scholarly journals Multiplexed CRISPR/Cas9-Mediated Knockout of Laccase Genes in Salvia miltiorrhiza Revealed Their Roles in Growth, Development, and Metabolism

2021 ◽  
Vol 12 ◽  
Author(s):  
Zheng Zhou ◽  
Qing Li ◽  
Liang Xiao ◽  
Yun Wang ◽  
Jingxian Feng ◽  
...  

Laccases are multicopper-containing glycoproteins related to monolignol oxidation and polymerization. These properties indicate that laccases may be involved in the formation of important medicinal phenolic acid compounds in Salvia miltiorrhiza such as salvianolic acid B (SAB), which is used for cardiovascular disease treatment. To date, 29 laccases have been found in S. miltiorrhiza (SmLACs), and some of which (SmLAC7 and SmLAC20) have been reported to influence the synthesis of phenolic acids. Because of the functional redundancy of laccase genes, their roles in S. miltiorrhiza are poorly understood. In this study, the CRISPR/Cas9 system was used for targeting conserved domains to knockout multiple genes of laccase family in S. miltiorrhiza. The expressions of target laccase genes as well as the phenolic acid biosynthesis key genes decrease dramatically in editing lines. Additionally, the growth and development of hairy roots was significantly retarded in the gene-edited lines. The cross-sections examination of laccase mutant hairy roots showed that the root development was abnormal and the xylem cells in the edited lines became larger and looser than those in the wild type. Additionally, the accumulation of RA as well as SAB was decreased, and the lignin content was nearly undetectable. It suggested that SmLACs play key roles in development and lignin formation in the root of S. miltiorrhiza and they are necessary for phenolic acids biosynthesis.

2020 ◽  
Author(s):  
Zheng Zhou ◽  
Qing Li ◽  
Yun Wang ◽  
Liang Xiao ◽  
Qitao Bu ◽  
...  

AbstractLaccases are multicopper-containing glycoproteins related to monolignol oxidation and polymerization. These properties indicate that laccases are involved in the formation of important medicinal phenolic acid compounds in Salvia miltiorrhiza such as salvianolic acid B (SAB), which is used for cardiovascular disease treatment. To date, 29 laccases have been found in S. miltiorrhiza, some of which influence the synthesis of phenolic acids. Because of the functional redundancy of laccase genes, their roles in S. miltiorrhiza are poorly understood. In this study, the CRISPR/Cas9 system was first used for dual gene locus targeting in S. miltiorrhiza to knock out multiple laccase family genes. The development of the roots was retarded, and root microstructure was abnormal in laccase mutant lines. Additionally, the accumulation of phenolic acid compounds as well as lignin was dramatically reduced. This study suggests that SmLACs are necessary for root development and phenolic acid compound metabolism in S. miltiorrhiza.


2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Wei Zhou ◽  
Min Shi ◽  
Changping Deng ◽  
Sunjie Lu ◽  
Fenfen Huang ◽  
...  

AbstractWater-soluble phenolic acids are major bioactive compounds in the medicinal plant species Salvia miltiorrhiza. Phenolic acid biosynthesis is induced by methyl jasmonate (MeJA) in this important Chinese herb. Here, we investigated the mechanism underlying this induction by analyzing a transcriptome library of S. miltiorrhiza in response to MeJA. Global transcriptome analysis identified the MeJA-responsive R2R3-MYB transcription factor-encoding gene SmMYB1. Overexpressing SmMYB1 significantly promoted phenolic acid accumulation and upregulated the expression of genes encoding key enzymes in the phenolic acid biosynthesis pathway, including cytochrome P450-dependent monooxygenase (CYP98A14). Dual-luciferase (dual-LUC) assays and/or an electrophoretic mobility shift assays (EMSAs) indicated that SmMYB1 activated the expression of CYP98A14, as well as the expression of genes encoding anthocyanin biosynthesis pathway enzymes, including chalcone isomerase (CHI) and anthocyanidin synthase (ANS). In addition, SmMYB1 was shown to interact with SmMYC2 to additively promote CYP98A14 expression compared to the action of SmMYB1 alone. Taken together, these results demonstrate that SmMYB1 is an activator that improves the accumulation of phenolic acids and anthocyanins in S. miltiorrhiza. These findings lay the foundation for in-depth studies of the molecular mechanism underlying MeJA-mediated phenolic acid biosynthesis and for the metabolic engineering of bioactive ingredients in S. miltiorrhiza.


2020 ◽  
Vol 155 ◽  
pp. 112826
Author(s):  
Min Shi ◽  
Yao Wang ◽  
Xiaorong Wang ◽  
Changping Deng ◽  
Wenzhi Cao ◽  
...  

2021 ◽  
Vol 22 (15) ◽  
pp. 7895
Author(s):  
Yao Cao ◽  
Rui Chen ◽  
Wen-Tao Wang ◽  
Dong-Hao Wang ◽  
Xiao-Yan Cao

Salvia miltiorrhiza is a renowned model medicinal plant species for which 15 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family genes have been identified; however, the specific functions of SmSPLs have not been well characterized as of yet. For this study, the expression patterns of SmSPL6 were determined through its responses to treatments of exogenous hormones, including indole acetic acid (IAA), gibberellic acid (GA3), methyl jasmonic acid (MeJA), and abscisic acid (ABA). To characterize its functionality, we obtained SmSPL6-ovexpressed transgenic S. miltiorrhiza plants and found that overexpressed SmSPL6 promoted the accumulation of phenolic acids and repressed the biosynthesis of anthocyanin. Meanwhile, the root lengths of the SmSPL6-overexpressed lines were significantly longer than the control; however, both the fresh weights and lateral root numbers decreased. Further investigations indicated that SmSPL6 regulated the biosynthesis of phenolic acid by directly binding to the promoter regions of the enzyme genes Sm4CL9 and SmCYP98A14 and activated their expression. We concluded that SmSPL6 regulates not only the biosynthesis of phenolic acids, but also the development of roots in S. miltiorrhiza.


2020 ◽  
Vol 331 ◽  
pp. 127365 ◽  
Author(s):  
Rong Fu ◽  
Min Shi ◽  
Changping Deng ◽  
Yi Zhang ◽  
Xichen Zhang ◽  
...  

Molecules ◽  
2011 ◽  
Vol 16 (3) ◽  
pp. 2259-2267 ◽  
Author(s):  
Jianglin Zhao ◽  
Jingfeng Lou ◽  
Yan Mou ◽  
Peiqin Li ◽  
Jianyong Wu ◽  
...  

Molecules ◽  
2014 ◽  
Vol 20 (1) ◽  
pp. 309-324 ◽  
Author(s):  
Bingcong Xing ◽  
Dongfeng Yang ◽  
Wanli Guo ◽  
Zongsuo Liang ◽  
Xijun Yan ◽  
...  

2016 ◽  
Vol 127 (1) ◽  
pp. 175-185 ◽  
Author(s):  
Dongfeng Yang ◽  
Zhicheng Huang ◽  
Bingcong Xing ◽  
Weibo Jin ◽  
Xijun Yan ◽  
...  

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