Biosynthesis of phytosterol esters: identification of a sterol o-acyltransferase in Arabidopsis

Fatty acyl esters of phytosterols are a major form of sterol conjugates distributed in many parts of plants. In this study we report an Arabidopsis (Arabidopsis thaliana) gene, AtSAT1 (At3g51970), which encodes for a novel sterol O-acyltransferase. When expressed in yeast (Saccharomyces cerevisiae), AtSAT1 mediated production of sterol esters enriched with lanosterol. Enzyme property assessment using cell-free lysate of yeast expressing AtSAT1 suggested the enzyme preferred cycloartenol as acyl acceptor and saturated fatty acyl-Coenyzme A as acyl donor. Taking a transgenic approach, we showed that Arabidopsis seeds overexpressing AtSAT1 accumulated fatty acyl esters of cycloartenol, accompanied by substantial decreases in ester content of campesterol and beta-sitosterol. Furthermore, fatty acid components of sterol esters from the transgenic lines were enriched with saturated and long-chain fatty acids. The enhanced AtSAT1 expression resulted in decreased level of free sterols, but the total sterol content in the transgenic seeds increased by up to 60% compared to that in wild type. We conclude that AtSAT1 mediates phytosterol ester biosynthesis, alternative to the route previously described for phospholipid:sterol acyltransferase, and provides the molecular basis for modification of phytosterol ester level in seeds.     

Metabolic engineering of soybean affords improved phytosterol seed traits

Different combinations of three rate‐limiting enzymes in phytosterol biosynthesis, the Arabidopsis thaliana hydroxyl methylglutaryl CoA1 (HMGR1) catalytic subunit linked to either constitutive or seed‐specific β‐conglycinin promoter, and the Glycine max sterol methyltransferase1 (SMT1) and sterol methyltransferase2‐2 (SMT2‐2) genes, under the control of seed‐specific Glycinin‐1 and Beta‐phaseolin promoters, respectively, were engineered in soybean plants. Mature seeds of transgenic plants displayed modest increases in total sterol content, which points towards a tight control of phytosterol biosynthesis. However, in contrast to wild‐type seeds that accumulated about 35% of the total sterol in the form of intermediates, in the engineered seeds driven by a seed‐specific promoter, metabolic flux was directed to Δ⁵‐24‐alkyl sterol formation (99% of total sterol). The engineered effect of end‐product sterol (sitosterol, campesterol, and stigmasterol) over‐production in soybean seeds resulted in an approximately 30% increase in overall sitosterol synthesis, a desirable trait for oilseeds and human health. In contradistinction, increased accumulation of cycloartenol and 24(28)‐methylencylartanol (55% of the total sterol) was detected in plants harbouring the constitutive t‐HMGR1 gene, consistent with the previous studies. Our results support the possibility that metabolic flux of the phytosterol family pathway is differentially regulated in leaves and seeds.     

Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis.

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.     

Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures

Squalene synthase (SS) dimerizes two molecules of farnesyl diphosphate to synthesize squalene, a shared precursor in steroid and triterpenoid biosynthesis in plants. The SS1 gene encoding SS from Arabidopsis thaliana was introduced in Withania coagulans under the control of the CaMV35S promoter together with the T-DNA of Agrobacterium rhizogenes A4. The engineered hairy roots were studied for withanolide production and phytosterol accumulation and the results were compared with those obtained from control roots harbouring only the T-DNA from pRiA4. The increased capacity of the engineered roots for biosynthesizing phytosterols and withanolides was strongly related with the expression level of the transgene, showing the effectiveness of overexpressing 35SS1 to increase triterpenoid biosynthesis.     

Identifying Mutations in Duplicated Functions in Saccharomyces cerevisiae: Recessive Mutations in Hmg-Coa Reductase Genes

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG1 and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMG1 gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other recovered in HMG1. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMG-CoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMG1. Intragenic complementation occurred with partial restoration of enzymatic activity. HMG1 was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.     

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.     

The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development

The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The Arabidopsis mutant tannin deficient seed 4 (tds4-1) has a reduced PA level and altered pattern PA accumulation. We identified the TDS4 gene as LDOX by complementation of the tds4-1 mutation either with a cosmid encoding LDOX or a 35S:LDOX construct. Independent Arabidopsis lines with a T-DNA insertion in the LDOX gene had a similar phenotype, and one was allelic to tds4-1. The seed phenotype of ban tds4 double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other TDS genes. Analysis of the PA intermediates in tds4-1 revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of Arabidopsis PA and its precursors indicates that Arabidopsis, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of tds4 were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that tds4 endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.     

Evidence for a Ustilago maydis steroid 5alpha-reductase by functional expression in Arabidopsis det2-1 mutants

We have identified a gene (udh1) in the basidiomycete Ustilago maydis that is induced during the parasitic interaction with its host plant maize (Zea mays). udh1 encodes a protein with high similarity to mammalian and plant 5alpha-steroid reductases. Udh1 differs from those of known 5alpha-steroid reductases by six additional domains, partially predicted to be membrane-spanning. A fusion protein of Udh1 and the green fluorescent protein provided evidence for endoplasmic reticulum localization in U. maydis. The function of the Udh1 protein was demonstrated by complementing Arabidopsis det2-1 mutants, which display a dwarf phenotype due to a mutation in the 5alpha-steroid reductase encoding DET2 gene. det2-1 mutant plants expressing either the udh1 or the DET2 gene controlled by the cauliflower mosaic virus 35S promoter differed from wild-type Columbia plants by accelerated stem growth, flower and seed development and a reduction in size and number of rosette leaves. The accelerated growth phenotype of udh1 transgenic plants was stably inherited and was favored under reduced light conditions. Truncation of the N-terminal 70 amino acids of the Udh1 protein abolished the ability to restore growth in det2-1 plants. Our results demonstrate the existence of a 5alpha-steroid reductase encoding gene in fungi and suggest a common ancestor between fungal, plant, and mammalian proteins.     

Past achievements,current status and future perspectives of studies on 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) in the mevalonate (MVA) pathway

Key message

HMGS functions in phytosterol biosynthesis, development and stress responses. F-244 could specifically-inhibit HMGS in tobacco BY-2 cells and Brassica seedlings. An update on HMGS from higher plants is presented.

Abstract

3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is the second enzyme in the mevalonate pathway of isoprenoid biosynthesis and catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce S-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Besides HMG-CoA reductase (HMGR), HMGS is another key enzyme in the regulation of cholesterol and ketone bodies in mammals. In plants, it plays an important role in phytosterol biosynthesis. Here, we summarize the past investigations on eukaryotic HMGS with particular focus on plant HMGS, its enzymatic properties, gene expression, protein structure, and its current status of research in China. An update of the findings on HMGS from animals (human, rat, avian) to plants (Brassica juncea, Hevea brasiliensis, Arabidopsis thaliana) will be discussed. Current studies on HMGS have been vastly promoted by developments in biochemistry and molecular biology. Nonetheless, several limitations have been encountered, thus some novel advances in HMGS-related research that have recently emerged will be touched on.     

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment

Aims:  Statins – inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs.
Methods and results:  We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins.
Conclusion:  The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes.
Significance and Impact of the Study:  These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.     

Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development

Anthocyanidin reductase encoded by the BANYULS (BAN) gene is the core enzyme in proanthocyanidin (PA) biosynthesis. Here, we analyzed the developmental mechanisms that regulate the spatiotemporal expression of BAN in the developing Arabidopsis seed coat. PA-accumulating cells were localized histochemically in the inner integument (seed body and micropyle) and pigment strand (chalaza). BAN promoter activity was detected specifically in these cells. Gain-of-function experiments showed that an 86-bp promoter fragment functioned as an enhancer specific for PA-accumulating cells. Mutations in regulatory genes of PA biosynthesis abolished BAN promoter activity (transparent testa2 [tt2], tt8, and transparent testa glabra1 [ttg1]), modified its spatial pattern (tt1 and tt16), or had no influence (ttg2), thus revealing complex regulatory interactions at several developmental levels. Genetic ablation of PA-accumulating cells targeted by the BAN promoter fused to BARNASE led to the formation of normal plants that produced viable yellow seeds. Importantly, these seeds had no obvious defects in endosperm and embryo development.     

Phenotypic changes in transgenic tobacco plants with an antisense form of the hmg1 gene

Tobacco plants were genetically transformed with the Arabidopsis thaliana heterologous hmg1 gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme involved in the metabolism of terpenoid compounds. The hmg1 gene was inserted under the control of the 35S RNA double promoter from the cauliflower mosaic virus (CaMV 35S) both in direct and reverse orientation relative to the promoter. DNA analysis by polymerase chain reaction (PCR) and Southern blotting confirmed the transgenic nature of the tobacco plants obtained. DNA-RNA hybridization revealed expression of the hmg1 gene in these tobacco plants. The plants transformed with the antisense copy of the hmg1 gene differed from the control plants in delayed development and in flower color and shape.