Characterization of Plant Homeodomain Transcription Factor Genes Involved in Flower Development and Multiple Abiotic Stress Response in Pepper

Plant homeodomain (PHD) transcription factor genes are involved in plant development and in a plant’s response to stress. However, there are few reports about this gene family in peppers (Capsicum annuum L.). In this study, the pepper inbred line “Zunla-1” was used as the reference genome, and a total of 43 PHD genes were identified, and systematic analysis was performed to study the chromosomal location, evolutionary relationship, gene structure, domains, and upstream cis-regulatory elements of the CaPHD genes. The fewest CaPHD genes were located on chromosome 4, while the most were on chromosome 3. Genes with similar gene structures and domains were clustered together. Expression analysis showed that the expression of CaPHD genes was quite different in different tissues and in response to various stress treatments. The expression of CaPHD17 was different in the early stage of flower bud development in the near-isogenic cytoplasmic male-sterile inbred and the maintainer inbred lines. It is speculated that this gene is involved in the development of male sterility in pepper. CaPHD37 was significantly upregulated in leaves and roots after heat stress, and it is speculated that CaPHD37 plays an important role in tolerating heat stress in pepper; in addition, CaPHD9, CaPHD10, CaPHD11, CaPHD17, CaPHD19, CaPHD20, and CaPHD43 were not sensitive to abiotic stress or hormonal factors. This study will provide the basis for further research into the function of CaPHD genes in plant development and responses to abiotic stresses and hormones.

With the rapid development of high-throughput sequencing technology, PHD family proteins are being identified and systematically studied in lots of plants.There are a large number of members of the PHD gene family in plants, with 67 in maize (Zea mays L.) [7], 73 in poplar (Populus trichocarpa) [8], 59 in rice (Oryza sativa L.) [9], 45 in tomato (Solanum lycopersicum L.) [10], and 106 in carrot (Daucus carota L.) having already been identified [11].The PHD-finger motif is a common structural motif found in plants and fungi, which has been reported to bind to nucleosomes and to function as a nuclear protein-interaction domain [12].The PHD zinc-finger family plays an important role in the growth and development of plants, with Arabidopsis PHD-domain ALFIN1-like proteins promoting seed germination [13] and the germostatin resistance locus 1 (GSR1) gene, which encodes a PHDfinger protein, regulating seed germination and dormancy through the auxin-signaling pathway [14].At the same time, PHDs can regulate SOC1/FT chromatin conformation to achieve the regulation of flowering [15].The MALE MEIOCYTE DEATH 1 (MMD1) gene in pepper (Capsicum annuum L.) encodes a PHD-type transcription factor that regulates the development of pollen and the tapetum.This gene is highly homologous to the Arabidopsis MALE STERILITY1 (MS1) gene, and MMD1 expression may occur during meiosis.An MMD1 knockout mutation can cause the death of male meiotic cells [16].ZmMs7 in maize encodes a PHD-finger protein, which is specifically expressed in maize anthers, causing transcriptional activation and playing a key role in tapetum development and pollen outer wall formation [17].The ms4 mutant locus was finely mapped to a 216 kb region in the soybean genome, which contains 23 protein-coding genes.Among them, Glyma.02G243200encodes a protein orthologous with MMD1 in Arabidopsis, which has a PHD domain that may be involved in the development of male sterility [18].In addition, MS4 in soybean (Glycine max) [18] and TDR INTERACTING PROTEIN3 [19] and PERSISTENT TAPETAL CELL1 [20] in rice also encode a PHD protein; they could regulate the formation of male sterility.
There are only a few reports that have shown that PHD proteins are involved in plant stress responses.For example, 15 of the 67 PHD family genes in maize responded to drought and high salt stress [7], whereas 9 of the PHD family genes in poplars showed differential expression under salt, drought, and cold stress conditions [8].The genomewide identification and characterization of the expression of the soybean PHD family genes showed that only six PHD family genes, GmPHD1-6, had been cloned and showed a response to drought stress.Among them, the expression of GmPHD4 and GmPHD5 was induced by cold stress [21], whereas that of GmPHD2 and GmPHD5 regulated the response of plants to salt stress [21,22].In addition, studies have shown that PHD proteins can participate in DNA methylation, playing an important role in this phenomenon [23].
The pepper belongs to the Solanaceae family, which originates from Central and South America and is cultivated all over the world.The world's pepper production exceeds 40 million tons (www.fao.org,(accessed on 15 November 2022)); the area of peppers planted annually in China is of the order of 1.5 to 2 million hectares, accounting for 8-10% of the total planted vegetable crop area.PHD genes have been identified in lots of plants, but the identification and functional analysis of PHD genes in pepper have not yet been studied.In this study, genes encoding PHD transcription factors were obtained by screening them from the pepper whole-genome database, and their sequence characteristics, chromosomal location, evolutionary relationship, protein conservation motifs, and gene structure were comprehensively analyzed to provide valuable information on the evolutionary mechanism of pepper PHD genes.By analyzing the expression of PHD family genes in different tissues at different developmental stages and under different stress treatments, the organ-specific expression and stress-responsive gene expression patterns of CaPHD genes were found, allowing us to preliminarily explore the biological functions of PHD family genes in pepper.The CaPHD genes identified in the current study could be the key genes involved in the flower development and stress response of pepper, and they will provide future insights for further research on the biological functions of CaPHD genes, as well as act as a theoretical reference for pepper molecular breeding in the future.

Identification of the PHD-Finger Family Genes in Pepper
The protein sequences of pepper "Zunla-1" (http://peppersequence.genomics.cn/)and Arabidopsis thaliana (https://www.arabidopsis.org/Blast/index.jsp,(accessed on 20 November 2022)) were obtained from the websites listed to construct the local protein database.Based on previous reports, the HMM model file (ID: PF00628) of the PHD proteins was downloaded from the Pfam database (http://pfam.xfam.org/,(accessed on 21 November 2022)), and the hmmsearch program of the HMMER software package was used to download proteins from the reference genome of "Zunla-1" and Arabidopsis thaliana.Possible PHD protein sequence IDs were screened from the protein database (E-value threshold < 1 × 10 −5 ).Using the Perl script to extract the sequence information of each candidate protein, the sequence of the preliminary protein file obtained was compared with that from the Pfam database to identify the characteristic domain contained in the candidate sequence and to select the protein sequence with a complete PHD domain to be a member of the PHD family of this species.
The online tool ExPASy (https://web.expasy.org/protparam/,(accessed on 21 November 2022)) was used to analyze the isoelectric point, the number of amino acids, and the molecular weight of each PHD gene family protein, and it was used to align the protein sequence of members of the PHD family of "Zunla-1" with the Arabidopsis database (https://www.arabidopsis.org/Blast/index.jsp,(accessed on 21 November 2022)) to identify the pepper genes with the greatest homology to the Arabidopsis PHD genes.

Analyses of Chromosomal Location, Phylogenetic Relationship, Gene Structure, Functional Domains, and Conserved Motifs of the CaPHD Gene Family
The online software tool Gene Structure Display Server 2.0 (GSDS) (http://gsds.cbi.pku.edu.cn/) was used to analyze the structure of the pepper PHD family genes, while Multiple Em for Motif Elicitation (MEME) (http://meme-suite.org/,(accessed on 25 November 2022)) was used to analyze the corresponding PHD proteins.The online tool ExPASy (https://web.expasy.org/protparam/) was used to analyze the isoelectric point, and the conserved PHD motif had an output value set at 10.The Mapgene2chrom (MG2C) online tool (http://mg2c.iask.in/mg2c_v2.0/)was used to locate each gene on a particular chromosome, with MEGA X software being employed to construct a phylogenetic tree using the Neighbor-Joining method.

Identification of Cis-Acting Promoter Elements and Gene Expression Patterns
TBtools was applied to the "Zunla-1" genomic data to extract the 2000 bp promoter sequence upstream of each CaPHD gene, and the cis-regulatory elements in the promoter region were predicted with PlantCARE [24].The expression data of the pepper inbred lines 6421, 9704A, and 9704B from the PHD gene family were obtained from PepperHub (http://www.hnivr.org/pepperhub/)and our previous research [25,26], and the gene expression heatmap was constructed using TBtools [24].

Plant Materials and Responses to Abiotic Stresses and Phytohormone Treatments
Using the expression data of the high-generation inbred line 6421 [25], the cytoplasmic male-sterile line 9704A, and the maintainer line 9704B to analyze the expression level of the CaPHD gene family during the different flower developmental periods and under different abiotic stresses.In order to ensure the accuracy and synchronicity of the data, floral buds were collected at different stages according to their size, and the expression level of each gene was calculated as the average FPKM of three biological replicates.Gene expression in 6421 was quantified in response to hormone treatments, including treatments with gibberellic acid (GA 3 ) (2 µM), abscisic acid (ABA) (30 µM), SA (2 mM), or methyl jasmonate (JA) (10 µM), or in response to exposure to abiotic stresses, such as mannitol (400 mM, to simulate drought), salt treatment (200 mM NaCl), heat stress (42 • C), or cold stress (10 • C).Control plants were mock-treated with nutrient solution only.Leaf and root tissues were collected from both the treated and control plants at 0, 0.5, 1, 3, 6, 12, and 24 h post-treatment [25].The flower development period was divided into three periods (S1, sporogenous celldivision stage; S2, uninucleate microspore stage; S3, mature pollen stage) in 9704A and 9074B based on the size of the flower: S1 (A1, B1 (2.0-3.0 mm)), S2 (A2, B2 (5.0-6.0 mm)), and S2 (A3, B3 (8.0-10.0mm)) [26].

qRT-PCR Analysis
Total RNA was isolated from the different samples of 9704A and 9704B using the RNAiso Plus reagent (TaKaRa) and treated with RNase-free DNase I. Subsequently, 0.5 µg of RNA was used for first-strand cDNA synthesis using a HiScript II 1st Strand cDNA Synthesis kit (Vazyme, Nanjing, China).qRT-PCR was performed using the LightCycler 96 (Roche, Basel, Switzerland) with the SYBR Green Premix Ex Taq™ II quantitative PCR system (TaKaRa, Dalian, China), and the primers of the DEGs and reference gene are listed in Table S1.The experiment was repeated to obtain three biological replicates, and three technical repeats were carried out for each biological repeat.Briefly, after 95 • C for 10 min, the amplifications were carried out with 35 cycles at 95 • C for 15 s and 60 • C for 30 s.The relative expressions of target genes were calculated using the 2 −∆∆CT method.

Genome-Wide Identification and Chromosomal Location of CaPHD Gene Family in Pepper
From the HMM file of the PHD proteins, 60 candidate members of the PHD gene family were identified from the genome data of the pepper "Zunla-1", and the PHD genes with incomplete sequences of the domain were removed via Pfam.In total, 43 PHD gene family members were ultimately identified from "Zunla-1" and numbered according to their chromosomal position and location on the chromosome (Table 1).The open reading frame size of the pepper PHD (CaPHD) gene family members was 405-7251 bp, with the number of amino acids encoded by the protein being in the range of 134-2416 and the isoelectric point being between 4.53 and 9.35 (Table 1).Based on the length of each "Zunla-1" chromosome and the distribution information of the CaPHD gene family members obtained from the genome, the MG2C online tool was used to draw the distribution map of the CaPHD genes on the 13 chromosomes.The 43 CaPHD genes were distributed across the "Zunla-1" chromosomes; chromosome 4 carried the fewest CaPHD genes (n = 1), whereas chromosome 3 carried the most genes (n = 5), with the remaining chromosomes, each containing between two and four PHD genes, and most of the CaPHD genes were located near the ends of chromosomes (Figure 1).The genome assembly proved to be incomplete, and four CaPHD genes that could not be mapped to any chromosome were placed on chromosome 0 (Figure 1).

Phylogenetic Analyses of the CaPHD Family
In order to better understand the evolutionary relationship between the CaPHD genes, we constructed a phylogenetic tree of the pepper PHD genes and their orthologs in

Phylogenetic Analyses of the CaPHD Family
In order to better understand the evolutionary relationship between the CaPHD genes, we constructed a phylogenetic tree of the pepper PHD genes and their orthologs in Arabidopsis, and the results showed that the PHD proteins were clearly divided into four categories (Figure 2).Most of the CaPHD genes were located in the d category, whereas the AtPHD genes were located in the b and c categories.Furthermore, most of the subfamilies had a different number of genes between Arabidopsis and pepper.

Gene Structure and Conserved Motif Analysis of the CaPHD Family
Determining the evolutionary relationship, structural analysis, and motif distribution of CaPHD gene family members can help us to better understand the similarity and diversity between the CaPHD protein family members.Cluster analysis showed that the 43 CaPHD members could be clearly divided into four groups, one of which (group b) contained only CaPHD11, CaPHD12, CaPHD21, and CaPHD28 (Figure 3).TBtools was used to map the structure of the pepper, and the results showed that most CaPHDs from the same subfamily shared similar gene structures.Conserved motifs of the CaPHD proteins were analyzed using MEME tools.All 43 CaPHD protein family members contained motif 1 and motif 2. Proteins CaPHD14, CaPHD22, CaPHD23, CaPHD25, CaPHD30, CaPHD34, and CaPHD41 each contained only motif 1 and motif 2, whereas at the other extreme, CaPHD15 contained 11 motifs.

Gene Structure and Conserved Motif Analysis of the CaPHD Family
Determining the evolutionary relationship, structural analysis, and motif distribution of CaPHD gene family members can help us to better understand the similarity and diversity between the CaPHD protein family members.Cluster analysis showed that the 43 CaPHD members could be clearly divided into four groups, one of which (group b) contained only CaPHD11, CaPHD12, CaPHD21, and CaPHD28 (Figure 3).TBtools was used to map the structure of the pepper, and the results showed that most CaPHDs from the same subfamily shared similar gene structures.Conserved motifs of the CaPHD proteins were analyzed using MEME tools.All 43 CaPHD protein family members contained motif 1 and motif 2. Proteins CaPHD14, CaPHD22, CaPHD23, CaPHD25, CaPHD30, CaPHD34, and CaPHD41 each contained only motif 1 and motif 2, whereas at the other extreme, CaPHD15 contained 11 motifs.

Analysis of Cis-Acting Regulatory Elements
In order to study the response of the expression of the CaPHD genes to hormones and stresses, cis-acting regulatory elements were predicted and identified in the promoter region that is 2 kb upstream of the coding sequence of the CaPHD genes.The prediction results showed that the promoter region of the CaPHD gene family could respond to a variety of hormones (namely ABA, GA3, SA, or MeJA) and abiotic stresses (drought, low/high temperatures, or oxidative or salinity stresses) (Figure 4).In the results, 36 upstream cis-elements of the CaPHD genes were responsive to ABA, 32 to GA3, 32 to MeJA, 23 to IAA, and 15 to SA (Figure 4).In addition, a number of stress-responsive elements were identified, of which 17 were able to respond to low-temperature stress, whereas all 43 genes were responsive to light.In addition, the expression of CaPHD genes also occurred in response to zein metabolism, wounding, MYB, and AT-rich DNA-binding proteins (ATBP-1), among others.These results indicate that CaPHD genes may be related to various signal transduction pathways that involve phytohormones, light response, and abiotic stresses, suggesting that they may be involved in regulating the development and stress response of plants.

Analysis of Cis-Acting Regulatory Elements
In order to study the response of the expression of the CaPHD genes to hormones and stresses, cis-acting regulatory elements were predicted and identified in the promoter region that is 2 kb upstream of the coding sequence of the CaPHD genes.The prediction results showed that the promoter region of the CaPHD gene family could respond to a variety of hormones (namely ABA, GA 3 , SA, or MeJA) and abiotic stresses (drought, low/high temperatures, or oxidative or salinity stresses) (Figure 4).In the results, 36 upstream cis-elements of the CaPHD genes were responsive to ABA, 32 to GA 3 , 32 to MeJA, 23 to IAA, and 15 to SA (Figure 4).In addition, a number of stress-responsive elements were identified, of which 17 were able to respond to low-temperature stress, whereas all 43 genes were responsive to light.In addition, the expression of CaPHD genes also occurred in response to zein metabolism, wounding, MYB, and AT-rich DNA-binding proteins (ATBP-1), among others.These results indicate that CaPHD genes may be related to various signal transduction pathways that involve phytohormones, light response, and abiotic stresses, suggesting that they may be involved in regulating the development and stress response of plants.

Tissue-Specific Expression Patterns of CaPHD Genes
In order to predict the function of the CaPHD genes in the flower development in pepper, gene expression levels were analyzed at three different stages of flower bud development in the near-isogenic cytoplasmic male-sterile 9704A and maintainer 9704B lines (Figure 5, Table S2).The gene expression and cluster analyses in the male-sterile line 9704A and the male-fertile maintainer line 9704B show that A3, B3, A2, and B2 are clustered into one category, with A1 and B1 being clustered into a separate category.TheT expression levels were significantly higher in the B1 stage than in A1 for CaPHD6, CaPHD15, CaPHD17, CaPHD22, CaPHD27, CaPHD28, CaPHD33, CaPHD37, CaPHD38, CaPHD40, and CaPHD42 (Figure 5).This indicates that the key period involved in flower development in pepper is the first stage.In addition, some differences between A3 and B3 were observed in the expression of several CaPHD genes.For example, the expression of CaPHD10 and CaPHD11 was significantly higher in B3.In total, 30 CaPHD genes exhibited obvious differences in their expression between the A1 and B1 period, being more highly expressed in the maintainer line than the male-sterile line.We speculate that CaPHD6, CaPHD15, CaPHD17, CaPHD22, CaPHD27, CaPHD28, CaPHD33, CaPHD37, CaPHD38, CaPHD40, and CaPHD42 are mainly involved in the early stage of flower bud development, whereas CaPHD4, CaPHD10, CaPHD14, CaPHD11, and CaPHD36 are predominantly involved in the late stage of flower bud development.These genes may also be involved in the formation of male sterility (Figure 5).

Tissue-Specific Expression Patterns of CaPHD Genes
In order to predict the function of the CaPHD genes in the flower development in pepper, gene expression levels were analyzed at three different stages of flower bud development in the near-isogenic cytoplasmic male-sterile 9704A and maintainer 9704B lines (Figure 5, Table S2).The gene expression and cluster analyses in the male-sterile line 9704A and the male-fertile maintainer line 9704B show that A3, B3, A2, and B2 are clustered into one category, with A1 and B1 being clustered into a separate category.TheT expression levels were significantly higher in the B1 stage than in A1 for CaPHD6, CaPHD15, CaPHD17, CaPHD22, CaPHD27, CaPHD28, CaPHD33, CaPHD37, CaPHD38, CaPHD40, and CaPHD42 (Figure 5).This indicates that the key period involved in flower development in pepper is the first stage.In addition, some differences between A3 and B3 were observed in the expression of several CaPHD genes.For example, the expression of CaPHD10 and CaPHD11 was significantly higher in B3.In total, 30 CaPHD genes exhibited obvious differences in their expression between the A1 and B1 period, being more highly expressed in the maintainer line than the male-sterile line.We speculate that CaPHD6, CaPHD15, CaPHD17, CaPHD22, CaPHD27, CaPHD28, CaPHD33, CaPHD37,

Expression Profiles of Stress-Related CaPHD Genes in Pepper
Previous studies have shown that the expression of PHD genes in other plant species can respond to drought, low temperatures, high temperatures, and salt stress by showing differences in expression under various stress treatments.We used the previous expression data to analyze the response of CaPHD genes in pepper leaves and roots to drought, low temperatures, high temperatures, and salt stress at different stages [25] and to identify CaPHD genes that could be involved in the regulation of the stress response (Figure 6, Table S3).Most of the CaPHD genes showed increased expression in roots and decreased expression in leaves under the simulated drought, low temperature, and salt stress, and this trend was most obvious in response to salt stress (Figure 6).For instance, CaPHD25 and CaPHD35 in the roots were both significantly upregulated under the four types of stress.The expression level of CaPHD25 and CaPHD34 was particularly sensitive to mannitol treatments, and changes in the expression of these two genes occurred mainly in the roots and reached the highest stage with 6 h treatment (Figure 6).The expression of CaPHD29 occurred in response to cold stress, heat stress, and simulated drought stress in the leaves, showing a significant increase in expression under the 6 h treatment.

Expression Profiles of Stress-Related CaPHD Genes in Pepper
Previous studies have shown that the expression of PHD genes in other plant species can respond to drought, low temperatures, high temperatures, and salt stress by showing differences in expression under various stress treatments.We used the previous expression data to analyze the response of CaPHD genes in pepper leaves and roots to drought, low temperatures, high temperatures, and salt stress at different stages [25] and to identify CaPHD genes that could be involved in the regulation of the stress response (Figure 6, Table S3).Most of the CaPHD genes showed increased expression in roots and decreased expression in leaves under the simulated drought, low temperature, and salt stress, and this trend was most obvious in response to salt stress (Figure 6).For instance, CaPHD25 and CaPHD35 in the roots were both significantly upregulated under the four types of stress.The expression level of CaPHD25 and CaPHD34 was particularly sensitive to mannitol treatments, and changes in the expression of these two genes occurred mainly CaPHD37 showed a unique response to heat stress, as it was upregulated by 7-fold after 6 h in the leaves and by 16-fold after 24 h in the roots.CaPHD39 and CaPHD42 were also involved in the response to heat stress in the roots (Figure 6).In addition, CaPHD41 always maintained a high level of expression, which suggests that it is an essential gene in the tolerance to abiotic stress.CaPHD9, CaPHD10, CaPHD11, CaPHD17, CaPHD19, CaPHD20, and CaPHD43 had a low expression and almost did not change after all of the stress treatments.We speculate that these genes are not involved in the response to drought, low temperatures, high temperatures, or salt stress.Comparing the four treatments, the CaPHD gene expression changed most obviously under high-temperature stress but was not sensitive to salt stress (Figure 6).
in the roots and reached the highest stage with 6 h treatment (Figure 6).The expression of CaPHD29 occurred in response to cold stress, heat stress, and simulated drought stress in the leaves, showing a significant increase in expression under the 6 h treatment.CaPHD37 showed a unique response to heat stress, as it was upregulated by 7-fold after 6 h in the leaves and by 16-fold after 24 h in the roots.CaPHD39 and CaPHD42 were also involved in the response to heat stress in the roots (Figure 6).In addition, CaPHD41 always maintained a high level of expression, which suggests that it is an essential gene CL, ML, NL, HL, and FL represent the leaf samples treated with control, mannitol, Nacl, heat, and cold, respectively; CR, MR, NR, HR, and FR represent the root samples treated with control, mannitol, Nacl, heat, and cold, respectively; 0, 1, 2, 3, 4, 5, and 6 represent 0, 0.5, 1, 3, 6, 12, and 24 h post-treatment, respectively.

Expression Response of CaPHD Genes to Phytohormone Treatment
We used previous expression data to analyze the expression pattern of CaPHD genes in pepper leaves and roots at six different time points (0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h) in response to a hormone treatment with ABA, JA, GA 3 , or SA (Figure 7, Table S4) [25].The results showed that the expression of CaPHD genes responded similarly to the ABA, JA, GA 3 , or SA treatment.The CaPHD genes exhibited low levels of expression in the leaves and high levels of expression in the roots after hormone treatment.The response of CaPHD to JA was earlier than that to the other phytohormones, and it was expressed highest in the roots after 0.5 h of JA treatment (Figure 7).CaPHD25, CaPHD34, and CaPHD35 exhibited the highest expression levels in roots in response to ABA, JA, GA 3 , and SA after 6 h of treatment, demonstrating that the above genes responded most significantly to hormone treatment after six hours (Figure 7).After treatment with any of the four hormones, the expression of CaPHD21, CaPHD27, CaPHD31, and CaPHD37 in leaves showed a downregulated trend, whereas in roots, the expression was upregulated.CaPHD41 always maintained a high level of expression, suggesting that this gene may play an important role in hormonal processing.CaPHD9, CaPHD10, CaPHD11, CaPHD17, CaPHD19, CaPHD20, and CaPHD43 were also found to have almost not changed with phytohormone treatment; we speculate that these genes are not involved in response to the ABA, JA, GA 3 , and SA treatment (Figure 7).

Identification and Phylogenetic Relationships of the CaPHD Gene Family in Pepper
Using the "Zunla-1" genome as the reference genome, a total of 43 PHD proteins were identified from pepper; this number was lower than that identified from maize (67), poplar (73), carrot (106), and cotton (Gossypium hirsutum) (108), but similar to the number identified from tomato (45).We speculate that this is due to the pepper and tomato both belonging to the Solanaceae family and thus having high levels of genomic similarity.According to the amino acid sequences encoded by the CaPHD genes, we carried out an evolutionary analysis of the 43 CaPHD proteins, where CaPHD was divided into four groups.The PHD proteins were categorized into eight groups in rice [9] and eleven or ten groups in maize [7,8].Analysis of the structure of the CaPHD genes in pepper showed that the 43 CaPHD genes exhibited big differences in structure, which was similarly found in maize and rice [7,9].Compared with previous studies on the evolution of PHD proteins in other species, our findings indicate that PHD proteins are more conserved in  Using the "Zunla-1" genome as the reference genome, a total of 43 PHD proteins were identified from pepper; this number was lower than that identified from maize (67), poplar (73), carrot (106), and cotton (Gossypium hirsutum) (108), but similar to the number identified from tomato (45).We speculate that this is due to the pepper and tomato both belonging to the Solanaceae family and thus having high levels of genomic similarity.According to the amino acid sequences encoded by the CaPHD genes, we carried out an evolutionary analysis of the 43 CaPHD proteins, where CaPHD was divided into four groups.The PHD proteins were categorized into eight groups in rice [9] and eleven or ten groups in maize [7,8].Analysis of the structure of the CaPHD genes in pepper showed that the 43 CaPHD genes exhibited big differences in structure, which was similarly found in maize and rice [7,9].Compared with previous studies on the evolution of PHD proteins in other species, our findings indicate that PHD proteins are more conserved in pepper than in other plants.The identification of motifs showed that two of the motifs were highly conserved as they were present in all 43 CaPHD proteins, suggesting that these two motifs may be associated with important functions of the PHD protein family.
Chromosomal localization indicated that most CaPHD genes were distributed in clusters, and most of them were located at the ends of the chromosomes.The distribution of CaPHDs may reflect a strategy for reducing variation and evolutionary divergence.Due to the large genome data on pepper, the diversity in gene structure is known to be closely related to the location of distribution and the range of functions it performs, and it can also predict the rate of evolution of the gene.Therefore, the distribution of PHD genes may provide a functional and structural framework for their role in reducing variation and evolution.Gene expression analysis revealed that CaPHD9, CaPHD10, CaPHD11, CaPHD17, CaPHD19, CaPHD20, CaPHD43, and other CaPHD genes exhibited minimal levels of expression in both the control plants and those exposed to various abiotic stresses.The coding regions of these CaPHD genes are shorter, and the lengths of amino acids are also small; these findings may be related to the evolution of the CaPHD genes in pepper.

CaPHDs Play Roles in Response to Developmental and Abiotic Stress
It has been reported that the PHD genes in plants are involved in the regulation of seed dormancy and in their subsequent germination [14], as well as being involved in the regulation of flowering [15].In addition, PHD genes have been found to be widely involved in regulating the development of anthers and are closely related to the development of male sterility [16][17][18].In the present study, most of the CaPHD genes were expressed differently between 9704A and 9704B in the early stage of flower buds.CaPHD6, CaPHD15, CaPHD17, CaPHD22, CaPHD27, CaPHD28, CaPHD33, CaPHD37, CaPHD38, CaPHD40, and CaPHD42 may be involved in the early development of microspores, whereas CaPHD4, CaPHD10, CaPHD14, CaPHD11, and CaPHD36 may be involved in regulating the fertility of pollen during the microspore maturation phase [26][27][28].Homology studies have shown that CaPHD17 is highly homologous to MS1 in rice, with the latter regulating tapetal programmed cell death and pollen exine formation through its PHD finger [16,29,30].We speculate that other CaPHD genes in the same group as CaPHD17 may also be involved in the development of male sterility.CaPHD10, CaPHD11, and CaPHD17 appeared to be involved in the development of flower buds, a phenomenon that may be functionally related to male sterility, but the expression of these genes was not sensitive to stress treatments.We speculate that the different expressions of CaPHD genes are significant in the early stage of bud development, and it may be the key point involved in the formation of male sterility.
CaPHD9, CaPHD10, CaPHD11, CaPHD17, CaPHD19, CaPHD20, and CaPHD43 responded negligibly to high-temperature, low-temperature, simulated drought, salt, and hormone treatments.Among these genes, CaPHD10, CaPHD11, and CaPHD17 are presumed to be involved in the development of flower buds, which could be closely associated with the development of male sterility.CaPHD25, CaPHD34, and CaPHD35 positively responded to ABA, JA, GA 3 , and SA phytohormone treatments.The expression of CaPHD21, CaPHD27, CaPHD31, and CaPHD37 in leaves following treatment with any of the four hormones was downregulated.These findings are the first to report the response of CaPHD gene expression to ABA, JA, GA 3 , or SA treatment, providing a foundation for the relationship between CaPHD genes in response to treatment with the above four hormones, although the specific function of the CaPHD gene family needs further research and confirmation.
Previous studies on the PHD gene family have focused mainly on their role in development, but PHD genes can also participate in plant stress responses, although this involvement is supported by only a few reports.One study showed that the expression of 15 PHD family genes in maize could respond to drought or high salt stress [7], whereas another study determined that nine PHD family genes in poplars showed differential expression under salt, drought, or cold stress [8], and other studies on soybeans also showed that PHD family genes could respond to drought or salt stress [21,22].The expression of PHD genes in upland cotton occurred in response to a range of abiotic stresses (and phytohormonal treatments) [31].However, there have been few reports on the response of CaPHD family genes to abiotic stresses and phytohormonal treatments in pepper.The present study focuses on the response of CaPHD genes to high temperatures, low temperatures, simulated drought, and high salt levels.CaPHD genes showed differential expression in different tissues and developmental stages, indicating that they have specific expression characteristics that may relate to the specific functions of individual gene family members.

Conclusions
In this present study, a total of 43 CaPHD proteins were identified in pepper, with these CaPHD proteins being divided into four categories.Different CaPHD genes showed different expression patterns in response to different stress or hormone treatments and in different tissues, with strong spatiotemporal specificity.The expression analysis results from different developmental stages of flower buds suggest that some CaPHD genes may be involved in the development of flower buds and may be functionally related to the development of male sterility, a phenomenon that may be of significance to the generation of F 1 hybrid cultivars.The expression analysis results of CaPHD genes in response to abiotic stress and hormone exposure also show that most CaPHD genes are involved in the regulation of stress tolerance, whereas a few genes are involved in only development but not in response to stress.Our research has provided a basis for identifying CaPHD gene family members and for screening for key genes with respect to particular functions while also providing a foundation for elucidating the functional mechanism by which CaPHD genes are involved in hormone response, organ development and abiotic stress response.

Figure 3 .
Figure 3.The structures of CaPHD genes and the conserved motifs of CaPHD proteins.The (a-d) stand for the subfamilies.

Figure 3 .
Figure 3.The structures of CaPHD genes and the conserved motifs of CaPHD proteins.The (a-d) stand for the subfamilies.

Figure 4 .
Figure 4. Predicted cis-regulatory elements in CaPHD promoters.Promoter sequences (2 kb upstream) of CaPHD genes were predicted using PlantCARE.The (a-d) stand for the subfamilies.

Figure 4 .
Figure 4. Predicted cis-regulatory elements in CaPHD promoters.Promoter sequences (2 kb upstream) of CaPHD genes were predicted using PlantCARE.The (a-d) stand for the subfamilies.

Figure 5 .
Figure 5. Expression patterns of CaPHD genes at different stages of flower bud development in 9704A and 9704B.A1-A3 and B1-B3 represent the sporogenous celldivision stage, uninucleate microspore stage, and mature pollen stage in 9704A and 9704B, respectively.

Figure 8 .
Figure 8. Nine CaPHD genes were selected to verify the expression level in 9704A and 9704B by qRT-PCR.S1-S3 represent the sporogenous celldivision stage，uninucleate microspore stage，and mature pollen stage in 9704A and 9704B; The significant differences test was performed using Duncanʹs test (p < 0.05), with different lowercase letters (a,b) indicating significant differences in mean values between different materials at the same stage.

Figure 8 .
Figure 8. Nine CaPHD genes were selected to verify the expression level in 9704A and 9704B by qRT-PCR.S1-S3 represent the sporogenous celldivision stage, uninucleate microspore stage, and mature pollen stage in 9704A and 9704B; The significant differences test was performed using Duncan's test (p < 0.05), with different lowercase letters (a,b) indicating significant differences in mean values between different materials at the same stage.

1 .
Identification and Phylogenetic Relationships of the CaPHD Gene Family in Pepper

Table 1 .
Detailed information on the pepper PHD gene family members.