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. 2023 Oct 9;24(19):15004.
doi: 10.3390/ijms241915004.

An Investigation of the JAZ Family and the CwMYC2-like Protein to Reveal Their Regulation Roles in the MeJA-Induced Biosynthesis of β-Elemene in Curcuma wenyujin

Yuyang Liu  1   2 , Shiyi Wu  1 , Kaer Lan  1 , Qian Wang  1 , Tingyu Ye  1 , Huanan Jin  1   2 , Tianyuan Hu  1   2 , Tian Xie  1   2 , Qiuhui Wei  1   2 , Xiaopu Yin  1   2
Affiliations

An Investigation of the JAZ Family and the CwMYC2-like Protein to Reveal Their Regulation Roles in the MeJA-Induced Biosynthesis of β-Elemene in Curcuma wenyujin

Yuyang Liu et al. Int J Mol Sci. .

Abstract

β-Elemene (C15H24), a sesquiterpenoid compound isolated from the volatile oil of Curcuma wenyujin, has been proven to be effective for multiple cancers and is widely used in clinical treatment. Unfortunately, the β-elemene content in C. wenyujin is very low, which cannot meet market demands. Our previous research showed that methyl jasmonate (MeJA) induced the accumulation of β-elemene in C. wenyujin. However, the regulatory mechanism is unclear. In this study, 20 jasmonate ZIM-domain (JAZ) proteins in C. wenyujin were identified, which are the core regulatory factors of the JA signaling pathway. Then, the conservative domains, motifs composition, and evolutionary relationships of CwJAZs were analyzed comprehensively and systematically. The interaction analysis indicated that CwJAZs can form homodimers or heterodimers. Fifteen out of twenty CwJAZs were significantly induced via MeJA treatment. As the master switch of the JA signaling pathway, the CwMYC2-like protein has also been identified and demonstrated to interact with CwJAZ2/3/4/5/7/15/17/20. Further research found that the overexpression of the CwMYC2-like gene increased the accumulation of β-elemene in C. wenyujin leaves. Simultaneously, the expressions of HMGR, HMGS, DXS, DXR, MCT, HDS, HDR, and FPPS related to β-elemene biosynthesis were also up-regulated by the CwMYC2-like protein. These results indicate that CwJAZs and the CwMYC2-like protein respond to the JA signal to regulate the biosynthesis of β-elemene in C. wenyujin.

Keywords: Curcuma wenyujin; MYC2-like protein; jasmonate ZIM-domain; transcriptional regulation; β-elemene.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1
Sequence logos and multiple sequence alignment of TIFY domains (A) and Jas domains (B) of CwJAZ proteins in C. wenyujin. The conserved TIFY/Jas/Degron/NLS motifs were outlined with a red straight line in sequence logos and a black straight line in alignment of the amino acid sequences, respectively. Each stack height indicates the conservation of the sequence at the corresponding position. Each letter height within each stack indicates the relative frequency of the corresponding amino acid. The amino acid residues with a black, purple, and gray background represent 100%, at least 80%, and at least 60% identity, respectively.
Figure 2
Figure 2
Phylogenetic relationships and motif compositions of JAZ proteins in C. wenyujin. (A) The phylogenetic tree of CwJAZ proteins was constructed using the neighbor-joining method with a bootstrap of 1000 replicates via MEGA 11. (B) Schematic diagrams of motif compositions. Different motifs for CwJAZ proteins were indicated by different colored boxes and numbered 1–10.
Figure 3
Figure 3
Phylogenetic tree of JAZ proteins from C. wenyujin, A. thaliana, O. sativa, and Z. mays. The phylogenetic tree was created using MEGA 11 software coupled with the neighbor-joining method with 1000 bootstrap replicates. Overall, 20 CwJAZs (red circle), 12 AtJAZs (blue triangle), 15 OsJAZs (pink rhombus), and 29 ZmJAZs (green square) were classified into four groups (groups I–IV).
Figure 4
Figure 4
Expression levels of CwJAZs under MeJA treatment. Three biological replicates and three technical replicates were performed. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant differences (* p < 0.05; ** p < 0.01).
Figure 5
Figure 5
Homo- and heteromeric interactions of 14 CwJAZ proteins via yeast two-hybrid assay. Recombinant plasmids were transformed into yeast strain AH109. Then, co-transformants were screened with SD/-Leu/-Trp medium (A) and SD/-Leu/-Trp/-His/-Ade medium with X-α-gal (C). (B) The co-transformants with pGADT7-LargeT and pGBKT7-P53 were used as positive controls, while those with pGADT7-LargeT and pGBKT7-LaminC were used as negative controls. Three independent experiments were performed.
Figure 6
Figure 6
Bioinformatics analysis of the CwMYC2-like protein. (A) A phylogenetic tree containing CwMYC2-like and 14 MYC2 proteins in other species. (B) Schematic diagram of the CwMYC2-like protein domain. (C) Amino acid sequence alignment between the CwMYC2-like protein and other species of MYC2 proteins. The outlined boxes indicate conserved domains. The amino acid residues with a black, purple, and gray background represent 100%, at least 80%, and at least 60% identity, respectively. The asterisk is the marker of an integer.
Figure 7
Figure 7
Analysis of expression pattern, transcriptional activation, and interactions of CwMYC2-like protein. (A) Expression level of CwMYC2-like gene in different tissues of C. wenyujin. Leave-3M and Flower-3M: leaves and flower obtained from 3-month-old C. wenyujin plant. Leave-6M and Rhizome-6M: leaves and rhizomes obtained from 6-month-old C. wenyujin plant. (B) Expression levels of CwMYC2-like gene in the leaf under MeJA treatment. Three biological replicates and three technical replicates were performed. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant differences (* p < 0.05; ** p < 0.01). (C) Analysis of the transactivation activity of CwMYC2-like protein in yeast. Recombinant plasmids were transformed into yeast strain AH109, and then the transformant strains were screened with SD/-Trp, SD/-His/-Trp/+X-a-gal and SD/-Trp/-His/-Ade/+X-a-gal media. (D) The interactions between CwJAZ proteins and CwMYC2-like protein. Three independent experiments were performed.
Figure 8
Figure 8
The biological function of CwMYC2-like protein in C. wenyujin. (A) Expression level of CwMYC2-like gene in control (EV) and overexpression (OE) lines. (B) Gas chromatography–mass spectrometry (GC-MS) analysis of β-elemene content extracted from EV and OE leaves. (C) The content of β-elemene in control and OE lines. Three biological replicates were performed. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant differences (* p < 0.05; ** p < 0.01).
Figure 9
Figure 9
Expression levels of structure genes related to β-elemene biosynthesis. Three biological replicates and three technical replicates were performed. Vertical bars refer to ±SE (n = 3). Asterisks indicate significant differences (* p < 0.05; ** p < 0.01). EV and OE lines represent A. tumefaciens GV3101 carrying the pBI121-GUS empty vector and GV3101 carrying the pBI121-CwMYC2-like-GUS recombinant plasmid, respectively. HMGS, 3-hydroxy-3-methylglutaryl-CoA; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; DXS, 1-deoxy-d-xylulose 5-phosphate synthase; DXR, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; HDS, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyldiphosphatereductase; FPPS, farnesyl diphosphate.
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