Overexpression of SrUGT76G1 in Stevia alters major steviol glycosides composition towards improved quality

Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP‐glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3‐β‐d ‐glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild‐type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S‐transferase‐tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.     

A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni)

The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.     

甜菊糖苷积累与其生物合成基因表达的关系

为探究甜叶菊叶片中甜菊糖苷积累与其合成途径上关键基因表达的关系,本研究分别检测了鑫丰3号苗期不同冠层和3个不同品种甜叶菊(守田3号、江甜3号、谱星1号)收获期混合叶片样品中多种糖苷的含量,同时定量检测对应样品中甜菊糖苷合成关键基因的表达水平。结果显示,总糖苷在鑫丰3号顶叶中最高,底叶中最低,而多数检测基因(6/8)表达水平也在顶叶最高底叶中最低;单一糖苷甜菊苷在顶叶中积累最高,而其催化酶编码基因Sr UGT74G1表达也在顶叶中最高;莱鲍迪苷A则在底叶中积累最多,其催化酶编码基因Sr UGT76G1表达水平也在底叶中表达最高。3个品种相比,总糖苷和莱鲍迪苷A的积累在江甜3号中最高,谱星1号中最低;甜菊苷的积累在守田3号中最高,江甜3号中最低,但基因表达水平与糖苷积累趋势一致的类似结果并未在不同品种间出现。由此可知,甜菊糖苷合成基因的表达水平可以影响总糖苷的积累,且在同一甜叶菊品种中单一糖苷合成调控基因的表达水平可以反映其调控的单糖苷的积累量。     

Cloning and expression analysis of ten genes associated with picrosides biosynthesis in Picrorhiza kurrooa

Picrorhiza kurrooa Royle ex Benth. is an economically important medicinal plant known to yield picrosides which have high medicinal value. Picroside I and picroside II are major picrosides associated with various bioactivities. The present work analyzed the expression of various genes of the picrosides biosynthesis pathway in different tissues of the plant in relation to the picrosides content. Eight full-length cDNA sequences namely, 1-deoxy-d-xylulose-5-phosphate synthase (2.317 kb), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (1.767 kb), 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (1.674 kb), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (1.701 kb), acetyl-CoA acetyltransferase (1.545 kb), 3-hydroxy-3-methylglutaryl coenzyme A reductase (2.241 kb), isopentenyl pyrophosphate isomerase (987 bp) and geranyl diphosphate synthase (1.434 kb), were cloned to full-length followed by expression analysis of ten genes vis-à-vis picrosides content analysis. There is maximum accumulation of picrosides in leaf tissue followed by the rhizome and root, and a similar pattern of expression was found in all the ten genes. The genes responded to the modulators of the picrosides biosynthesis. Picrosides accumulation was enhanced by application of hydrogen peroxide and abscisic acid, whereas methyl jasmonate and salicylic acid treatment decreased the content.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.     

2C-methyl- d- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene

Stevia [Stevia rebaudiana (Bertoni)] is a perennial herb which accumulates sweet diterpenoid steviol glycosides (SGs) in its leaf tissue. SGs are synthesized by 2C-methyl-d-erythritol 4-phosphate (MEP) pathway. Of the various enzymes of the MEP pathway, 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MDS) (encoded by MDS) catalyzes the cyclization of 4-(cytidine 5′ diphospho)-2C-methyl-d-erythritol 2-phosphate into 2C-methyl-d-erythritol 2,4-cyclodiphosphate. Complementation of the MDS knockout mutant strain of Escherichia coli, EB370 with putative MDS of stevia (SrMDS) rescued the lethal mutant, suggesting SrMDS to be a functional gene. Experiments conducted in plant growth chamber and in the field suggested SrMDS to be a light regulated gene. Indole 3-acetic acid (IAA; 50, 100?μM) down-regulated the expression of SrMDS at 4?h of the treatment, whereas, abscisic acid did not modulate its expression. A high expression of SrMDS was observed during the light hours of the day as compared to the dark hours. The present work established functionality of SrMDS and showed the role of light and IAA in regulating expression of SrMDS.     

Structure of the (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase from Plasmodium falciparum

Terpenoid precursor biosynthesis occurs in human and many pathogenic organisms via the mevalonate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways, respectively. We determined the X-ray structure of the Fe/S containing (E)-4-hydroxy-3-methyl-but-2-enyl-diphosphate reductase (LytB) of the pathogenic protozoa Plasmodium falciparum which catalyzes the terminal step of the MEP pathway. The cloverleaf fold and the active site of P. falciparum LytB corresponds to those of the Aquifex aeolicus and Escherichia coli enzymes. Its distinct electron donor [2Fe–2S] ferredoxin was modeled to its binding site by docking calculations. The presented structural data provide a platform for a rational search of anti-malarian drugs.     

Structure of the GcpE (IspG)-MEcPP complex from Thermus thermophilus

Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e., by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-d-erythritol-2,4-cyclo-diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55 Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60° rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP.     

乙烯对绞股蓝皂苷生物合成关键酶基因表达和皂苷含量的影响

以药食两用植物绞股蓝为材料,研究了植物激素乙烯对绞股蓝皂苷生物合成关键酶基因表达及皂苷含量的影响。该文采用荧光定量PCR技术,检测了乙烯利处理后绞股蓝不同器官中皂苷生物合成关键酶基因GpFPS、GpSS和GpSE的表达水平; 采用分光光度法及HPLC技术,测定了乙烯利处理对绞股蓝总皂苷和皂苷单体Rb₁、Rb₃和Rd含量的影响。结果表明:(1)外施乙烯利能够不同程度地上调GpFPS、GpSS和GpSE基因的表达水平,且3个基因的表达模式在不同器官间不同,而在同一器官中相似。(2)在乙烯利处理后3 d,所测各器官中的总皂苷含量与对照相比均有所上升,其中根、成熟叶和幼叶达到显著水平,但3个皂苷单体在不同器官中的增加或降低并不一致,以Rb₃含量最高。该结果为探索利用植物激素调控绞股蓝皂苷次生代谢提供了参考。     

Overexpression of a glycosyltransferase gene SrUGT74G1 from Stevia improved growth and yield of transgenic Arabidopsis by catechin accumulation

Steviol glycoside and gibberellin biosynthetic routes are known as divergent branches of a common origin in Stevia. A UDP-glycosyltransferase encoded by SrUGT74G1 catalyses the conversion of steviolbioside into stevioside in Stevia rebaudiana leaves. In the present study, transgenic Arabidopsis thaliana overexpressing SrUGT74G1 cDNA from Stevia were developed to check the probability of stevioside biosynthesis in them. However, stevioside accumulation was not evident in transgenics. Also, the transgenic Arabidopsis showed no change in GA₃ content on SrUGT74G1 overexpression. Surprisingly, significant accumulation of catechin was noticed in transgenics. The transgenics showed a considerable increase in shoot length, root length and rosette area. An increase in free radical scavenging activity of transgenics was noticed. Moreover, the seed yield of transgenics was also increased by 6–15 % than control. Additionally, variation in trichome branching pattern on leaf surface of transgenics was observed. The trichome branching pattern was also validated by exogenous catechin exposure (10, 50, 100 ng ml^?1) to control plants. Hence, present study reports the probable role of SrUGT74G1 from Stevia in catechin accumulation of transgenic Arabidopsis thaliana. Thus, detailed study in present perspective has revealed the role of Stevia SrUGT74G1 gene in trichome branching pattern, improved vegetative growth, scavenging potential and seed yield by catechin accumulation in transgenic Arabidopsis.     

Isoprenoid biosynthesis in plant chloroplasts via the MEP pathway: direct thylakoid/ferredoxin-dependent photoreduction of GcpE/IspG

In the methylerythritol phosphate pathway for isoprenoid biosynthesis, the GcpE/IspG enzyme catalyzes the conversion of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-enyl diphosphate. This reaction requires a double one-electron transfer involving a [4Fe-4S] cluster. A thylakoid preparation from spinach chloroplasts was capable in the presence of light to act as sole electron donor for the plant GcpE Arabidopsis thaliana in the absence of any pyridine nucleotide. This is in sharp contrast with the bacterial Escherichia coli GcpE, which requires flavodoxin/flavodoxin reductase and NADPH as reducing system and represents the first proof that the electron flow from photosynthesis can directly act in phototrophic organisms as reducer in the 2-C-methyl-d-erythritol 4-phosphate pathway, most probably via ferredoxin, in the absence of any reducing cofactor. In the dark, the plant GcpE catalysis requires in addition of ferredoxin NADP(+)/ferredoxin oxido-reductase and NADPH as electron shuttle.     

Plant isoprenoid biosynthesis via the MEP pathway: In vivo IPP/DMAPP ratio produced by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase in tobacco BY-2 cell cultures

Feeding tobacco BY-2 cells with [2-¹³C,4-²H]deoxyxylulose revealed from the ¹³C labeling that the plastid isoprenoids, synthesized via the MEP pathway, are essentially derived from the labeled precursor. The ca. 15% ²H retention observed in all isoprene units corresponds to the isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) ratio (85:15) directly produced by the hydroxymethylbutenyl diphosphate reductase, the last enzyme of the MEP pathway. ²H retention characterizes the isoprene units derived from the DMAPP branch, whereas ²H loss represents the signature of the IPP branch. Taking into account the enantioselectivity of the reactions catalyzed by the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase, the IPP isomerase and the trans-prenyl transferase, a single biogenetic scheme allows to interpret all labeling patterns observed in bacteria or plants upon incubation with ²H labeled deoxyxylulose.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.