Genome-Wide Identification of the KNOX Gene Family in Japanese Apricot (Prunus mume Sieb. et Zucc.) and Functional Characterization of PmKNAT2 Genes

The Knotted1-like Homeobox gene is crucial for plant morphological development and growth. Physicochemical characteristics, phylogenetic relationships, chromosomal localization, cis-acting elements, and tissue-specific expression patterns of the 11 PmKNOX genes found in the Japanese apricot genome in this study were examined. Proteins of 11 PmKNOX were soluble proteins with isoelectric points between 4.29 and 6.53, molecular masses between 15.732 and 44.011 kDa, and amino acid counts between 140 and 430. The identified PmKNOX gene family was split into three subfamilies by jointly constructing the phylogenetic tree of KNOX proteins in Japanese apricot and Arabidopsis thaliana. Combined outcomes of the analyzed conserved motifs and gene structures of the 11 PmKNOX genes from the same subfamily displayed comparable gene structure and motif patterns. The 11 PmKNOX members were distributed across six chromosomes, while two sets of PmKNOX genes were found to be collinear. Analysis of the 2000 bp promoter upstream of the coding region of the PmKNOX gene revealed that most PmKNOX genes might be involved in the physiological metabolism, growth and development processes of plants. The PmKNOX gene expression profile revealed that these genes were expressed at varying levels in different tissues, and most of them were linked to the meristems of leaf and flower buds, suggesting that PmKNOX may be involved in plants’ apical meristems. In Arabidopsis thaliana, functional validation of PmKNAT2a and PmKNAT2b revealed that these two genes might be involved in regulating leaf and stem development. In addition to laying the groundwork for future research on the function of these genes, understanding the evolutionary relationships between members of the PmKNOX gene family provides opportunities for future breeding in Japanese apricots.


Introduction
The Knotted1-like Homeobox genes are a class of transcriptional regulators consisting of homologous heterotypic cassettes that bind to target genes' cis-regulatory regions and play complex regulatory roles in eukaryotic individual development and cell differentiation. The first Homeobox gene isolated in plants was the KNOTTED1 (ZmKN 1 or KN1) gene of maize (Zea mays), which maintains the differentiation of the shoot apical meristem (SAM) and is essential for plant growth and development [1,2]. Subsequently, KNOTTED1-like genes were cloned in Arabidopsis, rice (Oryza sativa), barley (Hordeum vulgare), tomato (Solanum lycopersicum) and other plant species, and phylogenetic analysis revealed that these genes belong to the same gene family, known as the KNOX (KNOTTED I-like homeobox).
The KNOX gene family is classified into classes I, II and M based on structural features, phylogenetic relationships, and expression patterns in many plant species [3][4][5][6]. KNOX consists of four domains: KNOX1, KNOX2 at the conserved N-terminal region, and the ELK domain and homeodomain (HD) at the conserved C-terminal region. Class I KNOX

Plant Materials
The samples of Japanese apricot were collected from the National Field GeneBank for Prunus mume in Nanjing, Jiangsu Province, China, with the cultivar 'Longyan' as the sampled cultivar. The root stems, leaves, flower buds, and leaf buds of Japanese apricot were collected in early October 2022, while fruit samples were collected in June of the same year. The samples were immediately put in liquid nitrogen and then transported and stored in a refrigerator at −80 • C for subsequent experiments.
Sterilized Arabidopsis seeds (Col0 and transgenes) were sown in 1/2MS medium. After vernalization at 4 • C, the seedlings were placed in a plant light incubator (photoperiod: 16 h light and 8 h dark; Constant temperature: 22 • C; Humidity: 80%) until four true leaves had developed. Seedlings with good growth traits were selected and transferred to the nutrient soil mixed with perlite and vermiculite for further experiments.

Identification of PmKNOX Family Members
The Japanese apricot genome (GCF_000346735.1_P.mume_V1.0) file and its annotation file were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 20 October 2022). The hidden Markov model of KNOX (pfam03790, pfam03791, pfam03789 and pfam05920) was retrieved from PFAM (http://pfam.xfam.org/, accessed on 20 October 2022) as query domains for extracting KNOXs in P.mume local protein database. The candidate KNOXs (E value >  in Japanese apricot were screened using HMMER Software (http://hmmer.org/, accessed on 20 October 2022), and the KNOXs protein sequences were extracted by TBtools software [19]. After removing redundant and repeated sequences, the peach (Prunus persica) KNOX [20] was used as query sequences in a phylogenetic analysis with MAFFT v7.475 software [21]. We used the maximum likelihood (ML) method to construct phylogenetic trees to obtain the phylogenetic relationships. The ML analysis was conducted using IQ-TREE [22], with the best model selected by ModelFinder software [23]. The Bootstrap parameter is set to 1000 and beautified through iTOL software. Due to the location of KNOXs on the chromosome in P. mume, the genes were numbered PmKNOX1 to PmKNOX11. The protein properties of PmKNOXs, such as the molecular weight (MW), isoelectric point (pI) and grand average of hydropathicity (GRAVY) of each KNOX protein, were predicted by using ProtParam (https://web.expasy.org/protparam/, accessed on 21 October 2022). The BUSCA website (http://busca.biocomp.unibo.it/, accessed on 21 October 2022) was used to predict the subcellular localization.

Analysis of Structure and Conserved Motifs of PmKNOX Gene in Japanese Apricot
GSDS tool [24] was used to analyze and draw the gene structure map of PmKNOX gene family members. The protein sequences of PmKNOX genes were submitted to MEME (http://meme-suite.org/tools/meme, accessed on 22 October 2022) to search for conserved motifs of PmKNOX, setting the length to 6-50 and the number to 20 and other parameters were the default value and visualization of results by the software of TBtools.

Chromosomal Location and Syntenic Analysis
P.mume.gtf file and PmKNOX genes list was used as the input files, and TBtools software was used to analyze the location on the chromosomes of PmKNOX genes. Meanwhile, MCScanX software (http://chibba.pgml.uga.edu/mcsca n2/, accessed on 21 October 2022) was used to analyze the tandem and segmented replication events of PmKNOX family members.

Analysis of Cis-Acting Elements in PmKNOX Gene Upstream Promoter Region
The 2000 bp region upstream of the PmKNOX transcription start site (ATG) was extracted from the P. mume genome, and the cis-acting elements were identified by the PlantCARE tool [25], and then these cis-acting elements were analyzed and classified. On the basis of the PmKNOX gene family sequence, a phylogenetic analysis was conducted, and protein sequences were aligned using the MAFFT software with default parameters [21]. ML was used to construct a phylogenetic tree to determine the phylogenetic relationship. The best model was chosen by ModelFinder software and used in the ML study using IQ-TREE. The iTOL programmer is used to enhance the Bootstrap parameter, which is set to 1000.

Expression Profiling Analyses of PmKNOX Genes
The PmKNOX gene expression pattern in the root, stem, leaf, leaf bud, flower bud, flower and fruit were also analyzed. Total RNA was extracted from tissue with an RNA extraction kit (Tiangen, China), and the concentration of extracted RNA was determined, and agarose gel electrophoresis was used to ensure its quality. For the synthesis of cDNA, it was performed in reference to the operation manual of PrimeScript™ RT reagent Kit with gDNA Eraser. qRT-PCR was performed using ABI 7300 real-time PCR system (Applied Biosystems, Foster, CA, USA) and SYBR green real-time PCR Master Mix (Dongbao, Osaka, Japan); primers are shown in Supplementray Table S1. Each bioreactor had three replicates in each analysis. The housekeeping Japanese apricot DNA-directed RNA polymerases II (PmRP2, LOC103335337) gene was determined as an internal control [26][27][28]. Three repeated quantitative analysis was performed for each cDNA sample, and the relative gene expression level was calculated by the 2 −∆∆CT method.

PmKNAT2a and PmKNAT2b Cloning and Sequence Analysis
The reference sequences of PmKNOX2a (LOC103343109) and PmKNOX2b (LOC103321797) were found in NCBI, and the Japanese apricot cv 'Longyan' was used as the template for gene cloning. Primers (Supplementary Table S1) were designed by reference sequence information, and cloning experiments were performed to obtain PCR products, which were subcloned into the clone007 blunt simple vector and sequenced at TsingKe (Nanjing, China). The sequences obtained were compared using BioXM 2.7 software.

Vector Construction, Plant Transformation, and Identification of Transgenic Lines
PmKNAT2a and PmKNAT2b coding sections were subcloned and placed into the p2301-35SN vector. The flower-dip technique was used to genetically modify Arabidopsis (by Agrobacterium-mediated means). Genomic DNA level detection, GUS staining confirmation, and RNA level detection were used to identify the transgenic lines.

Determination of Lignin Percentage Content
The stems of transgenic and wild-type plants at the same growth stage were placed in an oven at 80 • C, dried to constant weight and ground to powder. 3 mg of powder was extracted for lignin according to the instructions of Solarbio (Beijing, China) (BC4205 kit), and the absorbance value A at 280 nm was measured using a microplate reader (recording A as sample), and A = A − A control was calculated. The percentage (%) of lignin = ∆A ÷ ε ÷ d × V detected ÷ (V supernatant ×W ÷ V acetylated) ÷ 1000 × 100% (V acetylated: acetylation reaction volume, 0.612 mL. ε: lignin extinction coefficient, 23.35 mL/mg/cm. d: light diameter of cuvette, 1 cm. V supernatant: supernatant volume, 0.012 mL. V detection: detection volume, 0.6 mL. W: sample quality, g; 1000: conversion factor, 1 g = 1000 mg).

Measurement of Leaf Area
The original method of counting grids (1 mm 2 /grid) was used in this study to obtain more accurate leaf areas because the leaves margin of transgenic plants is curled and not well fixed. After tracing the leaf traits along the leaf margin on the grid paper, the number of grids was counted. Three strains with high expression of heterologous PmKNAT2 were taken for the experiment, and three replicates were set up for each group of experiments.

Statistical Analyses
The student's t-test was performed by IBM SPSS Statistics 23 programmer (ANOVA). To discover whether the test was significantly different or not, a 0.05 p-value cutoff was used. Using GraphPad Prism 8 software, standard deviation (SD) analysis of biological replicates of independent samples was performed, and error bars and graphs were plotted.

Identification of KNOX Family Members in Japanese Apricot
The KNOX genes family conserved structural domains KNOX1 (pfam03790), KNOX2 (pfam03791), ELK (pfam03789) and Homeobox KN (pfam05920). A phylogenetic tree was constructed, including the candidate genes and ten KNOX genes from the peach (Prunus persica) (Figure 1) [29]. According to the division of Peach's PpKNOX gene family [20], independent samples was performed, and error bars and graphs were plotted.

Figure 1.
Phylogenetic tree of KNOX proteins from Japanese apricot and peach. The multiple protein sequences of 11 PmKNOX genes and 10 PpKNOX genes were aligned with the MUSCLE method, and the tree was built using the Maximum Likelihood method by using MAFFT v7.475 (bootstrap value: 1000). The tree was categorized into three subfamilies that include Class I (blue backdrop), Class II (yellow backdrop) and Class M (green backdrop). The PmKNOX proteins have been emphasized in red. The black font is the KNOX gene of peach.
The physical and chemical properties of PmKNOX family genes are shown in Table  1, including gene name, Gene ID, Protein ID, MW, pI, GRAVY value and subcellular location prediction. The average length of the CDS sequence of the PmKNOX gene is about 963 bp, of which the PmKNOX2 (1293 bp) gene sequence is the longest, and the PmKNOX9 (423 bp) sequence is the shortest.
The amino acid composition of PmKNOX family proteins is different, and their corresponding physical and chemical properties are also very different. The pI of the PmKNOX family was between 4.29 (PmKNOX4) and 6.53 (PmKNOX7). In general, all Figure 1. Phylogenetic tree of KNOX proteins from Japanese apricot and peach. The multiple protein sequences of 11 PmKNOX genes and 10 PpKNOX genes were aligned with the MUSCLE method, and the tree was built using the Maximum Likelihood method by using MAFFT v7.475 (bootstrap value: 1000). The tree was categorized into three subfamilies that include Class I (blue backdrop), Class II (yellow backdrop) and Class M (green backdrop). The PmKNOX proteins have been emphasized in red. The black font is the KNOX gene of peach. The physical and chemical properties of PmKNOX family genes are shown in Table 1, including gene name, Gene ID, Protein ID, MW, pI, GRAVY value and subcellular location prediction. The average length of the CDS sequence of the PmKNOX gene is about 963 bp, of which the PmKNOX2 (1293 bp) gene sequence is the longest, and the PmKNOX9 (423 bp) sequence is the shortest.
The amino acid composition of PmKNOX family proteins is different, and their corresponding physical and chemical properties are also very different. The pI of the PmKNOX family was between 4.29 (PmKNOX4) and 6.53 (PmKNOX7). In general, all PmKNOX proteins are rich in acidic amino acids (Pi < 7) and are hydrophilic(GRAVY < 0). The prediction of the subcellular location of the PmKNOX gene family showed that all the PmKNOX genes were localized in the nucleus.

Secondary Structure Analysis of PmKNOX Protein
The analysis of the structure of the proteins encoded by the PmKNOX transcription factor family showed ( Table 2) that there were some differences in the protein secondary structure among the members. The analysis of the protein secondary structure of protein allows us to determine the biological functions of PmKNOX proteins. Predictive analysis revealed that of the 11 PmKNOX protein sequences, six amino acid sequences (PmKNOX4, PmKNOX7, PmKNOX8, PmKNOX9, PmKNOX10 and PmKNOX11) were predominantly composed of α helix, and five amino acid sequences (PmKNOX1, PmKNOX2, PmKNOX3, PmKNOX5 and PmKNOX6) consisted mainly of random coils. β turn and Extended strands are scattered throughout each protein sequence.

Gene Structures, Protein Conserved Domain and Motif Compositions of PmKNOX Proteins
To investigate further the evolutionary relationships and gene structures among the PmKNOX members of Japanese apricot, we constructed a phylogenetic tree of the Pm-KNOX members (Figure 2A), mapped the PmKNOX gene structures ( Figure 2B), and analyzed the characteristics of the conserved domains of the protein sequences and the composition of Motif ( Figure 2C,D). The phylogenetic analysis showed that PmKNOX was divided into three subfamilies: PmKNOX1 was an independent subfamily (Subfamily I); PmKNOX2, PmKNOX10, PmKNOX8, PmKNOX4 and PmKNOX9 were clustered into a subfamily (Subfamily II); PmKNOX3, PmKNOX6, PmKNOX7, PmKNOX5 and PmKNOX11 formed a subfamily (Subfamily III). This indicates that PmKNOX1 may be genetically distant from the other PmKNOX genes. Structural analysis of the PmKNOX genes revealed that the number of exons in the PmKNOX genes ranged from 3 to 6, with PmKNOX4 and PmKNOX9 having three exons and PmKNOX6 and PmKNOX7 having four exons. The genes with the highest number of five exons were PmKNOX1, PmKNOX10, PmKNOX8, Pm-KNOX3, PmKNOX5, and PmKNOX11, and only one gene, PmKNOX2, had six exons. Most of the PmKNOX members have 5 UTR and 3 UTR, which contribute to the maintenance of mRNA stability and microRNA binding, with only PmKNOX7 lacking the 5 UTR structure and PmKNOX9 lacking the 3 UTR structure. The division of the PmKNOX subfamily was not significantly correlated with the structure of PmKNOX gene exons. However, PmKNOX genes classified into the same subfamily exhibited similar gene structure and Motif pattern. tant from the other PmKNOX genes. Structural analysis of the PmKNOX genes revealed that the number of exons in the PmKNOX genes ranged from 3 to 6, with PmKNOX4 and PmKNOX9 having three exons and PmKNOX6 and PmKNOX7 having four exons. The genes with the highest number of five exons were PmKNOX1, PmKNOX10, PmKNOX8, PmKNOX3, PmKNOX5, and PmKNOX11, and only one gene, PmKNOX2, had six exons. Most of the PmKNOX members have 5′UTR and 3′UTR, which contribute to the maintenance of mRNA stability and microRNA binding, with only PmKNOX7 lacking the 5′UTR structure and PmKNOX9 lacking the 3′UTR structure. The division of the PmKNOX subfamily was not significantly correlated with the structure of PmKNOX gene exons. However, PmKNOX genes classified into the same subfamily exhibited similar gene structure and Motif pattern.  The multiple protein sequences of the 11 PmKNOX gene were aligned with the MUSCLE method, and the tree was built using the neighbor-joining method by using MEGA-X. The length of the gene structure and motif components can be estimated by referring to the corresponding scales below.
As members of the KNOX gene family, all PmKNOX members contain the KNOX1 and KNOX2 conserved domains, whereas the majority of PmKNOX genes, except PmKNOX4 and PmKNOX9, also contain the Homeobox_KN and ELK conserved domains. MEME analysis of the PmKNOX gene family composition of Motif revealed that all PmKNOX family members contained Motif2, Motif3 and Motif8. Except for PmKNOX4 and Pm-KNOX9 contained Motif1, and Motif4, whereas no subfamily II members contained the motif5 pattern. In addition, PmKNOX10, PmKNOX8, and PmKNOX5 contained the largest number of Motifs, while PmKNOX4 and PmKNOX9 had only three Motifs. The above analysis indicates that the PmKNOX gene members have different gene functions among individuals. This is consistent with the previous description that class I and class II KNOX genes in angiosperms have different exon-intron structures [7].

Chromosomal Distributions Analysis of PmKNOX Genes
Based on the annotation information of the Japanese apricot genome, the chromosome mapping of 11 PmKNOX genes was obtained ( Figure 3). We found that the genes of PmKNOX were unevenly distributed on the chromosomes: PmKNOX1 was located on LG1, PmKNOX2-5 were all distributed on LG2, PmKNOX6 and PmKNOX7 were distributed on LG3 and LG4, respectively, PmKNOX8 and PmKNOX9 were distributed on LG7, Pm-KNOX10 on LG8, and no clear position of PmKNOX11 on the chromosome was obtained. On the Japanese apricot's chromosomes LG5 and LG6, no members of the PmKNOX gene family were found (consistent with the results in Table 1). The analysis revealed that there was no significant correlation between the length of chromosomes and the distribution of PmKNOX genes. Colinear analysis of the PmKNOX genes revealed two pairs of segmental repeat genes (PmKNOX2 and PmKNOX10, PmKNOX6 and PmKNOX7).
LG1, PmKNOX2-5 were all distributed on LG2, PmKNOX6 and PmKNOX7 were distributed on LG3 and LG4, respectively, PmKNOX8 and PmKNOX9 were distributed on LG7, PmKNOX10 on LG8, and no clear position of PmKNOX11 on the chromosome was obtained. On the Japanese apricot's chromosomes LG5 and LG6, no members of the PmKNOX gene family were found (consistent with the results in Table 1). The analysis revealed that there was no significant correlation between the length of chromosomes and the distribution of PmKNOX genes. Colinear analysis of the PmKNOX genes revealed two pairs of segmental repeat genes (PmKNOX2 and PmKNOX10, PmKNOX6 and PmKNOX7).

Cis-Acting Elements Analysis in PmKNOX Gene Promoter Region
In order to better understand the transcriptional mechanism of PmKNOX, we extracted and studied the 2000 bp promoter upstream of the coding sequence (CDS) of the eleven PmKNOX genes. The analysis revealed that the promoter region of PmKNOX genes contains a large number of cis-acting elements, which can be classified into three types: stressresponsive related elements type, hormone-responsive related elements type, and plant growth and development-related elements type ( Figure 4).

Cis-Acting Elements Analysis in PmKNOX Gene Promoter Region
In order to better understand the transcriptional mechanism of PmKNOX, we extracted and studied the 2000 bp promoter upstream of the coding sequence (CDS) of the eleven PmKNOX genes. The analysis revealed that the promoter region of PmKNOX genes contains a large number of cis-acting elements, which can be classified into three types: stress-responsive related elements type, hormone-responsive related elements type, and plant growth and development-related elements type ( Figure 4). Among all members of the PmKNOX gene family, the PmKNOX2 gene contains 36 cis-acting elements, and all other genes contain more than 16 cis-acting elements. Analysis of the distribution of elements on the promoters of PmKNOX genes showed that there were 161 growth-related elements, of which 134 were related to light response, accounting for 83.2% of the elements. There are 10 meristem expression-related elements, accounting for 6%, and the rest are zein metabolism regulation elements, cell cycle regulation ele- Among all members of the PmKNOX gene family, the PmKNOX2 gene contains 36 cis-acting elements, and all other genes contain more than 16 cis-acting elements. Analysis of the distribution of elements on the promoters of PmKNOX genes showed that there were 161 growth-related elements, of which 134 were related to light response, accounting for 83.2% of the elements. There are 10 meristem expression-related elements, accounting for 6%, and the rest are zein metabolism regulation elements, cell cycle regulation elements and flavonoid biosynthetic regulation elements, accounting for 10.8%. There were 91 hormone response-related elements, including 35 abscisic acid response elements, 32 methyl jasmonate response elements, 11 gibberellin response elements, 11 salicylic acid response elements, and 9 auxin response elements. 9 response elements, and 4 auxin response elements. There were 39 stress-related response elements, including anoxic specificity, anaerobic induction, drought-inducibility, low-temperature responsiveness, defense and stress. The above study indicates that PmKNOX genes are actively involved in plant growth and development and physiological and metabolic processes.

Expression Profiles Analysis of PmKNOX Genes in Different Tissues
To investigate the expression pattern of PmKNOX genes in different tissues of Japanese apricot, the expression levels of 11 PmKNOX genes were analyzed in the root (Rt), stem According to the findings, roots and leaves hardly ever expressed PmKNOX1, while flowers were the main organ. PmKNOX3 was almost exclusively expressed in the stems and barely in the leaves and fruits. Roots had the highest levels of PmKNOX4 expression compared to other tissues. PmKNOX6 was almost entirely absent in roots, leaves, and fruits and was mostly expressed in stems and flowers. PmKNOX7 was predominantly expressed in flower buds and leaf buds. The Japanese apricot's PmKNOX2, PmKNOX8, and According to the findings, roots and leaves hardly ever expressed PmKNOX1, while flowers were the main organ. PmKNOX3 was almost exclusively expressed in the stems and barely in the leaves and fruits. Roots had the highest levels of PmKNOX4 expression compared to other tissues. PmKNOX6 was almost entirely absent in roots, leaves, and fruits and was mostly expressed in stems and flowers. PmKNOX7 was predominantly expressed in flower buds and leaf buds. The Japanese apricot's PmKNOX2, PmKNOX8, and PmKNOX10 genes were expressed in every tissue. These PmKNOX genes may be involved in the growth and development of Japanese apricots and may have various activities based on the varying expression levels of these genes in various tissues. The PmKNOX gene expression profile again showed that these genes were expressed at different levels in different tissues. Most of them were associated with the meristems of leaves and flower buds, suggesting that PmKNOX may be involved in the apical meristem of plants; these results support the previous conclusion that most KNOX family genes are important for maintaining the state of apical meristem [30][31][32]. In the phylogenetic tree created in conjunction with peach (Prunus persica) KNOX genes, PmKNOX5 and PmKNOX11 belonged to the same homeobox protein knotted-1-like 2 ( Figure 1). PmKNOX5 expression was significantly higher in leaf buds and stemmed than in other tissues, and PmKNOX11 expression was also higher in leaf buds, indicating that these two genes may be crucial in the early stages of leaf and stem differentiation and merit further study.

Heterologous Expression of PmKNAT2 Genes in Arabidopsis
Since PmKNOX5 and PmKNOX11 are more closely related to PpKNAT2 of peach (Prunus persica) and belong to the Homeobox Knotted-1-like 2, these two genes were named PmKNAT2a (formerly PmKNOX11) and PmKNAT2b (formerly PmKNOX5) for simplicity for subsequent studies and classification. The CDS of PmKNAT2-a and PmKNAT2-b were obtained by cloning, respectively, and the CDS of these two genes were subcloned into the expression vector p2301-35SN ( Figure 6). buds, suggesting that PmKNOX may be involved in the apical meristem of plants; these results support the previous conclusion that most KNOX family genes are important for maintaining the state of apical meristem [30][31][32]. In the phylogenetic tree created in conjunction with peach (Prunus persica) KNOX genes, PmKNOX5 and PmKNOX11 belonged to the same homeobox protein knotted-1-like 2 ( Figure 1). PmKNOX5 expression was significantly higher in leaf buds and stemmed than in other tissues, and PmKNOX11 expression was also higher in leaf buds, indicating that these two genes may be crucial in the early stages of leaf and stem differentiation and merit further study.

Heterologous Expression of PmKNAT2 Genes in Arabidopsis
Since PmKNOX5 and PmKNOX11 are more closely related to PpKNAT2 of peach (Prunus persica) and belong to the Homeobox Knotted-1-like 2, these two genes were named PmKNAT2a (formerly PmKNOX11) and PmKNAT2b (formerly PmKNOX5) for simplicity for subsequent studies and classification. The CDS of PmKNAT2-a and PmKNAT2-b were obtained by cloning, respectively, and the CDS of these two genes were subcloned into the expression vector p2301-35SN ( Figure 6).  Figure S1). Phenotypic analysis revealed that HE-PmKNAT2a and HE-PmKNOX2b Arabidopsis leaves all became lobed (more pronounced in HE-PmKNOX2b) with curled leaf margins compared to the wild type (Figure 7). This is consistent with the phenotype of Arabidopsis heterologously transformed with KNAT2 from maize and Pinus pinaster [14,33,34]. The vector plasmids were transformed into Agrobacterium tumefaciens and then infested with Arabidopsis. Four heterologous expressions of PmKNAT2-a Arabidopsis lines (HE-PmKNAT2-a) and five heterologous expressions of PmKNAT2-b Arabidopsis lines (HE-PmKNAT2/6-b) were obtained by screening the transgenic strains (Supplementary Figure  S1). Phenotypic analysis revealed that HE-PmKNAT2a and HE-PmKNOX2b Arabidopsis leaves all became lobed (more pronounced in HE-PmKNOX2b) with curled leaf margins compared to the wild type (Figure 7). This is consistent with the phenotype of Arabidopsis heterologously transformed with KNAT2 from maize and Pinus pinaster [14,33,34]. In comparison with the wild type, we found an interesting phenomenon: heterologous expression of PmKNAT2a resulted in smaller leaf area near the base and basal leaves in Arabidopsis, whereas heterologous expression of PmKNAT2b resulted in larger leaves at the base and smaller leaves near the base in Arabidopsis (Table 3).

Effect of Heterologous Expression of PmKNAT2a and PmKNAT2b on Arabidopsis Stems
To investigate the effects of heterologous expression of PmKNAT2a (HE-PmKNAT2a) and PmKNAT2b (HE-PmKNAT2b) on the growth of Arabidopsis stems, we intercepted the stems of HE-PmKNAT2a, HE-PmKNAT2b, and wild-type plants (WT) near the base, respectively, and showed their stem phenotype ( Figure 8A). By observation, we found that the WT and the HE-PmKNAT2a stems exhibited a red color, while the HE-PmKNAT2b stems were green. Analysis of the leaf spacing of HE-PmKNAT2a and HE-PmKNAT2b revealed ( Table 4) that the leaf spacing of HE-PmKNAT2a was shorter than that of the wild type, and HE-PmKNAT2b was similar to the wild type only between the distance In comparison with the wild type, we found an interesting phenomenon: heterologous expression of PmKNAT2a resulted in smaller leaf area near the base and basal leaves in Arabidopsis, whereas heterologous expression of PmKNAT2b resulted in larger leaves at the base and smaller leaves near the base in Arabidopsis (Table 3).

Effect of Heterologous Expression of PmKNAT2a and PmKNAT2b on Arabidopsis Stems
To investigate the effects of heterologous expression of PmKNAT2a (HE-PmKNAT2a) and PmKNAT2b (HE-PmKNAT2b) on the growth of Arabidopsis stems, we intercepted the stems of HE-PmKNAT2a, HE-PmKNAT2b, and wild-type plants (WT) near the base, respectively, and showed their stem phenotype ( Figure 8A). By observation, we found that the WT and the HE-PmKNAT2a stems exhibited a red color, while the HE-PmKNAT2b stems were green. Analysis of the leaf spacing of HE-PmKNAT2a and HE-PmKNAT2b revealed ( Table 4) that the leaf spacing of HE-PmKNAT2a was shorter than that of the wild type, and HE-PmKNAT2b was similar to the wild type only between the distance from the base to the first leaf, while all other leaf spacing was shorter than that of the wild type. The measurement of stem diameter revealed that the stems of wild-type Arabidopsis plants were significantly thicker than those of transgenic Arabidopsis ( Figure 8B). Meanwhile, the plant height was measured, and it was found that the plants of wild-type Arabidopsis were significantly longer than the transgenic type ( Figure 8C). A similar phenotype was observed in Populus overexpressing Populus alba × P. glandulosa KNAT2/6b (PagKNAT2/6b) [35].
Genes 2023, 14, x FOR PEER REVIEW 13 of 19 from the base to the first leaf, while all other leaf spacing was shorter than that of the wild type. The measurement of stem diameter revealed that the stems of wild-type Arabidopsis plants were significantly thicker than those of transgenic Arabidopsis ( Figure 8B). Meanwhile, the plant height was measured, and it was found that the plants of wild-type Arabidopsis were significantly longer than the transgenic type ( Figure 8C). A similar phenotype was observed in Populus overexpressing Populus alba × P. glandulosa KNAT2/6b (PagKNAT2/6b) [35].  Since the class I KNOX genes have been shown to be involved in the lignin deposition process [36,37]. Therefore we tested the lignin percentage content of transgenic and wildtype Arabidopsis stems ( Figure 8D). It was discovered that lignin concentration was much reduced in plants with HE-PmKNAT2a and HE-PmKNAT2b compared to the wild type,  Since the class I KNOX genes have been shown to be involved in the lignin deposition process [36,37]. Therefore we tested the lignin percentage content of transgenic and wildtype Arabidopsis stems ( Figure 8D). It was discovered that lignin concentration was much reduced in plants with HE-PmKNAT2a and HE-PmKNAT2b compared to the wild type, indicating that PmKNAT2a and PmKNAT2b may be involved in the process of lignin deposition in plants.

Discussion
The Knotted1-like Homeobox (KNOX) genes are important regulators of meristem function, and their expression is tightly controlled by a complex network of transcription factors [38,39]. In the present study, 11 KNOX genes members have been identified in the genome of the Japanese apricot, with almost the same number as in tomato (8 members) [40], Arabidopsis (9 members) [11], coastal pine (6 members) [33], but less than apple (22 members) [4], Soybean (34 members) [3], etc. This result indicates that most PmKNOX genes have not been selectively eliminated by the environment, and no obvious gene family amplification events have occurred.
Phylogenetic analysis of KNOX proteins in Japanese apricot and Arabidopsis revealed that the Japanese apricot KNOX family is divided into three subfamilies: class I KNOX genes, class II KNOX genes and class M KNOX genes. The class I KNOX genes KNAT1, KNAT2 and KNAT6 in Arabidopsis are expressed in the flower meristem and affects the process of flower development [41,42]. The class I KNOX genes are specific in temporal and spatial expression patterns, have both redundant functions and their own specific effects in function, and eventually affect the morphogenesis of lateral organs by regulating cell differentiation [7,43]. The class II KNOX proteins operate as repressors in the transcriptional control of their target genes, in contrast to class I KNOX proteins, which appear to be activators [44]. The Class II KNOX genes include KNAT3, KNAT4, KNAT5, and KNAT7, which are expressed in all plant tissues and are associated with seed germination and secondary wall formation [45]. The class M KNOX genes are mainly expressed at the boundary between plant organ primordium and mature organs and may be involved in sensing or generating flowering signals [11]. Thus, the potential function and research direction of Japanese apricot KNOX clustered into the same group of KNOX genes in Arabidopsis can be inferred.
Combined gene structure and conserved Motif analysis revealed that PmKNOX belonging to the same subfamily exhibit similar gene structure and motif patterns, which may exercise similar biological functions. In general, the differences in the exon and intron structure of genes are the main reasons for the diversification of gene functions. The PmKNOX genes show similar intronic and exonic structures within the same subfamily, indicating high conservation of the family members in evolution, and the results were consistent with those of previous studies [7]. The predicted protein secondary structures of the 11 sequences showed that the PmKNOX protein sequence was mostly made up of random coils or α helices. Chromosome mapping results showed that 11 PmKNOX members were distributed in 6 chromosomes, and two pairs of genes PmKNOX genes had a collinear relationship.
The predicted cis-acting elements of PmKNOX genes showed that the promoter regions of these 11 PmKNOX gene family members were rich in a large number of growth and development-related elements, hormone response-related cis-acting elements and stressrelated elements. Therefore, the PmKNOX gene may affect plant growth, development and morphogenesis by influencing hormonal and metabolic pathways. According to RT-qPCR analysis, like in other plants, the majority of the PmKNOX family genes were slightly expressed differently in various tissues [46], indicating that PmKNOX is probably actively involved in a variety of plant growth and developmental processes [7,47]. At the same time, we noticed that PmKNOX was highly expressed in the leaf buds and flower buds of Japanese apricot, indicating that PmKNOX played an important role in maintaining the meristem potential, which is consistent with previous research findings [14,48].
PmKNOX5 and PmKNOX11 are phylogenetically related to the class I KNOX gene KNAT2 of peach ( Figure 1). Additionally, KNAT2 has been studied more intensively in many species, and several studies have shown that this class of genes regulates the formation and growth of lateral organs and can be expressed in the apical meristematic tissue to determine the fate of meristematic cells, which plays an important role in leaves, stems, etc. of plants [18,33,42,49]. However, functional studies on the PmKNAT2 gene in Japanese apricot have not been conducted. To perform a functional analysis of the PmKNAT2 gene of Japanese apricot in Arabidopsis, PmKNOX11 was named PmKNAT2a and PmKNOX5 named PmKNAT2b based on phylogenetic relationships, and the phenotypes of transgenic Arabidopsis observed. Leaf shape in plants is regulated by genetic, developmental, and environmental factors involving the regulation of a complex genetic network between hormonal signals and transcription factors. In Arabidopsis, leaves heterologously expressing PmK-NAT2a (HE-PmKNAT2a) and PmKNAT2b (HE-PmKNAT2b) had altered leaf phenotypes. Leaves of HE-PmKNAT2ah and HE-PmKNAT2b were twisted with lobed leaf margins, with HE-PmKNAT2b having more pronounced lobbing. It has been shown that leaves with deeper serrations or fissures have shorter heat transfer distances compared to full-rimmed leaves and are able to increase heat dissipation efficiency per unit leaf area, which has an important role in plant response to high-temperature stress [50].
In leaf area determination, heterologous expression of PmKNAT2a resulted in smaller leaf area near the base and basal in Arabidopsis, whereas heterologous expression of PmK-NAT2b resulted in larger leaves near the base and smaller leaves near the base in Arabidopsis. It has been shown that REDUCED COMPLEXITY (RCO) and class I KNOX genes in Cardamine hirsuta leaves increase leaf complexity (i.e., there will be various morphological and size-invariant leaves), similar to the findings herein, and demonstrates that PmKNAT2 may have a function in regulating leaf development. In addition, these two genes are barely expressed in leaves, suggesting that the period during which they are involved in leaf morphogenesis should be the period of leaf bud differentiation. These results seem to confirm the finding that class I KNOX genes regulate plant morphogenesis [51].
Lignin is commonly found in the xylem, parenchyma and cortical cells and is one of the main components of the xylem, and its biosynthesis plays an important role in plant cell growth and differentiation [52]. The class I KNOX genes may indirectly affect lignin synthesis by regulating the synthesis and metabolism of GAs or directly regulate the expression of lignin-synthesizing genes that affect the lignin deposition [53]. Combining these phenotypes of the phenomenon of shorter, thinner stems, shorter leaf spacing, and lower lignin content in HE-PmKNAT2a and HE-PmKNAT2b suggests that this may be related to class I KNOX gene expression promoting GAs synthesis [54]. GA20ox, a key enzyme in the plant GAs degradation process, is mainly expressed in the xylem and overexpression in poplar leads to stem elongation of the plant [55]. GA2ox, a key enzyme in plant GAs synthesis, is overexpressed in persimmon (Diospyros kaki) and leads to dwarfism of the plant [56]. These two genes work together in the plant to maintain the dynamic balance of active GAs. It has been demonstrated that class I KNOX genes in Rorippa aquatica, potato (Solanum tuberosum), and maize apical meristem tissues can directly bind to the active element on the GA20ox gene promoter, thereby inhibiting the expression of GA20ox and leading to dwarfing of the plant [57][58][59]. The PagKNAT2/6b gene of poplar is a homolog of Arabidopsis KNAT2 and KNAT6, and when this gene was overexpressed, the internode elongation of poplar was found to be inhibited, the content of GAs was significantly decreased, and anatomical analysis revealed that the length of epidermal cells, medullary cells, xylem fibers and ductal cells in transgenic poplar stems were significantly shorter [17]. Overexpression of class I KNOX genes in Arabidopsis leads to dwarf plants and leaf ruffling, and the aforementioned phenotypes make transgenic plants more drought tolerant, while studies have demonstrated that class I KNOX responds to drought-stressed environments by mediating the ability of stem cell division and differentiation in phloem tissues [60]. In this study, a preliminary exploration of the gene function of PmKNAT2 was conducted in Arabidopsis heterologously transformed with the PmKNAT2 gene, which was found to be potentially involved in leaf and stem development and lignin biosynthesis.

Conclusions
This study identified 11 KNOX gene family members on six chromosomes of the Japanese apricot genome and that PmKNOX can be divided into three subfamilies in phylogenetic tree analysis with KNOX proteins in Arabidopsis (I, II and M). The 11 Pm-KNOX gene structure and conserved Motif analysis showed that PmKNOX from other same subfamily had similar gene structure and motif patterns, thus suggesting that they have similar biological functions. The promoter analysis and expression profile analysis of the PmKNOX gene showed that it was actively involved in physiological metabolism, growth and development and may play a potential role in the apical meristem. Heterologous expression of PmKNAT2a and PmKNAT2b resulted in shorter plant height and abnormal leaf edge development in Arabidopsis and decreased lignin in stems. Hence these two genes may regulate leaf and stem development. This research advances our knowledge of the origin and biological significance of the KNOX gene family in the Japanese apricot and provides a theoretical foundation for the future application of PmKNOX functional gene studies.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14040939/s1, Table S1: Primer sequences used for qRT-PCR and PCR in Japanese apricot; Figure