Identification and Characterization of Jasmonic Acid Biosynthetic Genes in Salvia miltiorrhiza Bunge

Abstract

Jasmonic acid (JA) is a vital plant hormone that performs a variety of critical functions for plants. Salvia miltiorrhiza Bunge (S. miltiorrhiza), also known as Danshen, is a renowned traditional Chinese medicinal herb. However, no thorough and systematic analysis of JA biosynthesis genes in S. miltiorrhiza exists. Through genome-wide prediction and molecular cloning, 23 candidate genes related to JA biosynthesis were identified in S. miltiorrhiza. These genes belong to four families that encode lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), and 12-OPDA reductase3 (OPR3). It was discovered that the candidate genes for JA synthesis of S. miltiorrhiza were distinct and conserved, in contrast to related genes in other plants, by evaluating their genetic structures, protein characteristics, and phylogenetic trees. These genes displayed tissue-specific expression patterns concerning to methyl jasmonate (MeJA) and wound tests. Overall, the results of this study provide valuable information for elucidating the JA biosynthesis pathway in S. miltiorrhiza by comprehensive and methodical examination.

Keywords: Salvia miltiorrhiza; jasmonic acid; secondary metabolism; traditional Chinese medicine.

Figure 1

Schematic diagram of the JA biosynthesis pathway. Key enzymes are shown in boxes, with solid black lines and arrows indicating individual biosynthesis steps. The ellipsis indicates a variety of other products. α-LeA, α-linolenic acid; 9-HPOT, 9-hydroperoxylinolenic acid; 13-HPOT, 13-hydroperoxylinolenic acid; 12S, 13S-EOT, 12S, 13S-epoxy-linolenic acid; cis-(+)-12-OPDA, cis-(+)-12-oxo-phydienoic acid; OPC-8, 8-(3-oxo-2-(pent-2-enyl) cyclopentyl) octanoic acid; α-ketols, 13-hydroxy-12-oxo-octadecadienoic acid; γ-ketols, 9-hydroxy-12-oxo-octadecadienoic acid; 9-LOX, 9-lipoxygenase; 13-LOX, 13-lipoxygenase; AOS, allene oxide synthase; AOC, allene oxide cyclase; OPR3, 12-OPDA reductase3.

Figure 2

Phylogenetic relationships of JA synthesis pathway enzyme proteins. Amino acid sequences of different plant species were obtained from NCBI based on the accession numbers listed in Table S5. Red five-pointed stars indicate S. miltiorrhiza, and white five-pointed stars indicate other species.

Figure 3

Phylogenetic relationships, gene structures, and conserved motifs of genes related to the JA synthesis pathway in S. miltiorrhiza. (A) The phylogenetic tree on the left contains 23 enzyme gene proteins. (B) Motif patterns of 23 proteins, where each pattern is displayed in a different colored box. (C) Exon/intron structures of 23 enzyme genes. Yellow boxes represent exons, black lines represent introns, and blue boxes represent untranslated regions.

Figure 4

(A) Tissue specific expressions of JA biosynthesis related genes. The transcript levels in roots, stems, leaves, and flowers of S. miltiorrhiza were analyzed using the quantitative real-time reverse transcription-PCR method (qRT-PCR). Asterisks (p < 0.05) indicate significant differences compared with the control group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (B) Heat map showing expression profiles of 23 enzyme genes in four different tissues. The graph was generated using GraphPad software based on three repeated counts of log2-transformed RNA-Seq data. Red and blue boxes indicate high and low expressions, respectively.

Figure 5

(A) Predicted cis-elements in the prompters of JA biosynthesis related genes. Distribution of cis-elements in the 1 kb upstream promoter regions of JA biosynthesis related genes. Different cis-elements are represented by various colors. (B) Number of JA biosynthesis related genes containing various cis-acting elements. The cis-acting elements were identified with the online PlantCARE program using 1 kb upstream of transcription initiation sites of JA biosynthesis related genes. The graph was generated based on the presence of cis-acting elements in response to specific elicitors/conditions/processes (x-axis) in all JA biosynthesis related genes (y-axis).

Figure 6

Expressions of JA biosynthesis related genes were correlated at 0 h, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h following the S. miltiorrhiza injury treatment. The transcript levels were analyzed using the qRT-PCR method. Asterisks (p < 0.05) indicate significant differences compared with the control group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 7

Expression of JA biosynthesis related genes of S. miltiorrhiza treated with MeJA for 0 h, 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h. The transcript levels were analyzed using the qRT-PCR method. Asterisks (p < 0.05) indicate significant differences compared with the control group (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 8

Gene Ontology (GO) annotation of JA biosynthesis related proteins. The y-axis on the right side indicates the number of genes in a category and on the left side indicates the percentage of a specific category of genes in the main category. The proteins were divided into three independent categories, namely biological process, cellular components, and molecular function.