ZF-HD family.The result showed that the genetic relationship of ZF-HD gene family was closer with barley in rice and maize than that in Arabidopsis, which is consistent with the results of phylogenetic tree (Figure 2).In addition, HvZF-HD genes and orthologous genes in other species (soybean, rice and maize) were still highly conserved in a long evolutionary process, suggesting these ZF-HD genes might still maintain the similar functions.

Conserved Motifs and Gene Structure Analysis of HvZF-HDs
To identify the structure characteristic of HvZF-HD genes, we analyzed the conserved motifs and exon-intron structures, respectively (Figure 4).Conserved motif analysis showed that 10 motifs were identified in HvZF-HD protein, and all HvZF-HD proteins contained motif 1 and motif 4, which consisted of the core sequence of ZF domain (Figures 4A and 5A).In addition, Motif 2 and motif 3 comprised the typical HD domain (Figures 4A and 5B).The results of conserved domain analysis suggested that the HvZF-HD pro-

Conserved Motifs and Gene Structure Analysis of HvZF-HDs
To identify the structure characteristic of HvZF-HD genes, we analyzed the conserved motifs and exon-intron structures, respectively (Figure 4).Conserved motif analysis showed that 10 motifs were identified in HvZF-HD protein, and all HvZF-HD proteins contained motif 1 and motif 4, which consisted of the core sequence of ZF domain (Figures 4A and 5A).In addition, Motif 2 and motif 3 comprised the typical HD domain (Figures 4A and 5B).The results of conserved domain analysis suggested that the HvZF-HD proteins contained HD domain (HvZF-HD1, HvZF-HD2, HvZF-HD4, HvZF-HD5, HvZF-HD7 and HvZF-HD8) belonged to ZHD subfamily.Meanwhile, HvZF-HD3 and HvZF-HD6, which had no HD domain, belonged MIF subfamily (Figures 4A and 5B).The exon-intron structure of HvZF-HD genes was also analyzed, most HvZF-HD genes had no introns except HvZF-HD5 included one intron (Figure 4B).The difference of intron number indicates that the HvZF-HD gene family may have acquired or lost introns in the evolutionary process, and intron number also implies the potential ability of genes to form multiple splices.In conclusion, HvZF-HD genes with closer evolutionary relationships had similar gene structures (Figure 4B).

The Cis-Elements Analysis of HvZF-HD Promoters
The cis-elements in the gene promoter regions were related to gene expression, thus gene could perform different function in various environmental conditions [28].The ciselements in the promoter regions of the HvZF-HD genes were analyzed by PlantCARE [21], and the cis-elements identified were divided into four categories: light response element, hormone response elements, growth and development related elements, and stress response elements (Figure 4C and Table S4).The types and distribution of cis-element in the upstream of HvZF-HDs were diverse, suggesting that HvZF-HD gene family participated in the regulation of multiple signal pathways to respond to environmental stress.Each HvZF-HD promoter region contained at least one light response element, indicating that the expression of HvZF-HDs might be regulated by light.The diversity of cis-element type and number in the promoter of HvZF-HD genes showed that HvZF-HD genes participated in various hormones and stress response.

Expression Patterns of HvZF-HD Genes in Different Tissues
To identify tissue-specific expression profiles of HvZF-HD genes in barley, transcriptome data of inflorescence (1 cm), inflorescence (5 mm), internode, caryopsis (5 dpa), caryopsis (15 dpa), root (seedling), germinating embryo and shoot (seedling) were analyzed.The results showed that the transcript abundance of HvZF-HD genes was different in various tissues (Figure 6 and Table S5), suggesting that HvZF-HD family genes performed multiple functions in the growth and development of barley.For example, HvZF-HD6 was highly expressed in internode and shoot tissues, suggesting HvZF-HD6 might play important roles in vegetative tissues.HvZF-HD2 and HvZF-HD8 were highly expressed in both inflorescence, suggesting that these two genes play vital roles in the flowering process of barley (Figure 6 and Table S5).In addition, ZHD subfamily genes (HvZF-HD1, 2, 4, 5, 7, and 8) were higher expressed in inflorescence compared with MIF subfamily genes (HvZF-HD3 and HvZF-HD6) (Figure 6 and Table S5), indicating that the ZHD subfamily genes might be involved in regulating flower growth.

Expression Patterns of HvZF-HD Genes after PEG and Hormone Treatments
To explore the role of HvZF-HD genes in barley, the expression levels of seven HvZF-HD genes (HvZF-HD1-2, and HvZF-HD4-HvZF-HD8) after PEG and hormone (ABA, GA3 and MeJA) treatments in shoot tissues of barley at the seedling stage were detected by

Expression Patterns of HvZF-HD Genes after PEG and Hormone Treatments
To explore the role of HvZF-HD genes in barley, the expression levels of seven HvZF-HD genes (HvZF-HD1-2, and HvZF-HD4-HvZF-HD8) after PEG and hormone (ABA, GA3 and MeJA) treatments in shoot tissues of barley at the seedling stage were detected by real-time PCR (Figure 7).All genes except HvZF-HD1 were up-regulated after PEG stress for 36 h (Figure 7A).After ABA treatment, only HvZF-HD2 gene was down-regulated, and other six genes were highly induced by ABA treatment (Figure 7B).All HvZF-HD genes were up-regulated after GA3 treatment (Figure 7C).It is worth noting that the expression level of HvZF-HD6 was up-regulated (40-fold) at 36 h after GA3 treatment compared to control (Figure 7C).After MeJA treatment, the expression level of HvZF-HD5 was downregulated, and other HvZF-HD genes were up-regulated Figure 7C,D).In conclusion, most HvZF-HD genes were significantly induced after PEG, ABA, GA3 and MeJA treatments, therefore, we speculated that the HvZF-HD family might play important roles in stress defense response.
Genes 2024, 15, x FOR PEER REVIEW 9 of 12 real-time PCR (Figure 7).All genes except HvZF-HD1 were up-regulated after PEG stress for 36 h (Figure 7A).After ABA treatment, only HvZF-HD2 gene was down-regulated, and other six genes were highly induced by ABA treatment (Figure 7B).All HvZF-HD genes were up-regulated after GA3 treatment (Figure 7C).It is worth noting that the expression level of HvZF-HD6 was up-regulated (40-fold) at 36 h after GA3 treatment compared to control (Figure 7C).After MeJA treatment, the expression level of HvZF-HD5 was downregulated, and other HvZF-HD genes were up-regulated Figure 7C,D).In conclusion, most HvZF-HD genes were significantly induced after PEG, ABA, GA3 and MeJA treatments, therefore, we speculated that the HvZF-HD family might play important roles in stress defense response.

Discussion
The members of ZF-HD gene family have been detected in many plants, but a genome-wide analysis was not performed in barley.Previous studies have reported that ZF-HD family were classified into MIF and ZHD subfamilies [17], our results also confirm these result (Figure 2).Interestingly, the evolutionary relationship of MIF proteins was close with ZHD III proteins (Figure 2), suggesting the MIF proteins might originate from the deletion of the HD domain of ZHD proteins, or ZHD proteins might originate from the HD domain obtained by MIF proteins.
Collinear analysis showed ten orthologous gene pairs were found between barley and rice, fourteen orthologous gene pairs were found between barley and maize (Figure 3 and Table S3).Some HvZF-HD genes had two or more orthologous genes, such as HvZF-HD6 were collinear with three ZF-HD genes in maize, which might play crucial roles in the evolution of ZF-HD genes.(Figure 3 and Table S3).Moreover, only one orthologous gene pair of ZF-HD genes was found between barley and Glycine max (Figure 3B).This

Discussion
The members of ZF-HD gene family have been detected in many plants, but a genomewide analysis was not performed in barley.Previous studies have reported that ZF-HD family were classified into MIF and ZHD subfamilies [17], our results also confirm these result (Figure 2).Interestingly, the evolutionary relationship of MIF proteins was close with ZHD III proteins (Figure 2), suggesting the MIF proteins might originate from the deletion of the HD domain of ZHD proteins, or ZHD proteins might originate from the HD domain obtained by MIF proteins.
Collinear analysis showed ten orthologous gene pairs were found between barley and rice, fourteen orthologous gene pairs were found between barley and maize (Figure 3 and Table S3).Some HvZF-HD genes had two or more orthologous genes, such as HvZF-HD6 were collinear with three ZF-HD genes in maize, which might play crucial roles in the evolution of ZF-HD genes.(Figure 3 and Table S3).Moreover, only one orthologous gene pair of ZF-HD genes was found between barley and Glycine max (Figure 3B).This phenomenon might be caused by separate evolution of ZF-HD genes in monocotyledons and dicotyledonous plants.
ZF-HD genes regulate many biological processes and play an important role in plant growth, development and stress response [3,29].The result of tissue-specific expression profiles showed that HvZF-HD genes had different expression levels in different barley tissues (Figure 6), thus HvZF-HD genes might play various roles in plant growth and development.The HvZF-HD3 was not detected in barley tissues, suggesting that HvZF-HD3 might be expressed in specific tissues and environment conditions, or it was a pseudogene.HvZF-HD genes were mainly expressed in inflorescence, while most ZF-HD genes in Arabidopsis thaliana were universally expressed in flower tissue [4], indicating that ZF-HD genes might play a role in regulating flower development.Interestingly, the two MIF subfamily genes (HvZF-HD3 and HvZF-HD6) were not expressed in the inflorescence.Although HvZF-HD6 was not expressed in the inflorescence, it is highly expressed in other tissues, especially in the internodes, indicating that HvZF-HD6 may play an important role in the vegetative growth of barley.In addition, most HvZF-HD genes were significantly induced after PEG, ABA, GA3 and MeJA treatments, suggesting that the HvZF-HD genes played vital roles in plant growth, plant growth, development, and stress defense response (Figure 7).These results laid a foundation for future functional studies of HvZF-HDs.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes15050578/s1,Table S1.Real-time PCR primers of HvZF-HDs and the HvActin genes; Table S2.ZF-HD proteins used in the phylogenetic tree construction; Table S3.Synteny analysis of ZF-HD genes between barley and four species (Arabidopsis, rice, maize

Figure 2 .
Figure 2. The neighbor-joining (NJ) phylogenetic tree of ZF-HD proteins.The phylogenetic tree was constructed based on ZF-HD protein sequences from Hordeum vulgare (Hv), Brachypodium distachyon (Bd), Oryza sativa (Os) and Zea mays (Zm).Different groups of ZF-HD proteins are distinguished by different colors.

Figure 2 .
Figure 2. The neighbor-joining (NJ) phylogenetic tree of ZF-HD proteins.The phylogenetic tree was constructed based on ZF-HD protein sequences from Hordeum vulgare (Hv), Brachypodium distachyon (Bd), Oryza sativa (Os) and Zea mays (Zm).Different groups of ZF-HD proteins are distinguished by different colors.Genes 2024, 15, x FOR PEER REVIEW 6 of 12

Figure 3 .
Figure 3. Synteny analysis of ZF-HD genes between barley and other plant species.Gray lines in the background indicate the collinear blocks within the barley and other plant genomes, while the red lines highlight the syntenic ZF-HD gene pairs.The chromosomes of barley and four other species are painted in different colors, and their names are in the box.(A-D) Synteny analysis of ZF-HD family genes between Hordeum vulgare with Arabidopsis thaliana (A), Glycine max (B), Oryza sativa (C) and Zea mays (D).

Figure 3 .
Figure 3. Synteny analysis of ZF-HD genes between barley and other plant species.Gray lines in the background indicate the collinear blocks within the barley and other plant genomes, while the red lines highlight the syntenic ZF-HD gene pairs.The chromosomes of barley and four other species are painted in different colors, and their names are in the box.(A-D) Synteny analysis of ZF-HD family genes between Hordeum vulgare with Arabidopsis thaliana (A), Glycine max (B), Oryza sativa (C) and Zea mays (D).

Figure 4 .
Figure 4. Conserved motifs, gene structures and cis-elements analysis of ZF-HD genes in barley.The phylogenetic tree was constructed based on HvZF-HD proteins using MEGA 11 software.(A) The conserved motifs analysis of the barley HvZF-HD proteins.The motif 1-10 are displayed in different colored boxes.(B) Exon-intron structures of the barley HvZF-HD genes.Green boxes indicate 5′-and 3′-UTR; yellow boxes indicate exons; and black lines indicate introns.The ZF domain and HD domain are highlighted by red box and purple box, respectively.(C) The light response element, hormone response elements, growth and development related elements, and stress response elements are displayed in different colors.

Figure 4 .
Figure 4. Conserved motifs, gene structures and cis-elements analysis of ZF-HD genes in barley.The phylogenetic tree was constructed based on HvZF-HD proteins using MEGA 11 software.(A) The conserved motifs analysis of the barley HvZF-HD proteins.The motif 1-10 are displayed in different colored boxes.(B) Exon-intron structures of the barley HvZF-HD genes.Green boxes indicate 5 ′and 3 ′ -UTR; yellow boxes indicate exons; and black lines indicate introns.The ZF domain and HD domain are highlighted by red box and purple box, respectively.(C) The light response element, hormone response elements, growth and development related elements, and stress response elements are displayed in different colors.
phylogenetic tree was constructed based on HvZF-HD proteins using MEGA 11 software.(A) The conserved motifs analysis of the barley HvZF-HD proteins.The motif 1-10 are displayed in different colored boxes.(B) Exon-intron structures of the barley HvZF-HD genes.Green boxes indicate 5′-and 3′-UTR; yellow boxes indicate exons; and black lines indicate introns.The ZF domain and HD domain are highlighted by red box and purple box, respectively.(C) The light response element, hormone response elements, growth and development related elements, and stress response elements are displayed in different colors.

Figure 5 .
Figure 5.The conserved domain analysis of HvZF-HD protein in barley.(A) The ZF domain contain motif 1 and motif 4. (B) The HD domain contain motif 2 and motif 3.Figure 5.The conserved domain analysis of HvZF-HD protein in barley.(A) The ZF domain contain motif 1 and motif 4. (B) The HD domain contain motif 2 and motif 3.

Figure 5 .
Figure 5.The conserved domain analysis of HvZF-HD protein in barley.(A) The ZF domain contain motif 1 and motif 4. (B) The HD domain contain motif 2 and motif 3.Figure 5.The conserved domain analysis of HvZF-HD protein in barley.(A) The ZF domain contain motif 1 and motif 4. (B) The HD domain contain motif 2 and motif 3.

Figure 6 .
Figure 6.Tissue-specific expression patterns of the HvZF-HD genes in Hordeum vulgare.The transcriptome data of various tissues in barley were downloaded from Experiments database.The log2 of FPKM (fragments per kilobase of exon model per million mapped) values were calculated by RNA-seq data to show the expression levels of the HvZF-HD genes in barley.

Figure 6 .
Figure 6.Tissue-specific expression patterns of the HvZF-HD genes in Hordeum vulgare.The transcriptome data of various tissues in barley were downloaded from Experiments database.The log 2 of FPKM (fragments per kilobase of exon model per million mapped) values were calculated by RNA-seq data to show the expression levels of the HvZF-HD genes in barley.

Figure 7 .
Figure 7. Expression levels of HvZF-HD genes after PEG (A) and various hormone (ABA, GA3, MeJA) treatments (B-D) in barley.The expression level of the barley HvActin gene was used as the internal control to standardize the RNA samples for each reaction.The values are the mean ± SD from three samples.

Figure 7 .
Figure 7. Expression levels of HvZF-HD genes after PEG (A) and various hormone (ABA, GA3, MeJA) treatments (B-D) in barley.The expression level of the barley HvActin gene was used as the internal control to standardize the RNA samples for each reaction.The values are the mean ± SD from three samples.

Table 1 .
The HvZF-HD family genes in barley.