Isolation of a monocot 3-hydroxy-3-methylglutaryl coenzyme A reductase gene that is elicitor-inducible

The rice (Oryza sativa) phytoalexins, momilactones and oryzalexins, are synthesized by the isoprenoid pathway. An early step in this pathway, one that is rate-limiting in mammalian systems, is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). A gene that encodes this enzyme has been isolated from rice, and found to contain an open reading frame of 1527 bases. The encoded protein sequence of the rice HMGR appears to be conserved with respect to other HMGR proteins, and 1 or 2 membrane-spanning domains characteristic of plant HMGRs are predicted by a hydropathy plot of the amino acid sequence. The protein is truncated at its 5 end, and shows reduced sequence conservation in this region as compared to other plant sequences. The rice genome contains a small family of HMGR genes. The isolated gene, HMGR I, is expressed at low levels in both vegetative and floral organs of rice plants. It is not induced in plants by wounding, but is strongly and rapidly induced in suspension cells by a fungal cell wall elicitor from the pathogenMagnaporthe grisea, causal agent of rice blast disease. This suggests that HMGR I may be important in the induction of rice phytoalexin biosynthesis in response to pathogen attack, and therefore may play a key role as a component of the inducible defense mechanism in rice.     

Expression of the Hevea brasiliensis (H.B.K.) Mull. Arg. 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase 1 in Tobacco Results in Sterol Overproduction

A genomic fragment encoding one (HMGR1) of the three 3-hydroxy-3-methylglutaryl coenzyme A reductases (HMGRs) from Hevea brasiliensis (H.B.K.) Mull. Arg. (M.-L. Chye, C.-T. Tan, N.-H. Chua [1992] Plant Mol Biol 19: 473-484) was introduced into Nicotiana tabacum L. cv xanthi via Agrobacterium transformation to study the influence of the hmg1 gene product on plant isoprenoid biosynthesis. Transgenic plants were morphologically indistinguishable from control wild-type plants and displayed the same developmental pattern. Transgenic lines showed an increase in the level of total sterols up to 6-fold, probably because of an increased expression level of hmg1 mRNA and a corresponding increased enzymatic activity for HMGR, when compared with the level of total sterols from control lines not expressing the hmg1 transgene. In addition to the pathway end products, campesterol, sitosterol, and stigmasterol, some biosynthetic intermediates such as cycloartenol also accumulated in transgenic tissues. Most of the overproduced sterols were detected as steryl-esters and were likely to be stored in cytoplasmic lipid bodies. These data strongly support the conclusion that plant HMGR is a key limiting enzyme in phytosterol biosynthesis.     

Molecular characterization of three 3-hydroxy-3-methylglutaryl-CoA reductase genes including pathogen-induced Hmg2 from pepper (Capsicum annuum)

Sesquiterpene phytoalexins, a class of plant defense metabolites, are synthesized from the cytosolic acetate/mevalonate pathway in isoprenoids biosynthetic system of plants. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the synthesis of mevalonate, which is the specific precursor of this pathway, as a multi gene family. Three kinds of cDNA clones encoding HMGR were isolated from Korean red pepper (Capsicum annuum L. cv. NocKwang) and the HMGR2 gene (Hmg2) was especially obtained from a cDNA library constructed with Phytophthora capsici-infected pepper root RNAs. The Hmg2 encoding a 604-amino-acid peptide had typical features as an elicitor-induced isoform among HMGRs on its gene structure and had a predicted amino acid sequence homology. In addition, the expression of Hmg2 was rapidly induced within 1 h in response to a fungal pathogen and continuously increased up to 48 h. Together with sesquiterpene cyclase gene that was strongly induced 24 h after pathogen-infection, the Hmg2 and farnesyl pyrophosphate synthase gene were coordinately and sequentially regulated for the biosynthesis of defense-related sesquiterpene phytoalexins in pepper.     

Involvement of de Novo Protein Synthesis,Protein Kinase,Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.)

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested.     

Phytoalexins in defense against pathogens

Plants use an intricate defense system against pests and pathogens, including the production of low molecular mass secondary metabolites with antimicrobial activity, which are synthesized de novo after stress and are collectively known as phytoalexins. In this review, we focus on the biosynthesis and regulation of camalexin, and its role in plant defense. In addition, we detail some of the phytoalexins produced by a range of crop plants from Brassicaceae, Fabaceae, Solanaceae, Vitaceae and Poaceae. This includes the very recently identified kauralexins and zealexins produced by maize, and the biosynthesis and regulation of phytoalexins produced by rice. Molecular approaches are helping to unravel some of the mechanisms and reveal the complexity of these bioactive compounds, including phytoalexin action and metabolism.     

Targeting and topology in the membrane of plant 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate. This is the first committed step of isoprenoid biosynthesis. A common feature of all known plant HMGR isoforms is the presence of two highly conserved hydrophobic sequences in the N-terminal quarter of the protein. Using an in vitro system, we showed that the two hydrophobic sequences of Arabidopsis HMGR1S function as internal signal sequences. Specific recognition of these sequences by the signal recognition particle mediates the targeting of the protein to microsomes derived from the endoplasmic reticulum. Arabidopsis HMGR is inserted into the microsomal membrane, and the two hydrophobic sequences become membrane-spanning segments. The N-terminal end and the C-terminal catalytic domain of Arabidopsis HMGR are positioned on the cytosolic side of the membrane, whereas only a short hydrophilic sequence is exposed to the lumen. Our results suggest that the plant HMGR isoforms known to date are primarily targeted to the endoplasmic reticulum and have the same topology in the membrane. This reinforces the hypothesis that mevalonate is synthesized only in the cytosol. The possibility that plant HMGRs might be located in different regions of the endomembrane system is discussed.     

Hmg-coA reductase gene family in cotton (Gossypium hirsutum L.): unique structural features and differential expression of hmg2 potentially associated with synthesis of specific isoprenoids in developing embryos.

As a first step towards understanding the biosynthesis of isoprenoids that accumulate in specialized pigment glands of cotton at the molecular level, two full-length genes (hmg1 and hmg2) were characterized encoding hmg-coA reductase (HMGR; EC 1.1.1.34), the enzyme that catalyzes the formation of a key isoprenoid precursor. Cotton hmgr genes exhibited features typical of other plant genes, however, hmg2 encodes the largest of all plant HMGR enzymes described to date. HMG2 contains several novel features that may represent functional specialization of this particular HMGR isoform. Such features include a unique 42 amino acid sequence located in the region separating the N-terminal domain and C-terminal catalytic domain, as well as an N-terminal hydrophobic domain that is not found in HMG1 or other HMGR enzymes. DNA blot analysis revealed that hmg1 and hmg2 belong to small subfamilies that probably include homeologous loci in allotetraploid cotton (Gossypium hirsutum L.). Ribonuclease protection assays revealed that hmg1 and hmg2 are differentially expressed in a developmentally- and spatially-modulated manner during morphogenesis of specialized terpenoid-containing pigment glands in embryos. Induced expression of hmg2 coincided with a possible commitment to sesquiterpenoid biosynthesis in developing embryos, although other developmental processes also requiring HMGR cannot be excluded.     

Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae

3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum. In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens. Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms.     

A rice fungal MAMP‐responsive MAPK cascade regulates metabolic flow to antimicrobial metabolite synthesis

Plants recognize potential microbial pathogens through microbial‐associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti‐microbial secondary metabolites. Mitogen‐activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor‐induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4^DD) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4^DD also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4^DD‐induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4–OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP‐triggered defense responses.     

Regulation of Defense-related Gene Expression during Plant-Pathogen Interactions

Plants have evolved a broad array of defense mechanisms involved in disease resistance. These include synthesis of phytoalexin antibiotics and proteinase inhibitors, deposition of cell wall materials, and accumulation of hydrolytic enzymes such as chitinases. Resistance appears to depend on the ability of the host to recognize the pathogen rapidly and induce these defense responses in order to limit pathogen spread. Application of molecular technologies has yielded significant new information on mechanisms involved in pathogen recognition, signal transduction, and defense-related gene activation, and is leading to novel strategies for engineering enhanced disease resistance. We are using these approaches to analyze regulation of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR), a key enzyme mediating the production of terpenoid defense compounds. This enzyme is encoded by four genes in tomato; hmg2 gene expression is specifically associated with responses to pathogen or defense elicitors. Transgenic plants containing DNA constructs that fuse the hmg2 promoter to a reporter gene have been used to analyze both tissue specificity and patterns of defense-related expression. Because this gene is rapidly induced in tissues directly surrounding the site of ingress by a variety of pathogens, it may serve as a valuable tool in engineering new disease-resistance mechanisms.     

Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.     

Species-Specific Expansion and Molecular Evolution of the 3-hydroxy-3-methylglutaryl Coenzyme A Reductase (HMGR) Gene Family in Plants

The terpene compounds represent the largest and most diverse class of plant secondary metabolites which are important in plant growth and development. The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) is one of the key enzymes contributed to terpene biosynthesis. To better understand the basic characteristics and evolutionary history of the HMGR gene family in plants, a genome-wide analysis of HMGR genes from 20 representative species was carried out. A total of 56 HMGR genes in the 14 land plant genomes were identified, but no genes were found in all 6 algal genomes. The gene structure and protein architecture of all plant HMGR genes were highly conserved. The phylogenetic analysis revealed that the plant HMGRs were derived from one ancestor gene and finally developed into four distinct groups, two in the monocot plants and two in dicot plants. Species-specific gene duplications, caused mainly by segmental duplication, led to the limited expansion of HMGR genes in Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max after the species diverged. The analysis of Ka/Ks ratios and expression profiles indicated that functional divergence after the gene duplications was restricted. The results suggested that the function and evolution of HMGR gene family were dramatically conserved throughout the plant kingdom.     

Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway)

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of beta-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.     

次生代谢产物与植物抗病防御反应

次生代谢产物是由植物次生代谢产生的许多结构不同的小分子有机化合物,它们广泛参与植物的生长、发育、防御等生理过程。次生代谢产物在植物的抗病防御反应中发挥着重要作用,可以作为生化壁垒防御病原物侵染,还可以作为信号物质参与植物的抗病反应;在植物与病原物互作中,植物合成新的抗菌物质植保素,原有的抗菌物质也会增加。植物次生代谢产物的积累受到病原物、发育,环境等多种因素的调节。本文重点介绍次生代谢产物在植物抗病防御中的相关作用以及影响其合成的各种因素。     

Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115 bp containing a 1,695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones biosynthesis at the molecular level.     

Differential activation of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge.

Potato genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were expressed in response to pathogen, elicitor, and wounding. HMGR catalyzes the rate-limiting step in isoprenoid biosynthesis leading to accumulation of phytoalexins and steroid glycoalkaloids. Wounding caused increases in HMGR mRNA levels. A rapid and transient peak occurred 30 minutes after wounding, followed by a slower peak at 14 hours; both were correlated with increased enzyme activity. Induction of HMGR mRNA by the soft rot pathogen Erwinia carotovora subsp carotovora or arachidonic acid began 8 hours after challenge and continued through 22 hours. Potato HMGR is encoded by a gene family. An HMGR gene-specific probe was used to demonstrate that one isogene of the HMGR family is pathogen activated and is distinct from isogene(s) that are wound activated. This provides evidence that defense-related increases in HMGR activity are due to mRNA level increases and that HMGR isogenes are activated differentially by wounding or pathogen challenge.