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

Abstract

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.

Keywords: Japanese apricot; PmKNAT2a; PmKNAT2b; gene expression; lignin; phylogenetic analysis.

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.

Figure 2

The conserved protein domains, gene structures and conserved motifs of PmKNOX genes are based on phylogenetic relationships. The analysis of PmKNOX (A) Phylogenetic tree (I, II and III mean the subfamily). (B) Gene structures. (C) Protein conserved domains. (D) Motif compositions. 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.

Figure 3

The distribution of the PmKNOX gene on the chromosomes and syntenic relationships. The scale on the left represents the length of chromosomes in megabases (Mb). Numbers are on each chromosome in yellow, and segmentally duplicated genes are linked with red lines.

Figure 4

Predicted cis-elements in the PmKNOX promoters. (A) Analysis of the distribution of response elements on PmKNOX promoters. (B) Statistics on the classification and number of PmKNOX promoter response elements. The 2.0 kb sequence upstream from the coding sequence of PmKNOX genes was analyzed using the PlantCARE database.

Figure 5

Expression analysis of the PmKNOX genes in different tissues. Rt for root, St for the stem, L for leaf, L-b for leaf bud, F-b for flower bud, Fl for flower and Fr for fruit. The letter indicates a significant change in the variance analysis (p < 0.05).

Figure 6

Construction of expression vector of PmKNAT2-a and PmKNAT2-b. (a) Vector mapping of p2301-35SN; (b) Construction of p2301-PmKNAT-a vector. M is Marker; lane 1 is p2301-PmKNAT2/6-a vector plasmid; lane 2 is enzyme digestion assay (restriction enzyme: BgllI).

Figure 7

Phenotypic analysis of HE-PmKNAT2a and HE-PmKNAT2b. (A) Comparison of T1-generation transgenic plants with the highest heterologous expression and wild-type Arabidopsis. (B) Comparison of basal leaves of transgenic plants (left) and leaves on the main stem near the base (right) with WT plants. WT represents wild-type Arabidopsis, and HE-PmKNAT2a and HE-PmKNAT2b represent transgenic plants heterologously expressing the PmKNAT2a and PmKNAT2b genes in Arabidopsis, respectively.

Figure 8

Effects of HE-PmKNAT2a and HE-PmKNAT2b on stems. (A) Comparison of longitudinal stem sections of HE-PmKNAT2a, HE-PmKNAT2b and WT. (B) Comparison of the stem diameters of HE-PmKNAT2a, HE-PmKNAT2b and WT. (C) Comparison of plant height of HE-PmKNAT2a, HE-PmKNAT2b and WT. (D) Comparison of the lignin content percent of HE-PmKNAT2a, HE-PmKNAT2b and WT. WT represents wild-type Arabidopsis, and HE-PmKNAT2a and HE-PmKNAT2b represent transgenic plants heterologously expressing the PmKNAT2a and PmKNAT2b genes in Arabidopsis, respectively. The letter indicates a significant change in the variance analysis (p < 0.05).