Abstract
Zinc finger–homeodomain (ZF-HD) proteins, a family of plant-specific transcription factors, play an important role in regulating plant growth and development, as well as responses to stress. Although ZF-HDs have been investigated in several model plants, no systematic studies have been reported in apple (Malus ×domestica). In this study, 14 putative ZF-HD genes were identified in the apple genome and characterized using bioinformatics tools. All members harbored complete canonical structures of the ZF-HD motif. Phylogenetic analysis demonstrated that ZF-HD genes in the genome of apple could be classified into four subfamilies, with high intragroup similarities. Gene-structure analysis revealed that although 11 MdZHDs had only one exon, MdZHD6 and MdZHD13 had two exons and MdZHD8 had six exons, suggesting limited variation among the apple ZHD genes. The expression profiles of MdZHD genes revealed their involvement in the growth and development of different tissues. Numerous binding sites for transcription factors, such as MYB, bZIP, and AP2, were found in the promoter region of the putative MdZHD genes. Nearly all putative MdZHDs were predicted to localize in the nucleus. Finally, the expression levels of the MdZHD genes under abiotic stress were examined in apple rootstock Malus hupehensis and the results showed that the expression of 10 MdZHD genes was induced in response to three abiotic stress factors. Exceptionally, the expression of MdZHD11 was not induced in response to any of the abiotic stress treatments, MdZHD12 was only induced in response to salt stress, and MdZHD7 and MdZHD9 were induced in response to both drought and salt stress. The present results provide valuable insights into the putative physiological and biochemical functions of MdZHDs in apple.
When plants are subjected to adverse conditions, such as cold, drought, high salinity, pathogen infection, and oxidative stress, they can activate sophisticated signaling networks to sense and transmit environmental stimuli at the molecular or cellular level (Romeis, 2001). The transcription factors NAM, ATAF, CUC (NAC), WRKY, basic region/leucine zipper (bZIP), and v-myb avian myeloblastosis viral oncogene homolog (MYB), regulate the expression of stress-resistance genes and proteins in plants, which results in the production of various target metabolites and phenotypic adaptations (Gujjar et al., 2014).
Homeobox genes have a conserved structure and are abundant in eukaryotic genomes. They are ∼180 base pairs (bp) in length and can encode sequences of ∼60 amino acids. Most proteins containing a homeodomain (HD) are classified into six categories according to their sequence, size, location, and interaction with other structures; they include HD-Zip (Ariel et al., 2007), PHD-finger, bell-type HD, WUSCHEL-related homeobox (WOX) (Van der Graaff et al., 2009), knotted-related homeobox (KNOX), and zinc finger (ZF)-HD protein subfamilies. The functions of the corresponding genes vary in plants. Among them, WOX genes stabilize and maintain the fate of plant stem cells and can participate in early embryo differentiation. The PHD gene is important for the maturation of pollen (Rim et al., 2009). HD-ZIP can regulate plant apical meristem formation, root development, abscisic acid synthesis, and the response to various abiotic stresses (Hawker, 2004).
The ZF-HD transcription factor subfamily is unique to plants. Its ZF domain is a partial polypeptide structure comprising cysteine/histidine combined with zinc ions, in which the conserved cysteine residues help in forming homo- or heterodimers. Although the ZF domain does not participate in DNA binding, the HD domain can regulate the interaction with proteins and DNA (Wei et al., 2008; Windhövel et al., 2001).
ZF-HD genes were first discovered in chamomile (Matricaria recutita), a C4 plant, and were subsequently characterized in arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Chinese cabbage (Brassica campestris), and grape (Vitis vinifera) (Jain et al., 2008; Sun et al., 2021; Wang et al., 2016). The number of ZF-HD genes differs among species. For example, the ZF-HD gene family identified in arabidopsis includes 17 genes, among which 14 contain both ZF and HD domains, whereas three contain only ZF domains and are called mini zinc finger (MIF) genes. To date, 11 ZF-HD genes have been identified in rice, 17 in desert poplar (Populus euphratica), 20 in coconut (Cocos nucifera), 20 in Tartary buckwheat (Fagopyrum tataricum), and 22 in tomato.
ZF-HD transcription factors play an important role in the growth and development of plants, mediating the response to stressful events, controlling flower development, and regulating seed viability (Wei et al., 2008). In arabidopsis, ATHB25 is essential for ensuring the effect of gibberellins on seed longevity; whereas, ABF2 can regulate the expression of abscisic acid stress-related genes (Kim et al., 2004). ZHD5 gene can be induced by cytokinin, and when overexpressed, it can improve the regeneration efficiency, cell division, and stress resistance (Kim et al., 2019).
In China, apple is the fruit with the largest cultivation area and yield. Drought and salinization are the primary obstacles to the cost-effective cultivation of apple in arid and semiarid areas in the northern regions of the country. Successful sequencing of the apple genome enables genome-wide identification and classification of ZF-HD genes in this species. Previous studies have shown that ZF-HD proteins played an important role in regulating plant growth and development, as well as the response to abiotic stress in arabidopsis (Kim et al., 2004). These studies suggest that this family of proteins may provide a possibility for the generation of stress-tolerant transgenic plants. The present study aimed to perform a phylogenetic and classification analysis of the apple ZF-HD gene family using bioinformatics tools. Based on chromosomal location, physical and chemical properties, motif structure, gene structure, expression profiles, subcellular localization, and collinearity of genes, 14 putative ZF-HD genes were identified in apple.
Materials and Methods
Genome-wide identification of ZF-HD genes in apple.
To identify the ZF-HD family in the apple genome, the sequences of AtZF-HD cascade proteins were obtained from The Arabidopsis Information Resource (Lamesch et al., 2012). These sequences were used as queries to search against the apple protein databases using BLASTP [National Center for Biotechnology Information (NCBI), Bethesda, MD, USA] with an e-value of 1 × e−10 as the threshold. A hidden Markov model (HMM), built by the HMMER 3.0 software using all known ZF-HD protein sequences from arabidopsis, was used to search for all the potential ZF-HD–encoded protein sequences in the genome of apple (El-Gabali et al., 2019). Unique sequences were selected as candidate ZF-HD family protein sequences. Apple protein sequences can be downloaded from the apple genome database (Zhang et al., 2019).
The obtained candidate sequences were domain-annotated using the pfamscan software and Pfam A database (El-Gabali et al., 2019). Sequences containing only the PF04770 domain were selected as the final ZF-HD sequence in each case.
Sequence alignment and phylogenetic analysis.
The protein theoretical molecular weight, number of encoded amino acids, instability index, aliphatic index, and isoelectric point were predicted using compute pI/MW in the ProtParam tool (Gasteiger et al., 2005). Multiple alignments of nucleotide and amino acid sequences were performed using ClustalW (Hung and Weng, 2016). The phylogenetic tree was constructed based on the sequences of ZF-HD proteins from arabidopsis and apple using the neighbor-joining method with 1000 bootstrap replicates and visualized using MEGA 7.0 software (Arizona State University, Tempe, AZ, USA).
Chromosomal location, gene structure, and predicted subcellular localization studies.
The chromosomal location of MdZHD genes was determined based on identification results and visualized using Tbtools (Chen et al., 2020), according to the location of the MdZHD family genes on the chromosome. Intron/exon structure analysis of MdZHD genes was performed by comparing gene coding sequences using the Gene Structure Display Server (Guo et al., 2007). The MEME program was used to statistically identify conserved motifs in the complete amino acid sequences of MdZHD encoded proteins (Bailey et al., 2006). The subcellular localization of apple ZF-HD family members was predicated using the SoftBerry database (SoftBerry, Mount Kisco, NY, USA).
Cis-element analysis of putative promoter regions and SignalP prediction.
A region 2000 bp upstream of the putative MdZHD gene start site was defined as a promoter regulatory sequence, and transcription factor binding sites within this region were predicted via the PlantRegMap database (Tian et al., 2019). The species apple was selected, and the e-value was set to 1 × e−4. In the physical map of gene promoters, the position of the binding site was marked, and only the top 12 transcription factor families were displayed.
Potential signal peptide cleavage sites and their locations in the amino acid sequence of a ZF-HD family protein were predicted by the signal peptide prediction software, SignalP (Bendtsen et al., 2004). The prediction method is based on various artificial neural network algorithms.
Collinearity analysis of the MdZHD family.
Collinearity analysis was performed using the MCScanX software (Wang et al., 2012). We identified the collinearity caused by segmental duplicate events and tandem duplicates caused by gene duplication events in the data. In the alignment results, if a pair of alignment sequences was connected on the chromosome, MCScanX considered that one of the two sequences was produced by the duplication of the other sequence (i.e., there was a gene duplication event). Clusters also exist on chromosomes, and the MCScanX software can help to confirm that a group of multiple pairs of sequences is generated by the replication of another group (i.e., there is one fragment duplication event).
Tissue-specific expression analysis of MdZHD genes and responses to high salt, low temperature, and drought stress.
To study the physiological function of MdZHD genes, transcriptomic data were obtained from tissues at different developmental stages, including the apical bud, spur bud, flower, and apple fruit tissue (Supplemental Table 1). The raw reads from Illumina GA IIx (Illumina. Inc., San Diego, CA, USA) of all the analyzed samples were submitted as BioProject (PRJNA302879) to the NCBI Sequence Read Archive under accession number SRP066478 (Kumar et al., 2016). The fragments per kilobase million (FPKM) calculation method was applied to standardize paired-end data. After determining the gene accession number of the MdZHD family, the FPKM values of apple ZHD genes in different tissues were obtained. Subsequently, the log2 transformed FPKM values were used to generate a heat map.
In vitro tissue cultures of Malus hupehensis were subcultured on Murashige and Skoog solid medium with 0.5 mg·L−1 benzylaminopurine (BA) and 0.1 mg·L−1 naphthylacetate (NAA) at 25 °C under a 16/8-h light/dark photoperiod, as described by Cao et al. (2013). Then, 1-month-old apple tissue culture materials were treated with cold (4 °C), osmotic (10% polyethylene glycol 4000), and salt (200 mm NaCl) stress in the same sub-proliferation medium formulation as described previously, and subsequently leaves were collected at 0, 3, 6, 12, and 24 h for the expression analysis of 14 MdZHD genes. The expression of MdZHD genes was then examined using real-time quantitative PCR (qPCR) assays with gene-specific primers (Supplemental Table 2). The apple MdActin gene was used as a loading control, and three biological and three technical replicates were performed for each qPCR reaction.
Statistical analysis.
The mean values of treatment groups were compared by analysis of variance using statistical software (IBM SPSS Statistics ver. 18.0; IBM Corp., Armonk, NY, USA). Graphics were produced by scientific graphing software (Sigma Plot ver. 10.0; Systat Software, San Jose, CA, USA).
Results
Identification of ZF-HD family genes in apple.
To identify ZF-HD family genes from the apple genome, hidden HMM and BLAST searches were performed using arabidopsis ZF-HD proteins as query sequences. Sequence comparison between the candidate proteins obtained by BLAST and HMM yielded 14 ZF-HD proteins, with top hits for AtZHD homologs having an e-value cutoff of 1 × e−10. Sequences that encoded very short polypeptides, or those that did not contain known conserved ZF-HD motifs by phylogenetic and conserved domain analyses, were excluded. After multiple steps of screening and validation of the conserved domains, 14 putative ZF-HD genes were identified. The ZF-HD proteins were named according to the gene identity (ID) number from the apple genome as MdZHD1–MdZHD14.
To determine the chromosomal location of the 14 MdZHD genes, the physical location of their sequences on the apple chromosomes was investigated. The 14 genes were mapped to eight chromosomes. Chromosomes 1, 3, 9, 13, 16, and 17 contained the genes MdZHD3, MdZHD13, MdZHD9, MdZHD14, MdZHD1, and MdZHD12, respectively. Chromosome 8 contained the genes MdZHD5, MdZHD10, and MdZHD17, whereas chromosome 15 contained the genes MdZHD2, MdZHD4, MdZHD6, MdZHD7, and MdZHD8 (Fig. 1).
Physical location of MdZHD genes on the chromosomes of apple. The physical location map of the chromosome was drawn by TBtools (Chen et al., 2020), according to the location of the MdZHD family genes on the chromosome.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
Characteristics of putative ZHD genes in apple.
Based on the physical and chemical properties of apple ZF-HD family members (Gasteiger et al., 2005), the putative ZF-HD genes were predicted to encode proteins of 104 (MdZHD7) to 376 (MdZHD4) amino acids in length. Protein molecular weights were estimated to range from 11.60 kDa (MdZHD7) to 41.65 kDa (MdZHD4), and protein isoelectric points ranged from 6.42 (MdZHD7) to 9.6 (MdZHD13) (Table 1).
Physical and chemical characteristics and subcellular localization prediction of putative MdZHD proteins. The protein theoretical molecular weight, number of encoded amino acids, instability index, aliphatic index, and isoelectric point were predicted using compute pI/MW in the ProtParam tool (Gasteiger et al., 2005). Multiple alignments of nucleotide and amino acid sequences were performed using ClustalW (Hung and Weng, 2016). The subcellular localization of apple ZF-HD family members was predicated using the SoftBerry database (SoftBerry, Mount Kisco, NY, USA).
Analysis of phylogenetic relationships among ZHD genes in apple.
To evaluate the evolutionary relationships among ZHD proteins, a phylogenetic tree was constructed with amino acid sequences of the 14 putative MdZHD genes, 14 AtZHD genes, and three MIFs from arabidopsis (Liu et al., 2019). ZHD proteins in plants have diverged into six major subfamilies [I, II, III, IV, V, and MIF (Fig. 2)]. The phylogenetic analysis revealed that the 14 putative MdZHD genes could be divided into four distinct groups (I, II, III, and IV).
Phylogenetic relationship among putative ZHD genes in arabidopsis and apple. The phylogenetic tree was constructed based on the sequences of ZF-HD proteins from arabidopsis and apple using the neighbor-joining method with 1000 bootstrap replicates and visualized using the MEGA7.0 software (Arizona State University, Tempe, AZ, USA). Letters I–V indicate different groups of ZHDs.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
MdZHD2, MdZHD3, MdZHD5, MdZHD6, MdZHD11, and MdZHD13 were clustered into group I, together with well-characterized ZHD genes, such as AtZHD1, AtZHD2, AtZHD3, AtZHD5, AtZHD6, and AtZHD7. MdZHD4, MdZHD7, MdZHD8, and MdZHD10 were clustered into group II, which also included AtZHD3 and AtZHD4. Group III contained two genes, MdZHD9 and MdZHD12, whereas group IV included MdZHD1 and MdZHD14 (Fig. 2). No MdZHD genes were found to belong to group V, which contained only AtZHD13 and AtZHD14. The MIF group, containing three AtMIF genes, was separate from the other groups (Fig. 2).
Structural analysis of MdZHD genes in apple.
Subsequently, we sought to understand the distribution of the introns and exons and to analyze the structural characteristics of the MdZHD genes. The structures of the introns and exons were identified for each MdZHD gene by aligning the corresponding genomic DNA sequences, which confirmed the presence of various subgroups. MdZHD genes exhibited different intron and exon structures among groups, with a high degree of intragroup conservation (Fig. 3). Most of the putative MdZHD members, including MdZHD1, MdZHD2, MdZHD3, MdZHD4, MdZHD5, MdZHD7, MdZHD9, MdZHD10, MdZHD11, MdZHD12, and MdZHD14, had only one exon, whereas MdZHD6 and MdZHD13 had two exons. Notably, MdZHD8 exhibited a markedly different structure with six exons (Fig. 3).
Intron/exon structures of putative ZHD genes in apple. Intron/exon structure analysis of MdZHD genes was performed by comparing gene coding sequences using the Gene Structure Display Server (Guo et al., 2007). Orange boxes indicate exons [coding sequences (CDS)], and single lines indicate introns. Gene models are drawn to scale as indicated by the bar on the bottom.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
Analysis of conserved motifs and promoter regions in MdZHD genes.
The presence of similar motifs implies a similar function and 15 conserved motifs were identified in apple (Liu et al., 2019). Motifs 2 and 3 were present in all 14 MdZHDs. In addition, all of MdZHDs contained a combination of the 15 motifs. All members belonging to the same subfamily shared similar conserved motifs. For instance, MdZHD1 and MdZHD14 from group IV shared motifs 1, 2, 3, 6, 7, 10, and 11, whereas MdZHD9 and MdZHD12 from group III shared motifs 1, 2, 3, 5, 6, 7, 8, 11, and 12. Motif composition differed greatly among four MdZHDs in group II, although motifs 2, 3, and 4 were common to all its members (Fig. 4).
Conserved motifs of putative MdZHDs. All motifs were identified with the MEME program based on the complete amino acid sequences of the 14 MdZHDs (Bailey et al., 2006). Different colors of boxes represent different motifs at the corresponding position in each ZHD protein.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
To investigate the potential functions and transcriptional regulation of the putative MdZHD genes, cis-regulatory elements located within 2000 bp upstream of the transcriptional start site (ATG) were identified. Transcription factor binding sites for MYB, bZIP, and AP2, among others, were found (Supplemental Fig. 1), suggesting that the expression of MdZHD genes might be regulated by these transcription factors.
Predicted subcellular localization and signal peptide prediction of MdZHD family members.
Based on subcellular localization analysis of MdZHD family members, MdZHD7 and MdZHD8 were predicted to localize in the cytoplasm and extracellular space, respectively, whereas all other MdZHDs appeared to localize in the nucleus (Table 1).
The SignalP software predicts the presence of potential signal peptide cleavage sites and their location in a given amino acid sequence. SignalP prediction yielded no conspicuous peaks in any of the putative MdZHD proteins, suggesting the absence of signal peptide cleavage sites (Supplemental Fig. 2).
Collinearity analysis of the apple ZHD family.
Collinearity analysis of ZF-HD family members revealed no collinearity between arabidopsis and apple ZHD families. Furthermore, a comparison between MdZHD and AtZHD families suggested similarity between MdZHD1 and MdZHD14, MdZHD2 and MdZHD5, MdZHD4 and MdZHD10, and MdZHD9 and MdZHD12 in apple, as well as between AtZHD3 and AtZHD4, AtZHD11 and AtZHD12, AtZHD12 and AtZHD14, and among AtMIF1–3 in arabidopsis (Supplemental Fig. 3). Once again, members clustered in a subfamily were characterized by greater similarity. Some degree of similarity was observed among AtZHD8, AtZHD9, and MdZHD14.
Tissue-specific expression analysis of MdZHD genes and responses to high salt, low temperature, and osmotic stress.
To study the physiological function of MdZHD genes, transcriptomic data were obtained from different tissues at different developmental stages. Except for MdZHD10, the expression of the MdZHD genes exhibited similar trends in apical and spur buds. Among them, MdZHD4, MdZHD7, MdZHD9, and MdZHD13 exhibited low expression, whereas MdZHD1, 2, 3, 5, 6, 8, 11, 12, and 14 exhibited high expression. The expression of MdZHD10 was low in apical buds and high in lateral buds. In flowers, the expression of the MdZHD3, 7, 8, 9, 11, and 13 genes was relatively low, whereas that of MdZHD2, 4, 5, 10, 12, and 14 was relatively high. In fruit development, the expression trends of MdZHD7, 8, 10, and 13 were consistent, exhibiting continuous low or high expression. The expression of MdZHD9 was low in the early period of fruit development and high in the late period, and the remaining gene members (MdZH2, 3, 4, 5, 6, 11, 12, and 14) exhibited the opposite trend (Fig. 5).
Heatmap representing MdZHD genes at various developmental stages of bud, Flower and fruit tissues: apical bud (Map1–Map3), spur bud (Msp4–Msp8), flower (Mgt9, Mtc10, Mip11, Mfp12, Mff13, and Mpf14), and fruit (Mfs15–Mfs21). The raw reads from Illumina GA IIx of all the analyzed samples were submitted as BioProject (PRJNA302879) to the NCBI Sequence Read Archive under accession number SRP066478 (Kumar et al., 2016). The fragments per kilobase million (FPKM) calculation method was applied to standardize paired-end data. After determining the gene accession number of the MdZHD family, the FPKM values of apple ZHD genes in different tissues were obtained. Subsequently, the log2 transformed FPKM values were used to generate the heat map using MeV4 software (SourceForge, Boston, MA, USA).
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
To determine whether MdZHDs play an important role in abiotic stress responses, the expression levels of 14 MdZHD genes in apple were examined by reverse-transcriptase qPCR (RT-qPCR). The results showed significant differences in the expression levels of 14 MdZHD genes at different times under abiotic stress and in comparison with the control (0 h) as showed in Fig. 6. Based on a 2-fold expression level threshold, the expression of MdZHD11 could not be induced by any stress treatment, the expression of MdZHD12 could only be induced by salt stress, and the expression of MdZHD7 and MdZHD9 could be induced by both osmotic and salt stress. The expression of the remaining MdZHD genes could be induced by all of the abiotic stress factors. It is worth mentioning that the expression of MdZHD2, MdZHD4, and MdZHD5 was downregulated by osmotic stress, and the expression of MdZHD10 was downregulated by salt stress.
Effect of abiotic stress on the expression of 14 MdZHD genes in leaves of tissue culture materials of Malus hupehensi. These materials were treated with cold (4 °C), osmotic [10% polyethylene glycol 4000 (PEG)], and salt (200 mm NaCl) stress in the same sub-proliferation medium formulation. Subsequently, leaves were collected at 0, 3, 6, 12, and 24 h for the expression analysis of MdZHD genes by using real-time quantitative polymerase chain reaction (qPCR) assays with gene-specific primers. The apple MdActin gene was used as a loading control, and three biological and three technical replicates were performed for each qPCR reaction. Asterisks indicate values that are significantly different from those of the control group (*P < 0.05) according to analysis of variance.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
Discussion
Gene families derived from the same ancestor share similar structures and functions. The protein products of the same gene family can be closely arranged to form a gene cluster; however, most of the time, the genes are scattered at different positions along the same chromosome or on different chromosomes, whereby each follows a different expression regulatory mode.
Recently, the characterization of gene families has facilitated the study of their function. The reliability and accuracy of the evolutionary characterization of gene families depends largely on the availability of genomic sequences. The availability of the complete apple genome sequence has made it possible to identify all ZHD family members in this plant species (Velasco et al., 2010). In this study, 14 putative ZHD genes were identified in the apple genome, which is more than in rice (11 members) but less than in arabidopsis (17), desert poplar (17), coconut (20), and tomato (22).
The variation in the length of ZHD genes typically originates from the differences in the length of the ZHD domain or the number of introns. The full-length sequences of putative MdZHD protein products ranged from 104 to 376 amino acids. Structural analysis of ZHD genes in apple demonstrated that most of the putative MdZHD members, including MdZHD1, MdZHD2, MdZHD3, MdZHD4, MdZHD5, MdZHD7, MdZHD9, MdZHD10, MdZHD11, MdZHD12, and MdZHD14, had only one exon, whereas MdZHD6 and MdZHD13 had two exons, which is similar to other plants, including arabidopsis, rice, grape, and coconut. Only MdZHD8 stood out with a total of six exons. Besides, the structural analysis diagram cannot be annotated, because there are no untranslated regions (UTRs) (5' UTR and 3' UTR) on the structural annotation.
To assess the evolutionary relationships among ZHD proteins, a phylogenetic tree was constructed based on the amino acid sequences of the 14 putative MdZHDs. Fourteen MdZHDs could be divided into four distinct groups (I, II, III, and IV). In comparison, the ZF-HDs in arabidopsis have been classified into six subgroups, with MIF alone forming one subgroup. Previous studies have demonstrated that the AtZHD5 protein promotes shoot regeneration and confers other cytokinin-related phenotypes when overexpressed (Kim et al., 2019), whereas arabidopsis ZHD8 protein plays an important role in promoting the number and length of root hairs in plants.
The analysis of the conserved motifs of the putative ZHDs revealed that all identified MdZHDs contained motifs 2 and 3, indicating that all apple ZHDs share typical features of the ZHD family of proteins. In addition, most MdZHDs contained a combination of the 15 protein kinase motifs. Importantly, all members from the same subfamily shared similar conserved motifs.
To further investigate the potential functions and transcriptional regulation of the putative MdZHD genes identified in this study, cis-regulatory elements upstream of the start site, expression profiles, and MdZHD localization were determined. Various transcription factor binding sites were identified in the putative promoter regions of the MdZHD genes (Fig. 5), suggesting that the expression of MdZHD genes may be regulated by MYB, bZIP, and AP2 transcription factors. Predicted subcellular localization analysis results showed that most putative MdZHDs were present in the nucleus. Expression profiles suggested the involvement of MdZHDs in plant growth and development. Finally, to study their roles in response to abiotic stress, the expression of 14 apple MdZHD genes was examined, and the results showed that the expression of the 10 MdZHD genes could be induced by three abiotic stresses. Among them, we also found that the expression levels of MdZHD1, MdZHD3, MdZHD6, MdZHD8, MdZHD13, and MdZHD14 could be strongly upregulated by cold, osmotic, and salt stress, which means that they may play a positive regulatory role in response to abiotic stress. As such, these genes are suitable resistance-related candidate genes for studying their functions and regulatory mechanisms in response to abiotic stresses (e.g., drought and salt stress) in an apple callus transgenic system. In apple rootstocks that are transformed with selected MdZHD genes, the conferred resistance may be further characterized.
In conclusion, this study lays a foundation for analyzing the functions and regulation of the metabolic mechanisms of MdZHD genes in response to abiotic stresses. Moreover, it has identified MdZHD genes as potential resistance genes for the genetic improvement of resistant apple germplasm.
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Predicted transcription factor binding sites on the promoter of MdZHD genes. A region 2000 base pairs upstream of the putative MdZHDs start site was defined as a promoter regulatory sequence, and transcription factor binding sites within this region were predicted via the PlantRegMap database (Tian et al., 2019). The species apple was selected, and the e-value was set to 1 × e−4. In the physical map of gene promoters, the position of the binding site was marked, and only the top 12 transcription factor families were displayed. Different colors represent different transcription factor binding sites.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
Signal peptide prediction of MdZHD proteins. Potential signal peptide cleavage sites and their locations in the amino acid sequence of a ZF-HD family protein were predicted by the signal peptide prediction software SignalP (Bendtsen et al., 2004). The prediction method is based on various artificial neural network algorithms.
Note: S-score value represented each amino acid corresponds to 1 S value, which was used to determine whether the corresponding position was a signal peptide region. C-score represented value of the cutting site; each amino acid had a C value and the C value was highest at the cutting site. Y-score represented the geometric mean of the C-score and S-score. It helped to avoid multiple high-scoring C-score value pairs. Y-max provided a comprehensive representation of the S value and C value, which was more accurate than the C value alone, because the C value may have more than one peak in a sequence, but there was only one splicing site, the splicing site at this time was inferred from the Y-max value. This corresponded to the steep position of the S value and sites with high C values.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
Similarity results after BLAST comparison between the MdZHD and AtZHD families of proteins. Collinearity analysis was performed using the MCScanX software (Wang et al., 2012). Similarity ranges from weak to strong according to the following color-coding: blue, green, orange, and red. The bar chart above each sequence represents the number of times each color hits a specific part of the sequence.
Citation: Journal of the American Society for Horticultural Science 147, 6; 10.21273/JASHS05211-22
The sample abbreviation and their collection intervals of tissues at different developmental stages. The apical bud (Map1–3) was collected at 0, 38, and 70 d. Spur bud (Msp 4–8) was respectively collected at 0, 38, 70, 104, and 126 d. Flower (Mgt9, Mtc10, Mip11, Mfp12, Mff13, and Mpf14) was respectively collected at 0, 10, 15, 26, 30, and 39 d. Apple fruit tissue (Mfs15–Mfs21) was collected at 13, 28, 44, 83, 102, and 132 d.
Gene-specific primers were designed for the expression analysis of 14 MdZHD genes by real-time quantitative polymerase chain reaction.
