Characterization and chromosomal localization of ZF-HD genes in maize
The amino acid sequence of the ZF-HD dimer domain was used as a query to identify maize ZF-HD proteins from a local database, the candidate proteins lacking a ZF-HD domain were removed using the Pfam and InterPor databases. A total of 24 ZF-HD genes were accordingly detected in the maize genome. The predicted ZF-HD genes were named based on gene order on the chromosomes. (Table 1; Table S1 and S2).
Table 1
The characterization of ZF-HD proteins in maize
Gene ID | Gene name | Chromosome | Location (bp) | Subcellular localization | Protein properties |
Length (aa) | MW (kDa) | PI |
Zm00001d031840 | ZmZHD1 | 1 | 203354978–203355904 | Periplasmic | 308 | 31556.2 | 6.59 |
Zm00001d032175 | ZmZHD2 | 1 | 215598100–215599221 | Periplasmic | 373 | 38662.7 | 7.21 |
Zm00001d032177 | ZmZHD3 | 1 | 215697430–215697789 | Periplasmic | 119 | 12696.5 | 8.57 |
Zm00001d033791 | ZmZHD4 | 1 | 273799885–273800580 | Extracellular | 231 | 24075.7 | 8.35 |
Zm00001d003645 | ZmZHD5 | 2 | 51732292–51733194 | Periplasmic | 300 | 31130.8 | 6.72 |
Zm00001d005757 | ZmZHD6 | 2 | 186640920–186642005 | Periplasmic | 361 | 37714.3 | 7.73 |
Zm00001d005931 | ZmZHD7 | 2 | 193254454–193255314 | Periplasmic | 286 | 29912.6 | 6.96 |
Zm00001d041417 | ZmZHD8 | 3 | 118230787–118232606 | Cytoplasmic | 221 | 24695.4 | 6.70 |
Zm00001d041780 | ZmZHD9 | 3 | 137588464–137590201 | Periplasmic | 349 | 36904.5 | 8.55 |
Zm00001d044662 | ZmZHD10 | 3 | 234296141–234296843 | Extracellular | 100 | 10401.4 | 8.93 |
Zm00001d049000 | ZmZHD11 | 4 | 12135060–12136322 | Periplasmic | 420 | 43813.4 | 7.75 |
Zm00001d050443 | ZmZHD12 | 4 | 88621453–88622553 | Periplasmic | 366 | 38269.1 | 8.63 |
Zm00001d050452 | ZmZHD13 | 4 | 89089123–89089506 | Periplasmic | 127 | 13351.2 | 7.53 |
Zm00001d051573 | ZmZHD14 | 4 | 163144095–163145435 | Periplasmic | 446 | 48227.8 | 6.64 |
Zm00001d052395 | ZmZHD15 | 4 | 189092400–189092698 | Cytoplasmic | 86 | 9111.0 | 8.29 |
Zm00001d013409 | ZmZHD16 | 5 | 10540717–10541439 | Extracellular | 240 | 24975.7 | 7.16 |
Zm00001d017784 | ZmZHD17 | 5 | 206700640–206701893 | Periplasmic | 417 | 44774.1 | 6.56 |
Zm00001d039116 | ZmZHD18 | 6 | 170344563–170350619 | Periplasmic | 917 | 97551.7 | 8.74 |
Zm00001d020459 | ZmZHD19 | 7 | 115920523–115920858 | Cytoplasmic | 111 | 12390.1 | 9.10 |
Zm00001d020460 | ZmZHD20 | 7 | 116166449–116167561 | Periplasmic | 370 | 38611.5 | 8.21 |
Zm00001d020774 | ZmZHD21 | 7 | 132034507–132035328 | Periplasmic | 273 | 29154.5 | 7.07 |
Zm00001d009674 | ZmZHD22 | 8 | 75385981–75386700 | Periplasmic | 239 | 25995.4 | 9.47 |
Zm00001d023286 | ZmZHD23 | 10 | 2213583–2213879 | Periplasmic | 98 | 10100.1 | 6.87 |
Zm00001d023289 | ZmZHD24 | 10 | 2219925–2220221 | Periplasmic | 98 | 10113.1 | 7.59 |
The identified maize ZF-HD genes encoded proteins with lengths ranging from 86 to 917 amino acids, Mw from 9111 to 97551.7 kDa, and pI from 6.56 to 9.47. The distribution of the 24 maize ZF-HD genes on nine of the ten maize chromosomes was uneven (Fig. 1). Chromosome 4 harbored five ZF-HD genes, whereas only one gene was detected on each of chromosomes 6 and 8. Among the other genes, four were located on chromosome 1, three each on chromosomes 2, 3, and 7, and two each on chromosomes 5 and 10. Phylogenetic analysis of the maize ZF-HD genes revealed eight sister pairs showing a close relationship, namely, ZmZHD7/21, ZmZHD14/17, ZmZHD22/18, ZmZHD4/16, ZmZHD13/3, ZmZHD12/2, ZmZHD6/20, and ZmZHD23/24 (Fig. 2). Three pairs, (ZmZHD14/17, ZmZHD22/18, and ZmZHD4/16), detected in the same duplicated chromosomal blocks, were determined to have undergone segmental duplications. In addition, a further gene pair, ZmZHD23 and ZmZHD24, were located in the same duplicated chromosomal blocks. The physical distance between these two genes was only 6.3 kb, which indicates that this gene cluster has undergone tandem duplication. It is noteworthy that three of the sister pairs (ZmZHD13/3, ZmZHD12/2, and ZmZHD6/20) were found to be located in very close proximity to the duplicated chromosomal blocks. Moreover, they have similar gene structures and a close evolutionary relationship, indicating that they are putative segmentally duplicated genes. These findings tend to indicate that segmental duplication is an important mechanism in maize ZF-HD gene family expansion.
The chromosome numbers are indicated at the top of each bar. The segmentally duplicated genes are connected by red lines
Motifs were searched for with MEME using the amino acid sequences of maize ZF-HD genes. The constructed phylogenetic tree is shown on the left side of the figure. Motif sizes are indicated at bottom of figure. Different colors indicate motifs 1 to 15 at the bottom of the figure. For motif details, refer to Table 2.
Table 2
Motif sequences identified by MEME and annotate by InterPro
Motifs | Multilevel consensus sequences Predicted domains |
1 | WRYRECLKNHAASIGGHAVDGCCEFMPSG | ZF-HD homeobox protein, Cys/His-rich dimerisation domain |
2 | KKRFRTKFTQEQKERMLEFAERLGWRIQK | Homeobox domain-like |
3 | DEDIVQQFCDEIGVKRQVFKVWMHNNKH | Homeobox domain-like |
4 | IDALKCAACGCHRNFHRKEVE | ZF-HD homeobox protein, Cys/His-rich dimerisation domain |
5 | HHHHFSPYYRTPAGYFFHQ | Null* |
6 | EDDTDNDDEGSDYEEERSLSY | Null* |
7 | MEAMDVKYKPVMFPNGAGFKKPK | Null* |
8 | MDFDDHDDGDEEM | Null* |
9 | MMKRMIILRRCHPI | Null* |
10 | FNINGWA | Null* |
11 | QQQPQQ | Null* |
12 | IPLLLPPPHPHY | Null* |
13 | SNGGTATESSSEERG | Null* |
14 | PMPVSSSYDAP | Null* |
15 | LMDSAAFSRPLLPPNSSLVMQPPLPPPGFLPAHRQ | Null* |
*No conserved domains were predicted |
Identification Of Conserved Motifs In Maize Zf-hd Proteins
In order to investigate the sequence feature of maize ZF-HD proteins, the conserved sequence motifs of these proteins were obtained and annotated using MEME and Interpro, respectively. We accordingly identified 15 motifs in the maize ZF-HD proteins. The motif distribution was consistent with protein phylogenetic evolution, with members in the same clade generally sharing similar motifs. On the basis of motif distribution, the maize ZF-HD proteins can be divided into two clades (Fig. 2). In clade I, almost all of the maize ZF-HD proteins harbored motifs 1 and 4 (the exception being ZmZHD8, which has only motif 4). In clade I, 16 (66.7%) of the 24 ZF-HD proteins shared four common motifs (motifs 1, 2, 3, and 4), whereas the eight (33.3%) proteins in Clade II shared two common motifs (motifs 1 and 4). Motifs 2 and 3, which are located close to the C terminus, represent homeobox domain-like structures that are involved in DNA binding in the transcriptional regulation of target genes. Motifs 1 and 4 represent an indispensable basic conserved ZF-HD domain (Cys/His-rich dimerization domain), which is sufficient to confer homo- or heterodimer formation between proteins and plays a critical role in protein functions (Windhövel et al. 2001).
Zf-hd Phylogenetic And Gene Structure Analysis
To analyze the evolutionary history of the ZF-HD TF family, we performed a molecular phylogeny analysis of the 24 maize ZF-HD genes, in conjunction with 15 ZF-HD genes from rice and 17 from Arabidopsis. We accordingly found that these 56 ZF-HD genes could be clustered into six major groups: groups A, B, C, D, E, and F, containing 15, 3, 8, 13, 5, and 12 genes, respectively (Fig. 3). A notable feature of the constructed phylogenetic tree was that almost all the Arabidopsis ZF-HD genes were clustered together, whereas the ZF-HD genes in rice and maize were clustered together. In addition, many of the maize and rice ZF-HD genes fall into orthologous pairs, including ZmZHD1/OsZHD8, ZmZHD11/OsZHD13, ZmZHD9/OsZHD15, ZmZHD5/OsZHD4, and ZmZHD19/OsZHD9, which indicates that these ZF-HD orthologous genes have conserved functions, even though the maize and rice genomes have undergone markedly different recombination and replication events subsequent to their divergence from a common ancestor. We also performed structural analyses of the ZF-HD genes and found that most (46, 82.1%) of these genes contained no introns, which contrasts with the structure of other homeobox genes. Six of the genes contains a single intron (two genes each in maize, rice, and Arabidopsis). The general absence of introns in these genes tends to indicate that the structure and functions of these genes are highly conserved, as there would be no of alternative splicing in these genes.
The unrooted tree of ZF-HD protein family members and their gene structure constructed using MEGA 5.0 and GSDS 2.0, respectively. Numbers above or below branches of the tree represent bootstrap values. A, B, C, D, E and F indicate the respective groups. Exons, introns, and upstream/downstream sequences are represented by green boxes, black lines, and blue boxes, respectively. The colored boxes and black lines are scaled based on the length of genes (the short introns in ZmZHD9 and AtZHD1/11 are not shown in the figure).
Expression Pattern Of Maize Zf-hd Genes In Different Organs And At Different Developmental Stages
To explore the expression patterns of maize ZF-HD genes and obtain information for functional analyses, we investigated the expression of the 24 identified maize ZF-HD genes. The relative expression level of these ZF-HD genes was detected in 12 different tissues (Fig. 4). On the basis of cluster analysis, the maize ZF-HD genes were classified into two major clusters. Cluster A contained 16 genes, namely, ZmZHD4, 1, 12, 2, 5, 9, 17, 6, 11, 20, 18, 16, 14, 7, 21, and 22, and Cluster B contained eight genes, namely, ZmZHD8, 10, 19, 3, 13, 15, 23, and 24. We found that ZmZHD4 and ZmZHD6 were expressed at an extremely low level in all organs and at all developmental stages. All members of the gene family had relatively low expression levels in the ligule and sheath, whereas all genes in cluster A, with the exception of ZmZHD4, had extremely high expression levels in the embryo. Interestingly, four gene pairs, ZmZHD7/21, ZmZHD12/2, ZmZHD13/3, and ZmZHD23/24, had a high co-expression relationship consistent with the phylogenetic analysis, indicating that these genes might have conserved roles during growth and development in maize.
Red indicates a high expression value and green indicates a low expression value. The change in color from green to red indicates changes in expression value from low to high. The letters above the expression data indicate the following: a: whole anthers; b: B73 pollen; c: mature silk; d: mature leaf; e: seedling roots; f: embryo 14 days after pollination (DAP); g: endosperm 14 DAP; h: ovaries 1 DAP i: ear primordia; j: tassel primordia; k: P7 ligule; and i: P7 sheath
Many previous studies have reported that ZF-HD genes are exclusively expressed in floral tissues, indicating the potential involvement of ZF-HD TFs in regulating floral development (Wang et al. 2016). We therefore investigated the expression profile of ZF-HD genes in different maize tissues, the results of which showed that 14 genes in cluster A (with the exception of ZmZHD4 and ZmZHD18) had a high expression in ear and tassel primordia, indicating that they probably play regulatory roles in ear and tassel development. In addition, we found that ZmZHD5, 11, and 22 were highly expressed in mature silks and embryos at 14 DAP, indicating that these genes may play multiple roles in maize reproductive development. We found that the expression of cluster A genes tended to be exceptionally low in anthers and pollen, whereas cluster B genes such as ZmZHD3, 13, 15, 23 and 24 were highly expressed in these organs, indicating they also play critical roles during reproductive development in maize.
Expression Patterns Of Zf-hd Genes In Response To Exposure To Two Abiotic Stresses And Aba
Many ZF-HD genes, including Os09g29130, Os08g37400, Os11g13930, Os04g35500 in rice and At1g69600 in Arabidopsis, have been reported to be associated with resistance to stress (Figueiredo et al. 2012). When we investigated cis-elements in the promoter regions of the 24 maize ZF-HD genes, we found that 18 of these genes contain an ABA-responsive element (ABRE) (Table S3), thereby indicating that they might be involved in ABA signal transduction. This observation prompted us to investigate the expression pattern of the 24 identified maize ZF-HD genes in response to exposure to ABA, high salt, and drought treatments (Fig. 5). Gene specific primers were designed (Table S4) and gene expression patterns under different conditions were detected. Data analysis revealed that the expression of 11 genes (ZmZHD1, 3, 5, 7, 8, 9, 11, 12, 13, 16, and 18) was up-regulated under ABA treatment, and that three genes (ZmZHD9, 11, and 12), and seven genes (ZmZHD1, 2, 8, 11, 12, 19, and 23) were up-regulated under high salt and drought stress, respectively. Compared with the control (no treatment), the relative expression relative levels of ZmZHD11 were markedly increased at all-time points after exposure to high salt stress. Two genes (ZmZHD11 and 12) were all up-regulated under all three of the imposed stress conditions, indicating that these genes are likely to be involved in stress and ABA responses. In contrast, we also found that six genes (ZmZHD4, 14, 15, 17, 23, and 24), seven genes (ZmZHD10, 14, 15, 17, 22, 23 and 24), and eight genes (ZmZHD3, 4, 9, 10, 14, 15, and 17) were specifically down-regulated in response to ABA, high salt, and drought treatments, respectively.
Relative expression ratios for different treatments were calculated based on comparison with the actin reference gene. The gene name is written in the upper left corner of each bar chart. Error bars represent standard errors from three independent replicates