CCCH Zinc Finger Genes in Barley: Genome-Wide Identification, Evolution, Expression and Haplotype Analysis

doi:10.21203/rs.3.rs-1114922/v1

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Research Article

CCCH Zinc Finger Genes in Barley: Genome-Wide Identification, Evolution, Expression and Haplotype Analysis

https://doi.org/10.21203/rs.3.rs-1114922/v1

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published 15 Mar, 2022

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Background: CCCH transcription factors are important zinc finger transcription factors involved in the response to biotic and abiotic stress and physiological and developmental processes. Barley (Hordeum vulgare) is an agriculturally important cereal crop with multiple uses, such as brewing production, animal feed, and human food. The identification and assessment of new functional genes are important for the molecular breeding of barley.

Results: In this study, a total of 35 protein-encoding CCCH genes unevenly dispersed on seven different chromosomes were identified in barley. Phylogenetic analysis categorized the barley CCCH genes (HvC3Hs) into seven subfamilies according to their distinct features, and this classification was supported by intron–exon structure and conserved motif analysis. Despite the large genome size of barley, the lower number of CCCH genes in barley might be attributed to the low frequency of segmental and tandem duplication events. Furthermore, the HvC3H genes displayed distinct expression profiles for different developmental processes and in response to various types of stresses. The expression of HvC3H9 was significantly induced by multiple types of abiotic stress and/or phytohormone treatment, which might make it an excellent target for the molecular breeding of barley. Genetic variation of HvC3Hs was characterized using publicly available exome-capture sequencing datasets. Clear genetic divergence was observed between wild and landrace barley populations in HvC3H genes. For most HvC3Hs, nucleotide diversity and the number of haplotype polymorphisms decreased during barley domestication.

Conclusion: Overall, our study provides a comprehensive characterization of barley CCCH transcription factors, their diversity, and their biological functions.

Plant Physiology and Morphology

Barley

CCCH gene family

Expression pattern

Genetic variation

Haplotype analysis

Transcription factors (TFs), also known as trans-acting factors, are important regulatory proteins that activate or inhibit specific target genes by interacting with cis-regulatory elements [1]. They play a role in multiple biological processes, such as growth, development, signal transduction, cellular morphogenesis, and responses to environmental stress [2]. Zinc finger TFs are some of the most abundant TFs in plants and play key roles in regulating transcription and various biological functions [3]. They are characterized by the typical zinc finger motif and a compact three-dimensional finger-type structure formed by cysteine and/or histidine residues coordinated with a few zinc atoms, which are critical for their specific roles in binding to DNA, RNA, and proteins [4]. Zinc finger proteins can be further categorized into at least 14 families based on their structural and functional characteristics, such as DOF, ERF, LIM, ring-finger, and WRKY families [5–9].

Most of the zinc finger families are protein- or DNA-binding proteins; a recently identified group of zinc finger proteins referred to as the CCCH gene family exhibits RNA-binding and processing activity through their specific motifs in both animals and plants [10, 11]. The CCCH proteins typically contain 1–6 CCCH-type motifs characterized by three cysteine residues and one histidine residue. According to the number of amino acid spacers between cysteine and histidine residues, the consensus sequence of the CCCH motif was originally defined as C–X₆₋₁₄–C–X₄₋₅–C–X₃–H and later re-defined as C–X₄₋₁₇–C–X₄₋₆–C–X₃–H (C stands for cysteine, H for histidine, and X for any amino acid) [11–13]. The consensus sequence C-X₇₋₈-C-X₅-C-X₃-H is the most abundant motif of CCCH proteins in Arabidopsis and rice, suggesting that other motifs might be derived from these motifs [11].

An increasing number of studies have shown that CCCH-type zinc finger proteins play a role in cell fate specification and developmental processes in plants. For example, AtKHZ1 and AtKHZ2 are required for flowering and senescence in Arabidopsis [14]. AtC3H59/ZFWD3 plays an essential role in seedling development, seed germination, and development by interacting with the PPPDE gene family protein Desil [15]. In rice, OsDOS and OsTZF1 can act as repressors of leaf senescence [16, 17]. OsGZF1 affects glutelin accumulation during seed development [18]. GmZF351 and GmZF392 in soybean are involved in seed lipid accumulation [19, 20]. Functional studies have also shown that some CCCH proteins play a role in regulating plant growth and development through hormone signal transduction pathways. Specifically, AtTZF4/5/6 can act as negative regulators of light and gibberellins (GA) and positive regulators of abscisic acid (ABA)-mediated regulation of seed development, dormancy, and germination [21]. The CCCH-type zinc finger gene OsLIC is involved in the biosynthesis and/or signal transduction of brassinosteroid, which affects the architecture of rice plants [22]. In switchgrass, PvC3H69 can act as a negative regulator of leaf senescence by repressing ABA synthesis and ABA signaling pathways [23].

Several CCCH genes are implicated in the response to biotic and abiotic stress in plants. For example, OsC3H10, OsC3H47, and OsTZF5 are involved in the regulation of tolerance to drought stress in rice [24–26]. Overexpression of GhTZF1 significantly increases drought tolerance and delays drought-induced leaf senescence by negatively regulating the production and accumulation of reactive oxygen species (ROS) in transgenic Arabidopsis [27]. Arabidopsis AtZFP1 has been reported to confer salt tolerance by limiting oxidative and osmotic stress and maintaining an ionic balance [28]. Another non-tandem CCCH-type gene in Arabidopsis, AtC3H17, has pleiotropic effects in the response to salt stress via the ABA-dependent signaling pathway [29]. PvC3H72 was the first CCCH family gene identified to be involved in plant chilling and freezing tolerance. Overexpression of PvC3H72 improves cold tolerance in switchgrass, possibly via the ABA-mediated pathway [30]. More recently, DgC3H1 has been shown to confer cold tolerance in Chrysanthemum plants by regulating the osmotic and ROS system, as well as the expression of genes associated with the response to cold stress [31]. In addition, CCCH proteins have been shown to be involved in other adaptive processes, such as bacterial blight disease resistance [32], zinc homeostasis [33], and hydrogen peroxide [17] and oxidative stress [34]. These findings indicate that CCCH zinc finger proteins have various biological functions in model plants. However, few studies have evaluated the roles of CCCH gene family members in other plants, especially crop plants.

Genome assemblies have been used for the global identification and characterization of genes in numerous plant species. Genome-wide identification and characterization of CCCH genes have been carried out in Arabidopsis, rice [11], maize [13], poplar [35], tomato [36], Medicago truncatula [37], grape [38], citrus [39], switchgrass [40], Brassica rapa [41], and recently in the common bean [42], moso bamboo [43], wheat [44], and soybean [45]. However, CCCH genes have not yet been identified in barley (Hordeum vulgare), and their biological functions and evolutionary history remain poorly understood. Barley is one of the world’s earliest domesticated crops and ranks fourth among all cereal crops in both area and tonnage harvested. Barley is more adaptable to harsh and marginal environments than its wild relative wheat; it is thus widely used for animal feed and beer materials and provides a major source of calories in isolated regions [46]. The draft sequence and reference sequence V1 (Morex V1) [47] and V2 (Morex V2) [48] assemblies of the barley cultivar Morex have been released by the International Barley Sequencing Consortium (IBSC) [46]. The improved barley genome provides valuable information for the isolation, genetic cloning, and functional characterization of unknown genes. The aim of this study was to genomically identify and characterize barley CCCH genes (HvC3Hs). The phylogenetic relationships, distribution of motifs, intron-exon organization, and gene duplication events were comprehensively analyzed. We also conducted RNA-seq and qRT-PCR analysis to determine the possible function of HvC3H genes. Finally, genomic variation, genetic diversity, and selection on these genes during barley domestication were also investigated using barley resequencing data (including wild and landrace barley accessions). Our preliminary analysis provides new insight into the evolutionary history of CCCH genes and will aid future efforts to functionally characterize and genetically improve barley.

Identification of CCCH Proteins in Barley

The genomic proteins of barley Morex V2 were downloaded from the IPK database (https://doi.org/10.5447/ipk/2019/8). The CCCH protein sequences of Arabidopsis and rice were used as queries to search against the barley proteins with Basic Local Alignment Search Tool (BLAST) software. The Hidden Markov Model (HMM) of CCCH conserved domain (PF00642) was used as a query to search against the barley proteins by HMMER v3.0 with default parameters. The candidate CCCH proteins were further verified by Simple Modular Architecture Research Tool (SMART) (https://www.omicsclass.com/article/681), National Center for Biotechnology Information - Conserved Domains Database (NCBI-CDD) (https://www.omicsclass.com/article/310) and PFAM (http://pfam.xfam.org/) online databases. Putative proteins without CCCH domain were removed. A BLASTN search against barley expressed sequence tags (ESTs) was conducted to detect the existence of CCCH proteins. The computational physical and chemical properties of CCCH family members, including molecular weight (MW), theoretical isoelectric point (pI), instability index (II), and grand average of hydropathicity (GRAVY) were evaluated using the online tool ExPASy (http://web.expasy.org/protparam/). The subcellular location was predicted using the cello software (http://cello.life.nctu.edu.tw/).

Phylogeny, Gene Structure and Conserved Motif Analysis

The ClustalX v2.1 software was used to perform multiple alignments using the full-length CCCH protein sequences with default parameters. The phylogenetic analysis was carried out based on Neighbor-Joining (NJ) method with bootstrap value of 1000 replications using MEGA X software [99]. The intron-exon organization of HvC3H genes was generated by Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) based on the gene annotation Gene Transfer Format (GTF) file [100]. The Multiple Expectation Maximization for Motif Elicitation (MEME) (http://meme-suite.org/) was employed to analyze the conserved protein motifs with the following parameters: the number of all primitives was set to 10, the minimum and maximum width were set to 6 and 50, respectively [101]. The upstream 1.5kb genomic sequences of HvC3H genes were extracted and then submitted to the PlantCARE online database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to detect the potential cis-acting regulatory elements in the promoter region.

Chromosome Localization and Gene Synteny Analysis

The chromosomal locations of HvC3H genes were obtained from IPK database (https://doi.org/10.5447/ipk/2019/8), and the chromosome maps were visualized using MapChart v2.32. The MCScanX software was employed to analyze the synteny relationships of HvC3Hs in rice (Oryza sativa), wheat (Triticum aestivum), maize (Zea mays) soybean (Glycine max), tomato (Solanum lycopersicum) and Brassica rapa[102]. The gene duplication events of HvC3Hs were identified according to the genomic comparison. Tandem duplicated genes were defined based on the following criteria (1) located within the same chromosome; (2) <1 intervening gene [52]. The syntenic and duplicated gene pairs were visualized by the Circos v0.67 tool. The non-synonymous substitution (Ka) / synonymous substitution (Ks) ratio was calculated to estimate genes evolutionary rate using the PAL2NAL online tools (http://www.bork.embl.de/pal2nal/) [103]. Ka/Ks >1, =1 and <1 represent positive, neutral and purifying selection, respectively. The divergence time of syntenic and duplicated gene pairs was calculated based on the formula T= (Ks / 2λ) × 10⁻⁶ million years ago (MYA) (λ = 6.5 × 10−9) [104]. The BLAST and orthoVeen2 software employed to analyze the homologous genes between barley and other related species [105].

Expression Patterns of HvC3H Gene Members

In order to explore the expression patterns of HvC3Hs, the publicly available 148 RNA-seq samples were downloaded from the NCBI Sequence Reading Archive (SRA) database, including different developmental stages and tissues and different biotic and abiotic stresses. The accession number and sample information of RNA-seq were listed in Supplementary Table S1. The fragments per kilobase per million (FPKM) value was calculated by the HISAT2 v2.1.0 and StringTie v1.3.5 pipeline. The R package pheatmap was used to visualize the expression profiles using the log2 transformed FPKM value.

Plant Material, Stress Treatment, RNA Extraction and qRT-PCR Analysis

Seeds of barley Morex were obtained from the College of Agronomy, Northwest A&F University, and were used as the experimental material. the barley seeds were hydroponically grown in growth chamber under controlled conditions (23±1 °C, 16-h light/8-h dark cycle). The seedlings were processed for stress treatments at the three-leaf stage. For salt, drought, cold and ABA treatment, the plants were incubated in 150 mM NaCl, 19.2% (w/v) PEG-6000, 4°C and 100 μM ABA for 0, 1, 3, 6, 12, and 24 hours, respectively. Seedlings without any treatment at the same time point were considered as the control. Leaves of all these samples were randomly collected with three biological replications. The collected samples were immediately frozen in liquid nitrogen and stored at 80°C for RNA extraction.

To further reveal the possible functions of HvC3Hs, a total of 20 HvC3Hs were randomly selected to investigate their expression profile under various stresses by quantitative real-time PCR (qRT-PCR) analysis. HvACTIN (HORVU.MOREX.r2.5HG0378970) was used as the internal reference gene and the detail information of all the primers was listed in Supplementary Table S2. The total RNA was extracted by Plant RNA Kit reagent (Omega BioTek, USA), and cDNA synthesis was performed using 5X All-in-one RT MasterMix (ABM, Canada) according to the manufacturer’s instructions. The TB-Green ®Premix Ex Taq™ II kit (Takara, Dalian, China) was used to conduct qRT-PCR amplification in the Quant Studio™ Real-Time PCR system (Thermo Fisher, USA). The thermal cycling protocol was as follows: 95°C temperature for 30 seconds, followed by 40 cycles of 95°C for 3 seconds, and 30 seconds at 60°C. The relative expression level was calculated by the 2^−ΔΔCT method [106]. Three technical replications were applied for each treatment. The T-test was conducted to evaluate the significance of the results using R. One asterisk (*) indicate 0.05 significance level and double asterisk (^**) indicate 0.01 significance level, respectively.

Population Genetics Analysis of HvC3H-related Variants

The exome-captured resequencing data of 220 geographically-referenced barley accessions were retrieved from the NCBI SRA database (PRJEB8044/ERP009079) [93]. Raw reads were trimmed using Trimmonmatic v0.36 with default parameters [107]. The high-quality reads were mapped to the reference genome of barley Morex V2 using BWA-MEM v0.7.13r1126. The single nucleotide polymorphism (SNP) and insertion-deletion (InDel) was identified using the Picard-GATK pipeline [108]. The following criteria was used for SNPs filtration. (1) minor allele frequency (MAF) >0.05 and <0.95; (2) a maximum missing rate <0.1; (3) biallelic alleles. SNP and InDel were annotated using the SnpEff v4.3 according to the barley genome GTF file [109]. To better reveal the evolutionary relationships, barley accessions with SNP missing rate larger than 0.1 were excluded. Totally, the final collection included 95 landraces and 51 wild barley accessions (Supplementary Table S3). Only SNPs that located within the HvC3H genes were extracted for phylogenetic tree, population structure and principal component analysis (PCA). The HvC3H-related SNPs were used to construct NJ tree with the MEGA X. The phylogenetic tree was visualized by Interactive Tree of Life (iTOL). The population structure was quantified using ADMIXTURE v1.3.0 with predefined K values ranging from 2 to 4. The PCA was performed using the samrtpca sub-package implemented in EIGENSOFT v4.2. The nucleotide diversity (π) was estimated using vcftools v0.1.16. The DNAsp v6.12.01 was employed to calculate the haplotypes for each HvC3H genes. Finally, the media-joining haplotype networks were constructed using the PopART v1.7 [110].

Genome-wide Identification and Characterization of CCCH Proteins in Barley

The most updated barley Morex assembly was used for the identification of barley CCCH genes. A multifarious approach was used to obtain the most comprehensive results. First, the AtC3H and OsC3H proteins were used as queries to search against barley proteins as well as the HMM profile of the CCCH domain in Pfam (PF00642). A total of 52 putative CCCH-encoding genes were acquired from the barley genome. The protein sequences of HvC3H candidates were submitted to the Pfam, NCBI-CDD, and SMART online databases to ensure that the putative genes contained the CCCH domain. Only proteins that were verified by at least two databases were retained (Supplementary Table S4). A total of 17 genes were excluded from our dataset because no complete CCCH domain was verified by these online databases. A total of 35 high-confident HvC3H genes with complete open reading frames were identified, accounting for 1.07% of the total annotated protein-coding genes in barley (Table 1). Because there is no standard nomenclature for barley CCCH genes, the candidate HvC3Hs were designated as HvC3H1 to HvC3H35 according to their chromosomal number and location. A BLAST search against the barley ESTs indicated that 32 of the 35 HvC3Hs possessed EST records, which supported the existence of the HvC3Hs. Analysis of the physicochemical properties of HvC3H demonstrated that the amino acid length varied from 211 amino acids (HvC3H25) to 1,456 amino acids (HvC3H4), with an average length of 457.2 amino acids. The pI varied from 5.13 to 10.15, and the MW ranged from 4.501 kDa to 160.371 kDa. All of these CCCH proteins possessed negative GRAVY values (average value: -0.7715), indicating the hydropathic nature of HvC3Hs. Subcellular localization prediction showed that most of these proteins were located in the nucleus (29 HvC3Hs; 82.86%). In addition, three HvC3Hs were located in the chloroplast, and the other three were located in both the nucleus and chloroplast, which was consistent with their localization in Arabidopsis, rice, and wheat [11, 44].

Table 1

Characteristics of CCCH transcription factor gene family in barley.
Gene Name	Gene ID	Protein Length (aa)	Isoelectric Point	Molecular Weight (kDa)	Subcellular Location	Grand Average of Hydropathicity	ESTs Hit
HvC3H1	HORVU.MOREX.r2.1HG0072390	304	9.15	34.276	Nucleus	-1.163	11
HvC3H2	HORVU.MOREX.r2.1HG0074950	402	9.57	42.999	Nucleus	-0.305	2
HvC3H3	HORVU.MOREX.r2.1HG0074970	327	9.42	35.232	Nucleus	-0.389	6
HvC3H4	HORVU.MOREX.r2.1HG0078680	1456	5.13	160.373	Nucleus	-0.84	14
HvC3H5	HORVU.MOREX.r2.2HG0091490	697	6.26	73.624	Chloroplast	-0.384	25
HvC3H6	HORVU.MOREX.r2.2HG0140180	341	10.15	38.974	Nucleus	-1.009	22
HvC3H7	HORVU.MOREX.r2.2HG0166340	304	9.38	31.487	Nucleus	-0.623	33
HvC3H8	HORVU.MOREX.r2.2HG0176080	695	5.49	77.804	Nucleus	-1.149	11
HvC3H9	HORVU.MOREX.r2.3HG0196310	379	7.51	40.767	Nucleus	-0.53	57
HvC3H10	HORVU.MOREX.r2.3HG0200320	224	9.13	24.591	Nucleus	-0.81	0
HvC3H11	HORVU.MOREX.r2.3HG0200330	232	6.17	24.956	Nucleus	-0.616	1
HvC3H12	HORVU.MOREX.r2.3HG0210880	467	7.85	49.838	Nucleus	-0.493	53
HvC3H13	HORVU.MOREX.r2.3HG0210900	426	8.07	4.502	Nucleus	-0.658	16
HvC3H14	HORVU.MOREX.r2.3HG0225190	500	8.67	54.722	Nucleus	-0.648	32
HvC3H15	HORVU.MOREX.r2.3HG0228250	384	8.6	42.042	Nucleus	-0.419	9
HvC3H16	HORVU.MOREX.r2.3HG0230880	281	9.59	32.735	Chloroplast Nucleus	-1.177	64
HvC3H17	HORVU.MOREX.r2.3HG0258540	435	8.82	47.4	Nucleus	-0.564	61
HvC3H18	HORVU.MOREX.r2.4HG0318770	299	8.3	32.522	Nucleus	-0.595	2

Table 1

Characteristics of CCCH transcription factor gene family in barley (Continued)
Gene Name	Gene ID	Protein Length (aa)	Isoelectric Point	Molecular Weight (kDa)	Subcellular Location	Grand Average of Hydropathicity	ESTs Hit
HvC3H19	HORVU.MOREX.r2.4HG0325540	326	7.14	36.231	Nucleus	-1.003	11
HvC3H20	HORVU.MOREX.r2.5HG0362710	691	9.39	78.976	Nucleus	-1.218	5
HvC3H21	HORVU.MOREX.r2.5HG0374920	509	6.39	55.923	Nucleus	-0.702	23
HvC3H22	HORVU.MOREX.r2.5HG0377520	442	8.78	47.516	Nucleus	-0.504	20
HvC3H23	HORVU.MOREX.r2.5HG0407060	314	9.25	36.792	Chloroplast Nucleus	-1.241	36
HvC3H24	HORVU.MOREX.r2.5HG0429150	752	7.39	85.417	Nucleus	-1.256	5
HvC3H25	HORVU.MOREX.r2.6HG0475520	211	9.17	23.087	Nucleus	-0.813	0
HvC3H26	HORVU.MOREX.r2.6HG0475540	358	8.63	38.145	Chloroplast	-0.34	0
HvC3H27	HORVU.MOREX.r2.6HG0475570	308	9.49	31.997	Chloroplast	-0.541	39
HvC3H28	HORVU.MOREX.r2.6HG0505660	359	6.66	40.211	Nucleus	-0.46	75
HvC3H29	HORVU.MOREX.r2.6HG0515160	1001	8.81	110.273	Nucleus	-1.121	16
HvC3H30	HORVU.MOREX.r2.6HG0526270	647	5.23	71.397	Nucleus	-1.069	45
HvC3H31	HORVU.MOREX.r2.7HG0560290	489	9.46	55.275	Nucleus	-0.79	59
HvC3H32	HORVU.MOREX.r2.7HG0579580	363	6.85	38.791	Nucleus	-0.706	20
HvC3H33	HORVU.MOREX.r2.7HG0600900	297	9.64	30.833	Chloroplast Nucleus	-0.541	46
HvC3H34	HORVU.MOREX.r2.7HG0602740	407	8.56	44.684	Nucleus	-0.954	26
HvC3H35	HORVU.MOREX.r2.7HG0607870	375	9.32	42.524	Nucleus	-1.372	6
Supplementary materials

Ccch Domain Structure Analysis Of Hvc3hs

To evaluate the degree to which CCCH proteins in barley are evolutionarily conserved, the full-length proteins were used to characterize the domain organization of HvC3Hs. Significant differences in the domain organization of HvC3Hs were observed. A total of five domain organizations of 77 CCCH motifs (C-X_7-10-C-X_4-5-C-X₃-H) were identified, with an average of 2.2 CCCH motifs per protein. CCCH proteins have been shown to have one to six CCCH motifs (Figure 1) [11, 22, 49, 50], and the same pattern was observed in our study. However, no HvC3H proteins contained four CCCH motifs. Approximately 77.14% (27) of the HvC3H proteins had one or two CCCH motifs, 14.29% contained five copies, and 5.71% had six copies (Supplementary Table S5); similar patterns have also been observed in rice and Arabidopsis[51]. Although different frequencies of CCCH motifs have been identified among barley CCCH genes, two conventional CCCH motifs C-X₈-C-X₅-C-X₃-H and C-X₇-C-X₅-C-X₃-H were the two most common, suggesting that C-X_7-8-C-X₅-C-X₃-H might be ancestral to the other CCCH motifs (Supplementary Figure S1). Additionally, a total of four non-conventional CCCH zinc finger motifs, including 2 C-X₇-C-X₄-C-X₃-H, 1 C-X₉-C-X₅-C-X₃-H, and 1 C-X₁₀-C-X₅-C-X₃-H, were observed, which were previously identified to be abundant non-conventional CCCH motifs in Arabidopsis and rice. In contrast to other plants, the uncommon CCCH motifs C-X_4-6,11-15-C-X_4-5-C-X₃-H were not detected; no novel motif types were detected in barley CCCH proteins, suggesting that the CCCHs in barley are highly conserved. Aside from the CCCH zinc finger motifs, some HvC3H proteins also contained several other known functional domains, such as KH, RRM, and RING. Four (HvC3H7, -26, -27, and -33) and five (HvC3H1, -5, -16, -23, and -24) HvC3H members possessed the KH and RRM domains, respectively.

Phylogenetic Relationships, Gene Structure, and Conserved Domain Organization of HvC3H Genes

To determine the evolutionary relationships among HvC3Hs, a neighbor-joining (NJ) phylogenetic tree was constructed based on the alignment of the full-length CCCH protein sequences of barley. According to the criteria proposed by Wang and Peng et al. with slight modifications [11, 13], the 35 HvC3H family members within the phylogenetic tree were classified into seven subfamilies (group I to group VII) (bootstrap values >60%) (Figure 2A). Sixteen HvC3Hs formed eight sister gene pairs, seven of which possessed high bootstrap support (98%). Most of the phylogenetic clades had high bootstrap support; the relationships of some CCCH proteins (eight HvC3Hs) remained ambiguous because of their low bootstrap values at deep nodes. The number of CCCH proteins varied greatly for different subfamilies; group I and group II had seven and five members, respectively, whereas groups V and VI had only two members. We also constructed another phylogenetic tree based on the alignment of 170 CCCH proteins from Arabidopsis (68), rice (67), and barley (35) (Supplementary Figure S2). The phylogenetic tree revealed an alternating distribution of monocot and eudicot CCCH genes in most of the subfamilies. Within each subfamily, the HvC3H genes tended to cluster with their Arabidopsis and/or rice orthologs, suggesting that the orthologous genes might have evolved from a common ancestor when duplication events took place prior to species divergence. However, some groups consist of Arabidopsis and rice CCCH genes but lack barley genes. This indicates a presumed barley-specific loss of CCCH genes following the divergence of monocots and dicots.

The intron–exon gene structure not only provides important insights into the functional diversification of genes but also the evolutionary relationships within a gene family [52]. Unlike other TF family genes, which tend to lack introns, the average intron number of HvC3Hs was 3.97 (ranging from 0 to 12) (Figure 2B). In general, genes within the same subfamily had a similar structure of introns and exons. For example, genes from subfamily VII tended to be intron-less; subfamilies II, IV, and VI were nearly identical in their intron/exon lengths and structural organization. The intron–exon gene structure of subfamily I was highly variable. For example, HvC3H14 possessed 12 introns, whereas HvC3H12, HvC3H17, and HvC3H22 had six introns. HvC3H genes within subfamily I also had variable intron lengths.

To gain additional insights into the functional regions of HvC3Hs and their evolutionary relationships, the distribution of conserved motifs of HvC3Hs was visualized (Figure 2C). Consistent with the patterns in intron–exon gene structure, HvC3H proteins within the same subfamily tended to have a similar organization of motifs, and the patterns were highly variable among different phylogenetic clades. For example, the HvC3Hs in subfamily VII possessed two CCCH motifs and one RRM motif. Subfamilies IV, V, and VI had only one CCCH motif. Although the gene structure varied greatly among subfamily I members, the protein motif composition was conserved; there were five to seven CCCH motifs for each protein. The variation in gene structure and motif composition among subfamilies suggests prior sub-functionalization or neofunctionalization of these HvC3Hs.

Chromosomal Distribution And Gene Duplication

Chromosome location analysis revealed that the 35 HvC3H genes were unevenly located on the seven barley chromosomes, and chromosome 3H possessed the most abundant CCCH genes (nine HvC3Hs) (Supplementary Figure S3). By contrast, chromosomes 1H, 2H, and 4H had four, four, and two CCH genes, respectively. Chromosome 6H contained six CCCH genes, and chromosome 5H and 7H both had five. There was no significant correlation between the number of HvC3H genes and chromosome length (Pearson correlation r = 0.0278, p-value = 0.9528), demonstrating that longer chromosomes do not have more HvC3H genes.

Gene duplication is considered one of the primary drivers of gene family expansion in plants and plays an important role in the evolution of new gene functions and adaptation [53, 54]. A total of three duplicated gene pairs were identified (Figure 3). Two gene pairs (HvC3H25/HvC3H33 and HvC3H28/HvC3H31) were segmentally duplicated genes; all segmentally duplicated genes were located between chromosome 6H and 7H. HvC3H10/HvC3H11 were tandem duplicated genes and located on chromosome 3H. These duplicated genes were clustered in the same clade. Specifically, HvC3H10/HvC3H11 were clustered in subfamily III, HvC3H25/HvC3H33 were assigned to subfamily II, and HvC3H28/HvC3H31 belonged to subfamily VI. The aforementioned results indicated that the segmental and tandem duplication events might have played a role in the expansion of the HvC3H gene family.

To further evaluate the evolutionary constraints acting on HvC3Hs, the Ka/Ks ratios for each duplicated gene pair were calculated. The Ka/Ks ratios of the segmentally duplicated genes HvC3H28/HvC3H31 and HvC3H25/HvC3H33 and the tandem duplicated genes HvC3H10/HvC3H11 were all lower than 1, indicating purifying selection (Supplementary Table S6) [55]. The timing of duplication events was estimated to be approximately 22.83 – 808.04 MYA.

Syntenic relationships with six other representative species, including three monocots (Zea mays, Oryza sativa, and Triticum aestivum) and three dicots (Brassica rapa, Solanum lycopersicum, and Glycine max), were analyzed to determine the mechanisms underlying the evolution of HvC3Hs (Figure 4). A total of 63, 24, and 16 orthologous gene pairs between barley and Triticum aestivum, Zea mays, and Oryza sativa were identified, respectively. A total of 16 of 24 (66.7%) of the HvC3H genes were orthologous to three copies of CCCH genes in wheat, which might stem from the fact that the heterologous hexaploid wheat contained three distinct ancestral genomes, namely A, B, and D [56]. HvC3H11 on chromosome 3H was orthologous to four CCCH genes in wheat, which were located on chromosome 3A (one gene), chromosome 3B (one gene), and chromosome 3D (two genes). In addition, six HvC3Hs possessed two orthologous genes, and two HvC3Hs had one orthologous gene in wheat. This inconsistency might stem from gene loss and duplication events during the process of wheat polyploidization. However, the number of orthologous gene pairs between barley and three dicots (Glycine max, Brassica rapa, and Solanum lycopersicum) was ten, five, and one, respectively, which was much lower than those between barley and three monocots. This finding is consistent with the observed phylogenetic relationships between barley and these species. HvC3Hs are phylogenetically closer to CCCHs in Triticum aestivum, Zea mays, and Oryza sativa than CCCHs in Glycine max, Brassica rapa, and Solanum lycopersicum. Furthermore, we found that most of the HvC3H genes showed syntenic bias towards the chromosomes of these plants. For example, orthologous genes were on chromosome 1 in Oryza sativa, whereas the syntenic relationships in Zea mays tended to be located on chromosomes 3 and 8, suggesting that chromosomal rearrangements such as inversions or duplications might shape the organization and distribution of CCCH genes in these genomes. A total of seven HvC3H genes (HvC3H7, -11, -14, -15, -17, -18, and -31) were only syntenic with Triticum aestivum, Zea mays, and Oryza sativa, suggesting that these orthologous pairs may have formed after the divergence between monocotyledonous and dicotyledonous plants.

The Ka/Ks ratios were calculated for each orthologous gene pair to characterize selection on orthologous gene pairs. The overall Ka/Ks ratios (Triticum aestivum: 0.2733, Oryza sativa: 0.1682, Zea mays: 0.1873, Brassica rapa: 0.0149, Solanum lycopersicum: 0.0166, and Glycine max: 0.0295) of all the orthologous gene pairs were less than 1, suggesting that these HvC3Hs might have experienced strong purifying selection (Supplementary Table S7).

Cis -element Analysis of HvC3H Genes

Cis-elements play important roles in the transcriptional regulation of genes throughout the life cycle of plants. To obtain preliminary insights into the potential function and transcriptional regulation of HvC3H genes, the cis-regulatory elements in the promoter regions were analyzed. A total of 52 functional cis-elements were identified and grouped into five categories. A large number of light-responsive elements were detected in the promoter regions of HvC3Hs, which accounted for most of the putative cis-elements (Supplementary Table S8, Supplementary Figure S4). We also obtained a total of 11 types of hormone-responsive regulatory elements, such as auxin-responsive elements (AuxRR-core, TGA-box, and TGA-element), gibberellin-responsive elements (P-box, GARE-motif, and TATC-box), salicylic acid-responsive elements (TCA-element), MeJA-responsive elements (CGTCA-motif and TGACG-motif), ABA-responsive elements (ABRE), and ethylene-responsive elements (ERE). Several types of biotic and abiotic stress-related regulatory elements were observed in HvC3H promoters. Anaerobic induction elements (44 ARE and 30 GC-motif) were detected in 29 HvC3Hs, which were the most abundant cis-regulatory elements involved in the response to environmental stress. A total of 23 low temperature-responsive elements (LTR) and 28 drought-responsive elements (MBS, myeloblastosis ninding site) were detected in 15 and 17 HvC3Hs, respectively. A total of six HvC3H genes possessed wound-responsive elements (WUN-motif), suggesting that these genes might play essential roles in the response to biotic stress in plants. Additionally, eight types of plant organogenesis-related cis-elements were identified, such as the meristem expression-related element CAT-box (11 genes), zein metabolism regulation-related element O2-site (seven genes), endosperm expression-related element GCN4-motif (six genes), and seed-specific regulation element RY-element (four genes). These findings suggest that HvC3Hs might play an important role in barley plant growth and development, hormone signal transduction, and the response to biotic and abiotic stress.

Temporal-spatial And Stress-induced Expression Pattern Analysis

Analysis of tissue-/stage-specific expression profiles provided valuable insights into the potential functions of genes in plant species. To investigate the expression patterns of HvC3Hs in different tissues and developmental stages, publicly available RNA-seq data from 16 different tissues were used to estimate the expression levels of HvC3Hs, and the HvC3Hs were clustered into three major groups according to their expression levels (Figure 5). Distinct expression patterns were observed for the HvC3Hs, suggesting that these genes might have undergone significant differentiation and played various roles in particular stages during barley growth and development. The expression levels of HvC3Hs in group I were lower than those of genes in the other groups; eight genes were not expressed in most of the tissues/stages. By contrast, a total of eight genes in group II were highly expressed in all studied tissues/stages. HvC3H17 was predominantly expressed in LOD, CAR15, and EPI, whereas HvC3H1 and HvC3H12 showed high expression in INF2 and LOD, respectively. Genes in cluster III showed a medium expression level. Within this cluster, HvC3H5, -7, -9, -27, and -35 tended to be expressed in INF1 and INF2. These findings indicate that these HvC3Hs might be associated with the development of these tissues in barley. These genes might thus be interesting targets for barley breeding. The differentiation of subfamilies observed in the phylogenetic tree was not consistent with the expression clustering results, indicating that sequence similarity was not a strong predictor of expression patterns and functional similarity [57].

We analyzed the expression of HvC3Hs in response to different types of environmental stress. Under cold treatment, six HvC3Hs (HvC3H2, -3, -10, -11, -15, and -26) with FPKM values equal to 0 were excluded from the subsequent analysis (Figure 6A). The rest of the HvC3Hs were clustered into two major groups. Five HvC3H genes displayed increased expression patterns (>2.0-fold change). Among these genes, HvC3H18, HvC3H9, and HvC3H20 exhibited their highest level of expression under cold treatment, showing fold-changes of 9.21, 3.01, and 2.88, respectively. In addition, the expression of six genes was slightly down-regulated under cold treatment. Specifically, the expression of HvC3H4 decreased 0.65-fold, and HvC3H17 decreased 0.74-fold compared with the control, which was not exposed to low temperature. Salt stress induced differential expression patterns of HvC3H genes in the three root regions (Figure 6B). Compared with the unstressed control, a total of four, five, and three HvC3H genes were highly expressed in the meristematic, elongation, and maturation zones, respectively, especially HvC3H15, which exhibited a 23.16 and 32.50-fold increase in expression in the meristematic and elongation zones relative to the unstressed control. HvC3H17 was up-regulated in all tissues; its expression was increased 8.18-, 8.41-, and 2.11-fold in the meristematic, elongation, and maturation zones, respectively. Similarly, the expression of HvC3H22 and HvC3H28 was up-regulated in both the meristematic and elongation zones, and HvC3H9 was significantly up-regulated in the elongation and maturation zones. Under metal ion stress, the expression of HvC3H4, HvC3H9, and HvC3H18 was significantly up-regulated, and the up-regulation of HvC3H9 was induced by copper treatment (Figure 6C). Under zinc stress, HvC3H3 was up-regulated 2.67-fold.

Expression of HvC3Hs under Drought, Salt, Cold, and ABA Treatment by qRT-PCR Analysis

To investigate the expression of HvC3H genes in response to multiple treatments, 26 HvC3Hs were randomly subjected to qRT-PCR analysis. Under drought treatment, nine HvC3Hs were up-regulated at all time points (Supplementary Figure S5), and the expression of six of the nine HvC3Hs (HvC3H1, -5, -6, -12, -24, and -33) peaked at 24 h. The expression of HvC3H1 was approximately 54-fold larger than that of the control at 24 h. Several MBS cis-acting elements associated with drought inducibility were also identified within their promoter regions. However, no MBS cis-acting elements were detected in some genes, such as HvC3H5 and HvC3H6, suggesting that unknown regulatory elements related to drought stress might exist for these genes.

After salt treatment, the expression of HvC3H13 was suppressed compared with the control at all time points; the expression of 21 genes was significantly up-regulated, and the expression of these genes peaked at different times (Supplementary Figure S6). For example, the expression of HvC3H1 peaked at 3 h and was up-regulated 43-fold, whereas the expression of HvC3H5, -6, and -12 was initially slightly up-regulated and peaked at 24 h.

The expression levels of HvC3Hs after cold treatment were analyzed, and the expression of six genes (HvC3H4, -5, -7, -20, -25, and -27) was inhibited compared with the control; the expression of the other HvC3Hs was up-regulated at specific time points (Supplementary Figure S7). The expression of three HvC3Hs was up-regulated at 1 h (HvC3H23, -30, and -32), 3 h (HvC3H6, -28, and -33), and 6 h (HvC3H1, -17, and -22), suggesting that these HvC3Hs might primarily function in the initial stage in the response to cold injury. The expression of the other HvC3H genes peaked at 12 h or 24 h. The promoters of these genes were found to possess abundant LTR cis-elements, which might be responsible for their expression profiles under cold stress.

Plant CCCH proteins have been shown to be effective regulators of ABA-mediated stress responses [58]. qRT-PCR analysis showed that ABA treatment had a pronounced effect on the expression patterns of HvC3Hs, and a complex expression profile was observed (Supplementary Figure S8). For example, the expression of HvC3H1 was significantly up-regulated at 1 h and 3 h but down-regulated thereafter. By contrast, the expression of HvC3H24 was down-regulated before 12 h but significantly up-regulated at 24 h. With the exception of HvC3H5, -13, -20, -21, and -30, whose expression was suppressed relative to the control, the maximum expression levels of the other HvC3Hs peaked at different time points. Abundant ABRE cis-elements, which are important cis-acting elements associated with ABA-responsiveness, were detected in the promoter regions, including five ABRE cis-elements for HvC3H23, three for HvC3H33, and two for HvC3H17. Overall, our results demonstrated that ABA sensitivity might be a common feature of HvC3H genes.

Genetic Variation of CCCH Genes

We analyzed the sequence diversity of HvC3H genes at the population level based on exome-captured sequencing datasets. The single nucleotide polymorphism (SNP)-calling pipeline generated 331 high-confidence SNPs, 172 of which were in HvC3H21, followed by HvC3H14 (42) and HvC3H34 (23) (Supplementary Table S9; Supplementary Table S10). Most HvC3H-related SNPs were located within the intron regions (318); the rest of the SNPs were non-synonymous (8) and synonymous (5) SNPs, with a non-synonymous to synonymous ratio of 1.6. There were 277 InDels ranging from 1 to 55 bp in length, and short InDels 1 to 4 bp (76.90%) in length were more common than long InDels (Supplementary Table S11). Similarly, most HvC3H-related InDels were located in introns, which might be explained by the fact that the reading frame-independent variants were under weaker negative selection than frame-change variants.

To investigate the relatedness among the landraces and wild barley accessions worldwide, we carried out principal component analysis using HvC3H-related SNPs (Figure 7A,B; Supplementary Table S12). The first principal component was correlated with the biological differentiation between landrace from wild barley and explained 25.16% of the total genetic variance; the second and third principal components captured 5.09% and 5.00% of the genetic variance, respectively, and revealed geographical differentiation in barley accessions. These patterns were consistent with the topology of the NJ tree (Figure 7C). The phylogenetic tree revealed genetically divergent clusters associated with the contrast between barley wild accessions versus landraces rather than their geographical origins. ADMIXTURE analysis confirmed these findings (Figure 7D). When K=2, two groups coinciding with landraces and wild barley were observed. Increasing K to 4 provided additional insights. Within barley landraces, we detected two geographically distributed components from Europe and Africa, whereas the rest of the landraces from Mediterranean areas displayed signs of genetic admixture. Within wild barley accessions, accessions from the Southern and Northern Levant regions formed two distinct groups.

Genetic Diversity and Haplotypes of HvC3Hs in Wild and Domesticated Barley Populations

Population-based nucleotide diversity was calculated to assess the occurrence of prior genetic bottlenecks of HvC3H genes during barley domestication. The genetic diversity of HvC3Hs was further analyzed using exome-captured resequencing datasets; a total of 51 barley landraces and 95 wild barley accessions were included in the analysis. The total genetic diversity of HvC3H genes decreased by ~34.94% from the wild (π = 0.07622) to domesticated (π = 0.04959) barley population (Supplementary Figure S9).

A haplotype consists of a group of closely linked SNPs located in a specific chromosome region that determine the same trait. We constructed the haplotype network for each HvC3H gene using their SNPs. A total of 529 haplotypes belonging to 22 HvC3H genes were observed, with an average of 24.05 haplotypes per gene (Figure 8). Specific haplotypes represented in more than half of wild or landrace populations were defined as dominant haplotypes. Four HvC3H genes (HvC3H14, -21, -22, and -30) had no dominant haplotype, whereas 13 HvC3H genes had the same dominant haplotype in both wild and landrace populations. Nevertheless, clear genetic differentiation in haplotypes between wild and domesticated barley accessions was observed. HvC3H20 in wild barley mainly had the AAAAGGGGGGTTTTGGCC haplotype, but domesticated barley mainly had the AAAAGGGGAATTTTGGCC haplotype. The dominant haplotype in wild barley was AAGTTTTCCCTTGGGGAA, but haplotype AAGTTTTCTTTTGGGGTT was the most common in domesticated barley. Some rare haplotypes were also observed for specific HvC3H genes, such as HvC3H32, HvC3H34, and HvC3H35. The appearance of novel allelic variants greatly increased the degree of haplotype polymorphism of HvC3Hs. The rare haplotypes were mainly observed in the wild barley group, which was consistent with the results of the genetic diversity analysis indicating that these genes experienced a severe genetic bottleneck during barley domestication and that the haplotype diversity decreased in domesticated barley relative to the wild population. By contrast, the haplotype polymorphism of HvC3H14 and HvC3H20 in domesticated barley was higher than that in wild barley. We speculate that the process of artificial breeding has introduced various novel alleles and increased the degree of haplotype polymorphism.

Characterization of CCCH Genes in Barley

CCCH genes comprise a distinct zinc finger TF gene family that regulates gene expression through RNA binding and processing. CCCH domain-containing proteins are involved in various biological processes of plant growth, development, and adaptation. Given their important regulatory roles, many CCCH zinc finger family genes have been identified and functionally characterized in model plants and crops [45]. Barley is the fourth most abundant cereal crop worldwide in terms of both acreage and tonnage harvested, and its adaptability to a wide range of cultivation conditions might stem in part from its ability to withstand harsh environments [46]. However, there is limited information on CCCH proteins in agriculturally important cereal crops. The large genome size and high content of transposon elements have inhibited the assembly of the barley genome. With the advent of novel sequencing technologies (e.g., 10X genomics, chromosome conformation capture sequencing (Hi-C), and Bionano optical mapping) and computational algorithms (e.g., TRITEX workflow), barley genome assemblies, from the first draft reference genome to its subsequent revisions (Morex V1 and V2) have undergone multiple rounds of improvement [46–48]. The most recently updated barley reference genome assembly (Morex V3) based on PacBio HiFi sequence has near-complete coverage and high performance in the repeat-rich intergenic regions. Because the same transcriptomic resources were used for the previous genome assembly, the gene models of Morex V3 displayed near-complete alignments (≥95% alignment coverage, ≥99% identity) with the V2 assembly in the gene space [59]. Presently, we have determined the expression profile and characterized genetic variation using Morex V2 as the reference genome. Given the consistency in the gene models and computational calculations, comprehensive identification and characterization of HvC3Hs herein were performed using the barley Morex V2 assembly, which was considered to be a qualified reference genome for functional gene analysis [60–62].

A BLAST search [11, 41], HMM search [44, 45], or both [13, 35] were used to identify candidate CCCH genes in various species. Here, both search tools were adopted in our study based on the following criteria: BLAST e-value<1e-5 and HMMER e-value<0.001. The methodology was further validated by the SMART, NCBI-CDD, and Pfam online databases. Our results enhance our understanding of HvC3Hs. We identified 35 high-confident CCCH zinc finger genes within the barley genome through a comprehensive search. We found that the number CCCH proteins in barley was approximately half of those identified in Arabidopsis (67), rice (68), maize (68), and grape (69); a third of those in poplar (91), Brassica rapa (103), and switchgrass (103); and a similar number to those in Medicago truncatula (34), suggesting that species origin and genome size are not directly associated with the number of CCCH genes.

Plant CCCH proteins usually contain one to six CCCH motifs [11]. Similar to the other species, each barley CCCH protein has at least one CCCH motif, and the most common number of CCCH motifs was one (17 HvC3Hs) and two (10 HvC3Hs). Although plant CCCH proteins displayed a wide spacing pattern similar to C-X_4-15-C-X_4-6-C-X₃-H, two conventional CCCH motif types, C-X₇-C-X₅-C-X₃-H and C-X₈-C-X₅-C-X₃-H, were the most common in different species, suggesting that the consensus sequence C-X_7–8-C-X₅-C-X₃-H might be ancestral to the other CCCH motifs [35]. The CCCH motifs in barley showed similar patterns to the highly conserved consensus sequences, indicating that these domains might be important for the function of the C3H proteins. With the exception of the CCCH motifs, several other functional domains, such as KH, RRM, and RING, were also detected in barley CCCH proteins. Some studies indicate that KH and RRM are common RNA-binding domains in eukaryotes, and KH and RRM domain-containing proteins are involved in various aspects of RNA metabolism, such as pre-mRNA splicing, nucleocytoplasmic transport of mRNAs, polyadenylation, and transcriptional control [63, 64]. These results suggest that the four CCCH proteins harboring these specific domains might also function as RNA-binding proteins and play a role in RNA processing. In addition, HvC3H31 contained a RING domain, which suggests that it plays a key role in the response to various types of abiotic stress [65]. These additional domains might contribute to the neo-functionalization of HvC3H genes.

Expansion Patterns Might Affect the Gene Family Size of HvC3Hs

The 35 barley CCCH genes were clustered into seven subfamilies based on the phylogenetic analysis. Genes within the same subfamily tend to be similar in structure and domain composition, which supported the observed topology. The number of clades of HvC3Hs was slightly lower than the number of clades of HvC3Hs in Arabidopsis (11) and rice (8). Some genes could not be assigned to any groups because of their low sequence similarity. Therefore, a combined NJ tree was constructed to evaluate their evolutionary relationships using CCCH proteins from barley, rice and Arabidopsis. Most of the HvC3H genes displayed closer relationships with their orthologs than their paralogs, suggesting that these CCCH proteins might have originated from the common ancestor of these plants and then expanded independently in different species. Moreover, rice and/or Arabidopsis-specific clades without barley genes were also observed in our phylogenetic analysis, suggesting that a presumed barley-specific loss of CCCH genes may have occurred after the divergence between barley and other species.

A gene family is a group of similar genes that originated from a common ancestor, and gene duplications, such as segmental, tandem, whole-genome, or polyploidization duplications, contribute greatly to the rapid expansion and evolution of genes in a gene family [66–68]. In barley, duplication events were a dominant force contributing to the expansion of some gene families. For example, ten and seven pairs of HvGRASs have undergone segmental and tandem duplications, accounting for approximately 54.83% of the total number of HvGRAS genes [69]. Segmental duplication was the main force driving the expression of HvbHLH genes (42.55%) [70], whereas tandem duplication was largely responsible for the expansion of HvNRT2.1 (80.00%) [71]. Previous studies have indicated that duplication events are responsible for the expansion of the CCCH gene family. In wheat, allopolyploidization duplication stems from the combination of three distinct ancestral genomes, viz. A, B, and D, which significantly expands the number of CCCH gene members. Synteny analysis identified 63 gene pairs (comprising 24 HvC3Hs and 63 TaC3Hs) in the barley and wheat genomes. A total of 15 HvC3H genes were orthologous to three TaC3H genes corresponding to the three sets of sub-genomes, which confirmed the hexaploidy duplication of the wheat genome. Additionally, some CCCH genes of diverse species have undergone segmental and/or tandem duplications [11, 13, 38]. By contrast, only three gene pairs (one tandem duplication and two segmental duplications) were involved in the duplication of HvC3Hs, which was far less than the number of gene pairs in other species. Therefore, despite its large genome size, barley had fewer CCCH gene members, which might be attributed to the fact that its HvC3Hs have undergone fewer duplication events.

HvC3H s Might Play a Role in Plant Growth, Abiotic Stress, and Phytohormone Responses

CCCH proteins are an important class of zinc finger TFs that are involved in various plant growth processes and responses to stress, but their potential functions in barley are rather limited. Cis-acting regulatory elements within the promoter region are involved in the spatiotemporal regulation of gene expression [72]. In our current study, various cis-elements in the promoter regions of HvC3Hs were identified and divided into five groups based on their functions: structure and composition, hormone induction, plant growth development/tissue-specific, stress, and light response. These results indicated that HvC3Hs mediate various functions in multiple biological processes. Given that prediction of HvC3H cis-elements was carried out based on a bioinformatics approach, experiments need to be conducted to validate the function of predicted HvC3H genes.

The biological function of CCCHs has been well-documented in various plants, especially model plants [73]. The diversity of expression patterns of HvC3Hs in specific tissues or at specific stages provides insight into their possible functions at specific developmental stages. For example, HvC3H7, a KH domain-containing CCCH zinc finger gene, tended to be expressed highly in young inflorescences. In plants, the KH domain-containing genes FLK (Flowering Locus KH Domain) and PEP (PEPPER) regulate flowering by negatively and positively modulating the expression of FLC, respectively [74, 75]. The homologs of HvC3H17 in Arabidopsis are KHZ1 and KHZ2, which are two non-tandem CCCH zinc finger and K-homolog domain proteins that act as repressors of the splicing efficiency of FLC pre-mRNA and redundantly promote flowering and senescence [76, 77], suggesting that HvC3H17 might be involved in the transition from vegetative to reproductive development in barley. HvC3H15 was expressed highly in NOD, PAL, and LEM. Its orthologous gene AtC3H14 is the direct target of the MYB domain TF MYB46 and a master switch for cell elongation in Arabidopsis [78, 79], which suggests that HvC3H15 might have similar functions in regulating plant organogenesis in barley.

Given their sessile nature, terrestrial plants are often exposed to various adverse challenges such as drought, high salinity, extreme temperatures, and pathogen infection [80]. To adapt to such environmental conditions, plants have evolved complicated signal transduction pathways to perceive stress signals and coordinate plant growth and development [81]. Several regulatory components, including receptors, TFs, protein kinases, and plant hormones that simulate or repress other components [82], are needed to construct interconnected and sophisticated regulatory networks. To obtain preliminary insight into the potential functions of HvC3Hs in response to environmental stress, the expression patterns of HvC3Hs under cold, salt, and metal ion treatments were characterized based on publicly available RNA-seq datasets.

Soil salinization affects seed germination and crop growth and development and has become a major environmental barrier for agricultural production globally [83]. The expression of CCCH proteins has been reported to be induced by salt stress, indicating that CCCH proteins are involved in salt stress tolerance in plants [84]. For example, Arabidopsis AtSZF1 and AtSZF2, two closely related CCCH zinc finger genes, negatively regulate the expression of salt-responsive genes and modulate tolerance to salt stress [85]. In rice, OsTZF1 negatively regulates leaf senescence under salt conditions and confers stress tolerance by delaying stress-responsive phenotypes, possibly through post-transcriptional control of the RNA metabolism of salt stress-responsive genes [17]. In barley, HvC3H9 showed the highest homology among these genes, and its expression was significantly induced under salt stress according to both RNA-seq and qRT-PCR analysis, suggesting that HvC3H9 might have similar functions in response to salinity stress.

Recently, several lines of evidence from multiple approaches have shown that CCCH proteins directly increase the drought tolerance of plants [84]. In our study, HvC3H18 was the most up-regulated gene at 1 h, 6 h, and 12 h under drought stress. Homology analysis revealed that its orthologous gene OsC3H47 is involved in drought tolerance through its elevated sensitivity to ABA [25]. Another CCCH-tandem zinc finger protein OsTZF5, whose optimal BLAST hit was HvC3H9 in barley, promoted both drought avoidance and drought tolerance in rice [26]. Several MBS cis-acting elements associated with drought inducibility within the promoter regions of HvC3H18 and HvC3H9 genes were predicted, suggesting that these genes might play a potential role in the response to drought stress.

Low temperature greatly limits the geographical distribution, growth, and development, as well as the yield, quality, and storage properties of crops [86]. However, few studies have examined the molecular regulation of CCCHs in response to environmental temperatures. In Chrysanthemum, DgC3H1 enhances low temperature tolerance via regulation of the plant ROS system and the expression of downstream cold-related genes. However, the expression of HvC3H27, which is orthologous to DgC3H1, was not induced by cold stress for both RNA-seq and qRT-PCR analysis. Moreover, there was no cis-acting element, such as LTR, involved in low temperature responsiveness within the promoter region of HvC3H27 [31]. These results indicate that HvC3H27 might have functionally diversified in barley. Compared with the control, the expression of HvC3H9 changed approximately 3.01-fold under cold treatment for RNA-seq. qRT-PCR analysis also revealed significant up-regulation at 1 h, 3 h, and 6 h. Its orthologous gene in switchgrass PvC3H72 acts as an added signaling component by regulating the ICE1-CBF-COR regulon and ABA-responsive genes [30]. Overall, several candidate CCCH genes that could provide targets for subsequent genetic isolation and functional investigation in barley as well as in other cereal crops were identified. Experiments are needed to determine the functions of HvC3Hs.

Genetic Variation and Haplotype Polymorphism of HvC3Hs during the Domestication of Barley

SNPs are the most common type of genomic variants in the genome [87]. They have become an increasingly invaluable and powerful molecular marker for the high-resolution genetic mapping of traits, linkage disequilibrium analysis, and association studies [88, 89]. High densities of SNPs also provide invaluable resources for genome mapping, the generation of ultra-high-density genetic maps, the identification of haplotypes near a locus of interest, and the map-based positional cloning of genes. The application of next-generation sequencing technologies that increase the quantity of DNA and reduce the cost of sequencing has facilitated the discovery of DNA polymorphisms in silico [90]. In recent years, SNPs derived from resequencing data have been used to investigate the genetic relationships between wild and domesticated barley [91–94]. Genetic variation in HvC3H genes in barley populations was determined using publicly released exome capture datasets [93]. We obtained a large number of high-confident HvC3H-related SNPs and InDels in 95 landraces and 51 wild barley accessions. Most of the variants were enriched within the introns, possibly because of weak selection on the non-coding regions. The mutations also displayed significant base bias of C/T (39.27%) and A/G (32.33%). The overall transition/transversion (Ts/Tv) ratio was 2.52, which indicated the greater importance of transitions compared with transversions.

We also characterized the evolutionary history and genetic divergence of HvC3H genes in wild and domesticated barley populations. Two genetically divergent populations associated with domestic versus wild barley rather than barley populations with different geographic origins were observed, which was consistent with the deep phylogenetic split between wild and domesticated barley of HvmTERFs [60]. The greater effect of human-driven selection on the genomic regions of HvC3Hs relative to natural selection was confirmed by the genetic diversity analysis. The nucleotide diversity of HvC3Hs displayed a significant reduction characteristic of a genetic bottleneck between domesticated and wild barley. The estimated reduction by ~34.94% from wild to domesticated barley was higher compared with that estimated for barley adaptive genes (22.5%) [95], housekeeping genes (20%) [86], and disease resistance genes (18.2%) [95]. The estimated reduction was also much higher when compared with that reported at the whole-genome level (reduction of 22%) [93]. We concluded that these genes have undergone a genetic bottleneck from wild barley to cultivated barley and might be domestication-related genes.

Haplotype reconstruction and characterization of the haplotype distribution can shed light on the differentiation of important genes and the processes underlying their evolution [96]. Haplotype networks indicated that the haplotype composition of the HvC3H family in wild barley was rich compared with that in cultivated barley, which indicated that initial human selection was focused on the retention of specific haplotypes by screening out a large number of undesirable haplotypes during the process of domestication. Domestication is a plant–animal coevolutionary process driven by the human demands for certain morphological and physical characteristics of crops [97]; this results in a severe genetic bottleneck that reduces the nucleotide diversity of alleles [98]. Wild barley populations possessed specific haplotypes that were absent in domesticated populations, which suggests that the genetic traits controlled by HvC3Hs in domesticated barley could be enriched. Characterization of HvC3H haplotypes will not only provide new insight into the functional divergence between cultivated and wild barley but also help establish associations between genetic variants and important agronomic traits so that they can be used as molecular markers.

Despite the importance of CCCH genes in plant growth and development, the response to biotic and abiotic stress, and disease resistance, the precise roles of CCCH gene family members in barley have not yet been elucidated. Here, our genome-wide identification and characterization of HvC3H genes revealed the physical-chemical properties, phylogeny, intron–exon structure, and expansion patterns of these genes. The results of the expression profiling analysis suggest that HvC3H members might be associated with multiple physiological, metabolic, and developmental processes, especially in response to various types of biotic stress. The population structure based on the most recently released exome capture sequencing data revealed a deep phylogenetic split in the HvC3Hs between wild and domesticated barley. The nucleotide and haplotype diversity of most HvC3Hs indicated that these genes have undergone a severe genetic bottleneck during the transition from wild relatives to domesticated barley populations. Overall, these findings will aid future studies examining the evolutionary history of HvC3Hs as well as functional studies of candidate HvC3H genes for molecular breeding in barley.

ABA, Abscisic Acid; ABRE, Abscisic Acid Responsive Elements; ARE, Anaerobic Induction Elements; BLAST, Basic Local Alignment Search Tool; ERE, Ethylene Responsive Element; EST, Expressed Sequence Tag; FPKM, Fragments Per Kilobase per Million; GA, Gibberellins GRAVY, Grand Average of Hydropathicity; GSDS, Gene Structure Display Sever; GTF, Gene Transfer Format; HMM, Hidden Markov Model; HvC3H, Barley CCCH genes; IBSC, International Barley Sequencing Consortium; II, Instability Index; InDel, Insertion-Deletion; iTOL, Interactive Tree of Life; Ka, Non-synonymous substitution; Ks, Synonymous substitution; LTR, Low-Temperature Responsive; MAF, Minor Allele Frequency; MBS, Myeloblastosis Binding Site; MEME, Multiple Expectation Maximization for Motif Elicitation; MW, Molecular Weight; MYA, Million Years Ago; NCBI-CDD, National Center for Biotechnology Information - Conserved Domains Database; NJ, Neighbor-Joining; PCA, Principal Component Analysis; pI, Isoelectric Point; qRT-PCR, Quantitative Real-time PCR; ROS, Reactive Oxygen Species; SMART, Simple Modular Architecture Research Tool; SRA, Sequence Read Archive; SNP, Single Nucleotide Polymorphism; TF, Transcription Factor; π, Nucleotide diversity.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Author Contributions

YL and LC designed and supervised the project. QA and WP performed the data analysis, WP and YZ performed the experiments. QA and LC drafted the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This research was mainly funded by the National Natural Science Foundation of China (Grant No. 32060458), Jiangxi Natural Science Foundation (Grant No. 20202BAB215002), and partially supported by the Science and Technology Research Project of Jiangxi Provincial Department of Education (Grant No. GJJ180241). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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No competing interests reported.

FigureS1.jpg
Fig. S1. Sequence logos for common CCCH zinc finger motifs in different species. (A) C-X8-C-X5-C-X3-H motifs of Hordeum vulgare, Arabidopsis thaliana, Oryza sativa and Zea mays. (B) C-X7-C-X5-C-X3-H motifs of Hordeum vulgare, Arabidopsis thaliana, Oryza sativa and Zea mays.
FigureS2.jpg
Fig. S2. Phylogenetic tree of CCCH protein in barley, rice and Arabidopsis.
FigureS3.jpg
Fig. S3. Localization of 35 HvC3H genes on 7 barley chromosomes. The scale on the left is in mega-bases.
FigureS4.jpg
Fig. S4. Distribution of important cis-acting regulatory elements in the promoter regions of HvC3H genes.
FigureS5.jpg
Fig. S5. qRT-PCR analysis of HvC3H genes in response to drought treatment. Error bar represent the standard error of the mean. One asterisk (*) and double asterisk (**) indicates 0.05 and 0.01 significance level, respectively.
FigureS6.jpg
Fig. S6. qRT-PCR analysis of HvC3H genes in response to salt treatment. Error bar represent the standard error of the mean. One asterisk (*) and double asterisk (**) indicates 0.05 and 0.01 significance level, respectively.
FigureS7.jpg
Fig. S7. qRT-PCR analysis of HvC3H genes in response to cold treatment. Error bar represent the standard error of the mean. One asterisk (*) and double asterisk (**) indicates 0.05 and 0.01 significance level, respectively.
FigureS8.jpg
Fig. S8. qRT-PCR analysis of HvC3H genes in response to ABA treatment. Error bar represent the standard error of the mean. One asterisk (*) and double asterisk (**) indicates 0.05 and 0.01 significance level, respectively.
FigureS9.jpg
Fig. S9. Nucleotide diversity analysis of HvC3Hs in wild and domesticated barley populations. The nucleotide diversity was estimated based on all the 331 HvC3H-related SNPs. The boxplot was drawn using the R package ggplot2. The horizontal line inside the box refers to the median of the nucleotide diversity distribution, the top and bottom line of the box corresponds to the first and third quartiles, and the scattered points outside the box can be considered as outliers.
TableS1.xlsx
Table S1. Sample information and accession number of the RNA-seq data downloaded from the NCBI SRA database.
TableS2.xlsx
Table S2. The primer sequences used in qRT-PCR analysis in this experiment.
TableS3.xlsx
Table S3. Passport information of 95 landraces and 51 wild barley accessions.
TableS4.xlsx
Table S4. Validation of the CCCH domains using the NCBI-CDD, SMART and HMMER online databases.
TableS5.xlsx
Table S5. Detailed information of CCCH protein motifs in barley.
TableS6.xlsx
Table S6. Duplicated HvC3H gene pairs and Ka/Ks ratios across the whole genome in barley.
TableS7.xlsx
Table S7. The Ka/Ks ratios between barley and other six species (Triticum aestivum, Oryza sativa, Zea mays, Glycine max, Brassica rapa, Lycopersicon esculentum).
TableS8.xlsx
Table S8. Characteristics of cis-acting regulatory elements in the promoter region of HvC3Hs.
TableS9.xlsx
Table S9. The genetic variation information (VCF format) of HvC3H-related SNPs across the 51 wild and 95 domesticated barley accessions.
TableS10.xlsx
Table S10. SNP distribution, haplotype and genetic diversity of the 22 HvC3Hs in wild and domesticated barley.
TableS11.xlsx
Table S11. Genomic distribution and putative function of HvC3H-related InDels within various genomic regions.
TableS12.xlsx
Table S12. Tracy-Widom (TW) test for the first ten eigenvalues of the PCA based on HvC3H-related SNPs.

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CCCH Zinc Finger Genes in Barley: Genome-Wide Identification, Evolution, Expression and Haplotype Analysis

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Genome-wide Identification and Characterization of CCCH Proteins in Barley

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