Dataset Information

Molecular Cloning and Analysis of an Acetyl-CoA C-acetyltransferase Gene (EkAACT) from Euphorbia kansui Liou.

ABSTRACT: Terpenoids are the largest class of natural products and are essential for cell functions in plants and their interactions with the environment. Acetyl-CoA acetyltransferase (AACT, EC2.3.1.9) can catalyze a key initiation step of the mevalonate pathway (MVA) for terpenoid biosynthesis and is modulated by many endogenous and external stimuli. Here, the function and expression regulation activities of AACT in Euphorbia kansui Liou (EkAACT) were reported. Compared with wild-type Arabidopsis, the root length, whole seedling fresh weight and growth morphology of EkAACT-overexpressing plants were slightly improved. The transcription levels of AtAACT, AtMDC, AtMK, AtHMGR, and AtHMGS in the MVA pathway and total triterpenoid accumulation increased significantly in transgenic Arabidopsis. Under NaCl and PEG treatment, EkAACT-overexpressing Arabidopsis showed a higher accumulation of total triterpenoids, higher enzyme activity of peroxidase (POD) and superoxide dismutase (SOD), increased root length and whole seedling fresh weight, and a decrease in the proline content, which indicated that plant tolerance to abiotic stress was enhanced. Thus, AACT, as the first crucial enzyme, plays a major role in the overall regulation of the MVA pathway.

SUBMITTER: Wang M

PROVIDER: S-EPMC9229008 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Molecular Cloning and Analysis of an Acetyl-CoA C-acetyltransferase Gene (EkAACT) from Euphorbia kansui Liou.

Wang Meng M Zheng Zhe Z Tian Zheni Z Zhang Hao H Zhu Chenyu C Yao Xiangyu X Yang Yixin Y Cai Xia X

Plants (Basel, Switzerland) 20220609 12

Terpenoids are the largest class of natural products and are essential for cell functions in plants and their interactions with the environment. Acetyl-CoA acetyltransferase (AACT, EC2.3.1.9) can catalyze a key initiation step of the mevalonate pathway (MVA) for terpenoid biosynthesis and is modulated by many endogenous and external stimuli. Here, the function and expression regulation activities of AACT in Euphorbia kansui Liou (EkAACT) were reported. Compared with wild-type Arabidopsis, ...[more]

PMID: 35736690

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Project description:Sandalwood (Santalum album L.) is famous for its unique fragrance derived from the essential oil of heartwood, whose major components are santalols. To understand the mechanism underlying the biosynthesis of santalols, in this study, we cloned two related genes involved in the mevalonate pathway in S. album coding for acetyl-CoA C-acetyl transferase (AACT) and 3-hydroxy-3-methyglutary-CoA synthase (HMGS). These genes were characterized and functionally analyzed, and their expression profiles were also assessed. An AACT gene designated as SaAACT (GenBank accession No. MH018694) and a HMGS gene designated as SaHMGS (GenBank accession No. MH018695) were successfully cloned from S. album. The deduced SaAACT and SaHMGS proteins contain 415 and 470 amino acids, and the corresponding size of their open-reading frames is 1538 bp and 1807 bp, respectively. Phylogenetic trees showed that the SaAACT protein had the closest relationship with AACT from Hevea brasiliensis and the SaHMGS proteins had the highest homology with HMGS from Siraitia grosvenorii. Functional complementation of SaAACT and SaHMGS in a mutant yeast strain deficient in these proteins confirmed that SaAACT and SaHMGS cDNA encodes functional SaAACT and SaHMGS that mediate mevalonate biosynthesis in yeast. Tissue-specific expression analysis revealed that both genes were constitutively expressed in all examined tissues (roots, sapwood, heartwood, young leaves, mature leaves and shoots) of S. album, both genes showing highest expression in roots. After S. album seedlings were treated with 100 μM methyl jasmonate, the expression levels of SaAACT and SaHMGS genes increased, suggesting that these genes were responsive to this elicitor. These studies provide insight that would allow further analysis of the role of genes related to the sandalwood mevalonate pathway in the regulation of biosynthesis of sandalwood terpenoids and a deeper understanding of the molecular mechanism of santalol biosynthesis.

Project description:BACKGROUND:Terpenoids, the largest class of natural products in the plant kingdom, have been widely used in medicine. The precursors of terpenoids, isoprene phosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), were synthesized from a mevalonate (MVA) pathway and a 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway respectively. The acetyl-CoA acetyltransferase (AACT) is the initial enzyme in MVA pathway and is considered presently to be essential for terpenoid backbone biosynthesis. The basic research on cytochemistry of terpenoid metabolic enzymes is important for understanding the mechanisms underlying major metabolic processes. However, compartmentalization of AACT in plants is in controversy. Euphorbia helioscopia L. containing laticifers in the whole plant is a famous ancient folk medicine for tumor treatment, and the terpenoid is an active ingredient. Furthermore, the laticifer cell is the main synthesizing and storing site for terpenoids. RESULTS:The gene of AACT was cloned successfully from E. helioscopia, and named as EhAACT. The EhAACT expression has no significant difference among roots, stems and leaves. However, compared with the roots and stems, the EhAACT expression level is slightly higher in leaves. In addition, EhAACT recombinant protein was expressed by procaryotic expression system and anti-EhAACT antibody was prepared, the molecular weight is about 43 kDa. Western blotting results illustrated that the EhAACT antibodies specifically recognized the endogenous proteins in E. helioscopia laticifers. At last, the subcellular localization of EhAACT in E. helioscopia laticifers was observed by using colloidal gold immune-electron microscopy. EhAACT was found to mainly distribute in the endoplasmic reticulum (ER), vacuoles originated from ER and cytosol aound vacuoles originated from ER. CONCLUSIONS:As a result, we speculated that in E. helioscopia laticifers, EhAACT located in cytosol would be transferred to small vacuoles dilated from ER, and the precursors of terpenoids were synthesized in these small vacuoles, then terpenoids were further synthesized into latex particles. This result would provide theoretical basis for regulating and controlling of terpenoid biosynthesis in laticifers.

Dataset Information

Molecular Cloning and Analysis of an Acetyl-CoA C-acetyltransferase Gene (EkAACT) from Euphorbia kansui Liou.

Publications

Molecular Cloning and Analysis of an Acetyl-CoA C-acetyltransferase Gene (<i>EkAACT</i>) from <i>Euphorbia kansui</i> Liou.

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