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Characterization and Function of 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Populus trichocarpa: Overexpression of PtHMGR Enhances Terpenoids in Transgenic Poplar.

ABSTRACT: In the mevalonic acid (MVA) pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis. In this study, we cloned a full-length cDNA from Populus trichocarpa with an open reading frame of 1,734 bp. The deduced PtHMGR sequence contained two HMG-CoA motifs and two NADPH motifs, which exhibited homology with HMGR proteins from other species. Subsequently, truncated PtHMGR was expressed in Escherichia coli BL21 (DE3) cells, and enzyme activity analysis revealed that the truncated PtHMGR protein could catalyze the reaction of HMG-CoA and NADPH to form MVA. Relative expression analysis suggests that PtHMGR expression varies among tissues and that PtHMGR responds significantly to abscisic acid (ABA), NaCl, PEG6000, hydrogen peroxide (H2O2), and cold stresses. We used polymerase chain reaction (PCR) analysis to select transgenic Nanlin 895 poplars (Populus× euramericana cv.) and quantitative reverse-transcription PCR (qRT-PCR) to show that PtHMGR expression levels were 3- to 10-fold higher in transgenic lines than in wild-type (WT) poplars. qRT-PCR was also used to determine transcript levels of methylerythritol phosphate (MEP)-, MVA-, and downstream-related genes, indicating that overexpression of PtHMGR not only affects expression levels of MVA-related genes, but also those of MEP-related genes. We also measured the content of terpenoids including ABA, gibberellic acid (GA), carotenes, and lycopene. PtHMGR overexpression significantly increased ABA, GA, carotene, and lycopene content, indicating that PtHMGR participates in the regulation of terpenoid compound synthesis.

SUBMITTER: Wei H

PROVIDER: S-EPMC6872958 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Characterization and Function of 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Populus trichocarpa: Overexpression of PtHMGR Enhances Terpenoids in Transgenic Poplar.

Wei Hui H Xu Chen C Movahedi Ali A Sun Weibo W Li Dawei D Zhuge Qiang Q

Frontiers in plant science 20191115

In the mevalonic acid (MVA) pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis. In this study, we cloned a full-length cDNA from Populus trichocarpa with an open reading frame of 1,734 bp. The deduced PtHMGR sequence contained two HMG-CoA motifs and two NADPH motifs, which exhibited homology with HMGR proteins from other species. Subsequently, truncated PtHMGR was expressed in Escherichia coli BL21 (DE3) ...[more]

PMID: 31803212

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Project description:The mevalonate pathway has emerged as a promising target for several solid tumors. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of this pathway, and are commonly used to treat patients with hypercholesterolemia. Pleiotropic antitumor mechanisms of statins have been demonstrated for several human cancer types. However, cancer cells differ in their individual statin sensitivity and some cell lines have shown relative resistance. In this study we demonstrate, that the human breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and T47D are differentially affected by statins. Whereas the vitality of MDA-MB-231 and MDA-MB-468 cells was reduced by up to 60% using atorvastatin, simvastatin, or rosuvastatin (p < 0.001), only marginal effects were seen in T47D and MCF-7 cells following exposure to statins. Statin treatment led to an upregulation of HMGCR mRNA and protein expression by up to sixfolds in the statin-resistant cells lines (p < 0.001), but no alterations of HMGCR were observed in the statin-sensitive MDA-MB-231 and MDA-MB-468 cells. The knockdown of HMGCR prior to statin treatment sensitized the resistant cell lines, reflected by a 70% reduction in vitality, increased apoptotic DNA fragmentation (sixfold) and by accumulation of the apoptosis marker cleaved poly-ADP ribose polymerase. Statins induced a cleavage of the sterol-regulatory element-binding protein (SREBP)-2, a transcriptional activator of the HMGCR, in T47D and MCF-7 cells. The inhibition of SREBP-2 activation by co-administration of dipyridamole sensitized MCF-7 and T47D cells for statins (loss of vitality by 80%; p < 0.001). Furthermore, assessment of a statin-resistant MDA-MB-231 clone, generated by long-term sublethal statin exposure, revealed a significant induction of HMGCR expression by up to 12-folds (p < 0.001). Knockdown of HMGCR restored statin sensitivity back to levels of the parental cells. In conclusion, these results indicate a resistance of cancer cells against statins, which is in part due to the induction of HMGCR.

Project description:BackgroundCCCH zinc finger proteins contain a typical motif of three cysteines and one histidine residues and serve regulatory functions at all stages of mRNA metabolism. In plants, CCCH type zinc finger proteins comprise a large gene family represented by 68 members in Arabidopsis and 67 in rice. These CCCH proteins have been shown to play diverse roles in plant developmental processes and environmental responses. However, this family has not been studied in the model tree species Populus to date.ResultsIn the present study, a comprehensive analysis of the genes encoding CCCH zinc finger family in Populus was performed. Using a thorough annotation approach, a total of 91 full-length CCCH genes were identified in Populus, of which most contained more than one CCCH motif and a type of non-conventional C-X(11)-C-X(6)-C-X(3)-H motif was unique for Populus. All of the Populus CCCH genes were phylogeneticly clustered into 13 distinct subfamilies. In each subfamily, the gene structure and motif composition were relatively conserved. Chromosomal localization of these genes revealed that most of the CCCHs (81 of 90, 90 %) are physically distributed on the duplicated blocks. Thirty-four paralogous pairs were identified in Populus, of which 22 pairs (64.7 %) might be created by the whole genome segment duplication, whereas 4 pairs seem to be resulted from tandem duplications. In 91 CCCH proteins, we also identified 63 putative nucleon-cytoplasm shuttling proteins and 3 typical RNA-binding proteins. The expression profiles of all Populus CCCH genes have been digitally analyzed in six tissues across different developmental stages, and under various drought stress conditions. A variety of expression patterns of CCCH genes were observed during Populus development, of which 34 genes highly express in root and 22 genes show the highest level of transcript abundance in differentiating xylem. Quantitative real-time RT-PCR (RT-qPCR) was further performed to confirm the tissue-specific expression and responses to drought stress treatment of 12 selected Populus CCCH genes.ConclusionsThis study provides the first systematic analysis of the Populus CCCH proteins. Comprehensive genomic analyses suggested that segmental duplications contribute significantly to the expansion of Populus CCCH gene family. Transcriptome profiling provides first insights into the functional divergences among members of Populus CCCH gene family. Particularly, some CCCH genes may be involved in wood development while others in drought tolerance regulation. Our results presented here may provide a starting point for the functional dissection of this family of potential RNA-binding proteins.

Dataset Information

Characterization and Function of 3-Hydroxy-3-Methylglutaryl-CoA Reductase in Populus trichocarpa: Overexpression of PtHMGR Enhances Terpenoids in Transgenic Poplar.

Publications

Characterization and Function of 3-Hydroxy-3-Methylglutaryl-CoA Reductase in <i>Populus trichocarpa</i>: Overexpression of <i>PtHMGR</i> Enhances Terpenoids in Transgenic Poplar.

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