[Objective] Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. The aim of this study was to identify CAD gene family members in pomegranate and its expression correlation with seed hardness. [Methods] Based on the reported CAD sequence of Arabidopsis, the CAD gene family of pomegranate was identified by homologous comparison, and then phylogenetic, molecular characterization, and expression profile analysis were performed. [Results] Pomegranate CAD gene family has 25 members, distributed on seven chromosomes of pomegranate. All pomegranate CAD proteins have similar physical and chemical properties. We divide the family into four groups based on evolutionary relationships. The member of group I, called bona fide CAD, was involved in lignin synthesis. Most of the members of group II were involved in stress resistance. The functions of groups III and IV need to be explored. We found four duplicated modes (whole genome duplication or segmental (WGD), tandem duplication (TD), dispersed duplication (DSD), proximal duplication (PD) in this family; TD (36%) had the largest number of them. We predicted that 20 cis-acting elements were involved in lignin synthesis, stress resistance, and response to various hormones. Gene expression profiles further demonstrated that the PgCAD gene family had multiple functions. [Conclusions] Pomegranate CAD gene family is involved in lignin synthesis of hard-seeded cultivar Hongyushizi and Baiyushizi, but its role in seed hardness of soft-seeded cultivar Tunisia needs to be further studied.
Anthocyanidin Synthase (ANS) is a key enzyme in the later stages of the anthocyanin biosynthetic pathway, and its role is to convert colorless leucoanthocyanidins to colored anthocyanidins. In this study, a total of 75 members of the pomegranate ANS family were identified and divided into four groups (Group I, Group Ⅱ, Group Ⅲ and Group Ⅳ) based on evolutionary relationships. The 75 ANS gene family members were unevenly distributed on seven of the eight chromosomes of pomegranate. The results of the physical and chemical property analysis showed that 93.33% of the proteins were acidic proteins, 6.67% were alkaline proteins, 28% of the proteins were stable proteins and 72% were unstable proteins. Protein secondary structure analysis showed that α-Spiral and irregular curl are the main structural elements. Analysis of the conserved structural domains of the proteins showed that all 75 ANS family members contained one DIOX -N subfamily structural domain and one 2OG-FeII_Oxy subfamily structural domain. The results of subcellular localization showed that all 75 ANS family members of pomegranate were localized in the cytoplasm. Analysis of the transcriptome data showed that the expression of the pomegranate ANS genes were variety-specific and period-specific.
R2R3-MYB TFs represent one of the most extensive gene families in plants and play a crucial role in regulating plant development, metabolite accumulation, and defense responses. Nevertheless, there has been no systematic investigation into the pomegranate R2R3-MYB family. In this study, 186 R2R3-MYB genes were identified from the pomegranate genome and grouped into 34 subgroups based on phylogenetic analysis. Gene structure analysis showed that the PgR2R3-MYB family in the same subgroup had a similar structure. Gene duplication event analysis revealed that the amplification of the PgMYB family was driven by Whole Genome Duplication (WGD) and dispersed duplication. In the upstream promoter sequence of the PgMYB gene, we identified a large number of plant hormones and environmental response elements. Using phylogenetic analysis and RNA-seq analysis, we identified three PgMYB TFs that may be involved in the regulation of lignin synthesis. Their expression patterns were verified by qPCR experiments. This study provides a solid foundation for further studies on the function of the R2R3-MYB gene and the molecular mechanism of lignin synthesis.
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