Genome-Wide Analysis and Characterization of the SDR Gene Superfamily in Cinnamomum camphora and Identification of Synthase for Eugenol Biosynthesis

Abstract

Short-chain dehydrogenase/reductases (SDRs) are the largest NAD(H)-dependent oxidoreductase superfamilies and are involved in diverse metabolisms. This study presents a comprehensive genomic analysis of the SDR superfamily in Cinnamomum camphora, a species that is one of the most significant woody essential oil plants in southern China. We identify a total of 222 CcSDR proteins and classify them into five types based on their cofactor-binding and active sites: 'atypical', 'classic', 'divergent', 'extended', and 'unknown'. Phylogenetic analysis reveals three evolutionary branches within the CcSDR proteins, and further categorization using the SDR-initiative Hidden Markov model resulted in 46 families, with the CcSDR110C, CcSDR108E, and CcSDR460A families being the most populous. Collinearity analysis identified 34 pairs of CcSDR paralogs in C. camphora, 141 pairs of SDR orthologs between C. camphora and Populus trichocarpa, and 59 pairs between C. camphora and Oryza sativa. Expression profile analysis indicates a preference for the expression of 77 CcSDR genes in specific organs such as flowers, bark, twigs, roots, leaves, or fruits. Moreover, 77 genes exhibit differential expression patterns during the four developmental stages of leaves, while 130 genes show variance across the five developmental stages of fruits. Additionally, to explore the biosynthetic mechanism of methyl eugenol, a key component of the leaf essential oil in the methyl eugenol chemotype, this study also identifies eugenol synthase (EGS) within the CcSDR460A family through an integrated strategy. Real-time quantitative PCR analysis demonstrates that the expression of CcEGS in the leaves of the methyl eugenol chemotype is more than fourfold higher compared to other chemotypes. When heterologously expressed in Escherichia coli, it catalyzes the conversion of coniferyl acetate into a mixture predominantly composed of eugenol (71.44%) and isoeugenol (21.35%). These insights pave the way for future research into the functional diversity of CcSDR genes, with a focus on secondary metabolism.

Keywords: Cinnamomum camphora; SDR460A family; eugenol synthase; expression profiles; short-chain dehydrogenase/reductase (SDR) superfamily.

Figure 1

Paralogous genes of the SDR gene superfamily arising from segmental duplications in Cinnamomum camphora. Red lines represent duplicated gene pairs from the C type; green lines represent duplicated gene pairs from the A type; purple lines represent duplicated gene pairs from the U type; light slate blue lines represent duplicated gene pairs from the E type; and orange lines represent duplicated gene pairs from the D type.

Figure 2

Synteny analysis of SDR orthologous genes in the genomes between C. camphora and other plants. (A) SDR orthologous genes in C. camphora and Populus trichocarpa are highlighted by purple lines; (B) SDR orthologous genes in C. camphora and Oryza sativa are highlighted by dark cyan lines. The synteny blocks are shown in gray lines.

Figure 3

Identification of ten motifs within the CcSDR gene superfamily.

Figure 4

Phylogenetic tree analysis of SDR proteins from C. camphora. The CcSDR proteins were divided into three folds based on the clustering of the protein sequence. The proteins from the A, C, D, E, and U types are presented in blue, red, light blue, green, and light green, respectively.

Figure 5

CcSDR genes with preferential expression in the leaves, fruits, flowers, roots, bark, and twigs of C. camphora. The expression levels in the heat map were adjusted based on the log2 transformation of FPKM values and subsequent normalization.

Figure 6

Transcriptional dynamics of CcSDR genes in leaves across four developmental stages. (A) Samples representing four stages of leaf development were analyzed using RNA sequencing. (B) Cluster analysis of DEGs in leaves across four developmental stages.

Figure 7

Expression variation of CcSDR genes in fruit across five developmental stages. (A) Fruits at each of the five developmental stages were collected and underwent RNA-seq. (B) Cluster analysis of DGEs in fruits across five developmental stages.

Figure 8

Characterization of eugenol synthase (EGS) from C. camphora. (A) Fresh leaf essential oil profiles of methyleugenol-type, linalool-type, borneol-type, and camphor-type in camphor tree. (B) Evaluation and comparison of expression levels of candidate CcEGSs in leaves among methyleugenol-type, linalool-type, borneol-type, and camphor-type in camphor tree. (C) Gas chromatography–mass spectrometry analysis of products formed in E. coli spent medium by CcEGS. (D) Subcellular localization of CcEGS in Nicotiana benthamiana leaves. The adaxial leaf surface was observed with laser confocal microscopy (Bars = 50 µm). GFP, green fluorescent protein fluorescence image; chlorophyll, chlorophyll autofluorescence image; bright, transmission image; merged, all channels (GFP, chlorophyll, and bright) combination.