Cloning and characterization of a potato StAN11 gene involved in anthocyanin biosynthesis regulation

Anthocyanins are a class of products of plant secondary metabolism and are responsible for tubers color in potato.The biosynthesis of anthocyanins is a complex Researchbiological process,in which multiple genes are involved including structural genes and regulatory genes.In this study,StAN11,a WD40-repeat gene,was cloned from potato cultivar Chieftain(Solanum tuberosum L.).StAN11(HQ599506)contained no intron and its open reading frame(ORF)was 1,029 bp long,encoding a putative protein of 342 amino acids.In order to verify its role in anthocyanin biosynthesis,StAN11 was inserted behind the CaMV-35S promoter of pCMBIA1304 and the recombination vector was introduced into the potato cultivar Désirée plants by Agrobacterium-mediated transformation.The color of transgenic tuber skin was significantly deepened,compared to the wild-type control,which was highly consistent with the accumulation of anthocyanin and expression of StAN11 in transgenic lines tuber skin.Further analysis on the expression of Flavonone-3-hydroxylase(F3H),Dihydroflavonol reductase(DFR),Anthocyanidin synthase(ANS),and Flavonoid 3-O-glucosyl transferase(3GT)in transgenic plants revealed that only DFR was upregulated.This result suggested that StAN11 regulated anthocyanin biosynthesis in potato by controlling DFR expression and accumulation of anthocyanin could be increased through overexpression of StAN11 in the tubers with the genetic background of anthocyanin biosynthesis.     

Genetic and Proteomic Analyses of a Xanthomonas campestris pv. campestris purC Mutant Deficient in Purine Biosynthesis and Virulence

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.     

Computational identification of sweet wormwood (Artemisia annua) microRNA and their mRNA targets

Despite its efficacy against malaria, the relatively low yield (0.01%-0.8%) of artemisinin in Artemisia annua is a serious limitation to the commercialization of the drug. A better understanding of the biosynthetic pathway of artemisinin and its regulation by both exogenous and endogenous factors is essential to improve artemisinin yield. Increasing evidence has shown that microRNAs (miRNAs) play multiple roles in various biological processes. In this study, we used previously known miRNAs from Arabidopsis and rice against expressed sequence tag (EST) database of A. annua to search for potential miRNAs and their targets in A. annua. A total of six potential miRNAs were predicted, which belong to the miR414 and miR1310 families. Furthermore, eight potential target genes were identified in this species. Among them, seven genes encode proteins that play important roles in ar- temisinin biosynthesis, including HMG-CoA reductase (HMGR), amorpha-4,11-diene synthase (ADS), farnesyl pyrophosphate synthase (FPS) and cytochrome P450. In addition, a gene coding for putative AINTEGUMENTA, which is involved in signal transduction and development, was also predicted as one of the targets. This is the first in silico study to indicate that miRNAs target genes encoding enzymes involved in artemisinin biosynthesis, which may help to understand the miRNA-mediated regulation of artemisinin biosynthesis in A. annua.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.     

Plant Terpenoids： Biosynthesis and Ecological Functions

Among plant secondary metabolites terpenolds are a structurally most diverse group; they function as phytoalexins In plant direct defense, or as signals In Indirect defense responses which involves herbivores and their natural enemies. In recent years, more and more attention has been paid to the Investigation of the ecological role of plant terpenolds. The biosynthesis pathways of monoterpenes, sesquiterpenes, and diterpenes Include the synthesis of C5 precursor isopentenyl diphosphate （IPP） and Its allylic isomer dlmethylallyl dlphosphate （DMAPP）, the synthesis of the immediate diphosphate precursors, and the formation of the diverse terpenoids. Terpene synthases （TPSs） play a key role In volatile terpene synthesis. By expression of the TPS genes, significant achievements have been made on metabolic engineering to Increase terpenoid production. This review mainly summarizes the recent research progress In elucidating the ecological role of terpenoids and characterization of the enzymes Involved in the terpenold biosynthesis. Spatial and temporal regulations of terpenoids metabolism are also discussed.     

A Rice Panicle Mutant Created by Transformation with an Antisense cDNA Library

A rice （Oryza sativa L.） mutant displaying defects in panicle development was identified among transformants in a transgenic mutagenlzed experiment using an antlsense cDNA library prepared from young rice panicles. In the mutant, the average splkelet number was reduced to 59.8 compared with 104.3 in wild-type plants. In addition, the seed-setting rate of the mutant was low （39.3%） owing to abnormal female development. Genetic analysis of T1 and T2 progeny showed that the traits segregated In a 3 （mutant） ： 1 （wild type） ratio and the mutation was cosegregated with the transgene. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses showed that the mutant had a single T-DNA insertion on chromosome 5, where no gene was tagged. Sequencing analysis found that the transgenic antisense cDNA was derived from a gene encoding an F-box protein in chromosome 7 with unidentified function. This and another four homologous genes encoding putative F-box proteins form a gene cluster. These results indicate that the phenotyplc mutations were most likely due to the silencing effect of the expressed transgenic antisense construct on the member（s） of the F-box gene cluster.     

Molecular cloning and functional analysis of an organ-specific expressing gene coding for farnesyl diphosphate synthase from <Emphasis Type="Italic">Michelia chapensis</Emphasis> Dandy

Farnesyl diphosphate synthase (FPS; EC 2.5.1.1/EC 2.5.1.10) catalyzes the synthesis of farnesyl diphosphate, a key intermediate in the biosynthesis of sesquiterpenes. This present study described the cloning and characterization of a cDNA encoding FPS from leaves of Michelia chapensis Dandy (designated as McFPS, GenBank accession number: GQ214406) for the first time. McFPS was 1,432 bp and contained an open reading frame (ORF) of 1,056 bp, encoding a protein of 351 amino acids with a calculated molecular mass of 40.52 kDa. Bioinformatic analysis revealed that the deduced McFPS had high homology with FPSs from other plant species. Phylogenetic tree analysis indicated that McFPS belonged to the plant FPS group and had the closest relationship with FPS from Chimonanthus praecox. Southern blot analysis revealed that there were at most two copies of McFPS gene existed in M. chapensis genome. The organ expression pattern analysis showed that McFPS expressed strongly only in leaves, and there were no expression in stems and roots, implying that McFPS was an organ-specific expressing gene. Functional complementation of McFPS in a FPS-deficient yeast strain demonstrated that cloned cDNA encoded a farnesyl diphosphate synthase.     

Molecular characterization and expression analysis of a gene encoding for farnesyl diphosphate synthase from Euphorbia pekinensis Rupr

The root of Euphorbia pekinensis as a traditional herbal medicine has been recorded in Chinese pharmacopoeias for the treatment of oedema, gonorrhea, migraine and wart cures. In this work, we reported on the cDNA cloning and characterization of a novel farnesyl diphosphate synthase (FPS) from E. pekinensis. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPSs, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed that there exists a small FPS gene family in E. pekinensis. Expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. In callus, expression of EpFPS gene and biosynthesis of triterpenoids were strongly induced by Methyl jasmonate and slightly induced by Salicylic acid. Functional complementation of EpFPS in an ergosterol auxotrophic yeast strain indicated that the cloned cDNA encoded a functional farnesyl diphosphate synthase.     

Cloning and functional analysis of a cDNA encoding Ginkgo biloba farnesyl diphosphate synthase

Farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2. 5.1.10) catalyzes the synthesis of farnesyl diphosphate, and provides precursor for biosynthesis of sesquiterpene and isoprenoids containing more than 15 isoprene units in Ginkgo biloba. Here we report the cloning, characterization and functional analysis of a new cDNA encoding FPS from G. biloba. The full-length cDNA (designated GbFPS) had 1731 bp with an open reading frame of 1170 bp encoding a polypeptide of 390 amino acids. The deduced GbFPS was similar to other known FPSs and contained all the conserved regions of trans-prenyl chain-elongating enzymes. Structural modeling showed that GbFPS had the typical structure of FPS, the most prominent feature of which is the arrangement of 13 core helices around a large central cavity. Southern blot analysis revealed a small FPS gene family in G. biloba. Expression analysis indicated that GbFPS expression was high in roots and leaves, and low in stems. Functional complementation of GbFPS in an FPS-deficient strain confirmed that GbFPS mediates farnesyl diphosphate biosynthesis.     

Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) Urban

A cDNA encoding farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2.5.1.10) was isolated from Centella asiacita (L.) Urban, using degenerate primers based on two highly conserved domains. A full-length cDNA clone was subsequently isolated by rapid amplification of cDNA ends (RACE) PCR. The sequence of the CaFPS (C. asiatica farnesyl diphosphate synthase) cDNA contains an open reading frame of 1029 nucleotides encoding 343 amino acids with a molecular mass of 39.6 kDa. The deduced CaFPS amino acid sequence exhibits 84, 79, and 72%, identity to the FPSs of Artemisia annua, Arabidopsis thaliana, and Oryza sativa, respectively. Southern blot analysis suggested that the C. asiatica genome contains only one FPS gene. An artificially expressed soluble form of the CaFPS was identified by SDS-PAGE. It had high specific activity and produced farnesyl diphosphate as the major isoprenoid.     

Two different farnesyl diphosphate synthase genes exist in the genome of the green peach aphid, Myzus persicae.

Farnesyl diphosphate synthase (FPS; EC 2.5.1.1, 2.5.1.10) catalyzes biosynthesis of farnesyl diphosphate, which is important to insects as the precursor of juvenile hormone and the substrate for (E)-beta-farnesene synthase. Here, two FPS cDNAs were isolated from the green peach aphid, Myzus persicae (EU334430 and EU334431). Their shared identity within the coding region is approximately 82%. The deduced amino acid sequences of the two M. persicae FPS cDNAs have the highly conserved motifs characteristic of most known FPSs. Phylogenetic analyses showed that they are closely related to other insect FPSs. Homology modeling of structures suggested a very good fit between the three-dimensional structures of the two putative M. persicae FPSs (designated as MpFPS1 and MpFPS2) and the avian FPS crystal structure. The corresponding genomic DNA sequences were subsequently determined (EU429295 and EU429296). Sequence comparisons revealed a different splicing pattern between the two MpFPS genes. Furthermore, the two MpFPS genes exhibited a seemingly very primitive gene-splicing pattern at 5' ends but a gene-splicing style similar to mammalian FPS genes at 3' ends. These data, combined with results of Southern blotting, suggest that M. persicae contains two different FPS genes. This is the first report that two different FPS genes exist in a hemipteran insect.     

The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis,and complete loss of FPP synthase activity causes early developmental arrest

Farnesyl diphosphate (FPP) synthase (FPS) catalyses the synthesis of FPP, the major substrate used by cytosolic and mitochondrial branches of the isoprenoid pathway. Arabidopsis contains two farnesyl diphosphate synthase genes, FPS1 and FPS2, that encode isozymes FPS1L (mitochondrial), FPS1S and FPS2 (both cytosolic). Here we show that simultaneous knockout of both FPS genes is lethal for Arabidopsis, and embryo development is arrested at the pre‐globular stage, demonstrating that FPP‐derived isoprenoid metabolism is essential. In addition, lack of FPS enzyme activity severely impairs male genetic transmission. In contrast, no major developmental and metabolic defects were observed in fps1 and fps2 single knockout mutants, demonstrating the redundancy of the genes. The levels of sterols and ubiquinone, the major mitochondrial isoprenoid, are only slightly reduced in the single mutants. Although one functional FPS gene is sufficient to support isoprenoid biosynthesis for normal growth and development, the functions of FPS1 and FPS2 during development are not completely redundant. FPS1 activity has a predominant role during most of the plant life cycle, and FPS2 appears to have a major role in seeds and during the early stages of seedling development. Lack of FPS2 activity in seeds, but not of FPS1 activity, is associated with a marked reduction in sitosterol content and positive feedback regulation of 3‐hydroxy‐3‐methylglutaryl CoA reductase activity that renders seeds hypersensitive to the 3‐hydroxy‐3‐methylglutaryl CoA reductase inhibitor mevastatin.     

法呢基焦磷酸(FPP)的生物合成及其分子调控

法呢基焦磷酸合酶作为异戊二烯途径中的重要调节酶,是许多萜类物质的合成前体。FPS的cDNA克隆在许多生物体中也已得到了分离并进行了表达特性研究。从FPP的生物合成途径入手,对FPP生物学特性、FPS酶基因调控的相关信息进行了综述,同时对FPS在基因工程方面的应用进行了展望。     

Engineering linear,branched‐chain triterpene metabolism in monocots

Triterpenes are thirty‐carbon compounds derived from the universal five‐carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large‐scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T₀ mature plants) were obtained using the cytosolic‐targeting strategy. Plastid‐targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid‐targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.     

LEFPS1, a tomato farnesyl pyrophosphate gene highly expressed during early fruit development

Farnesyl pyrophosphate synthase (FPS) catalyzes the synthesis of farnesyl pyrophosphate, a key intermediate in sterol and sesquiterpene biosynthesis. Using a polymerase chain reaction-based approach, we have characterized LeFPS1, a tomato (Lycoperscion esculentum cv Wva 106) fruit cDNA, which encodes a functional FPS. We demonstrate that tomato FPSs are encoded by a small multigenic family with genes located on chromosomes 10 and 12. Consistent with farnesyl pyrophosphate requirement in sterol biosynthesis, FPS genes are ubiquitously expressed in tomato plants. Using an LeFPS1 specific probe, we show that the corresponding gene can account for most of FPS mRNA in most plant organs, but not during young seedling development, indicating a differential regulation of FPS genes in tomato. FPS gene expression is also under strict developmental control: FPS mRNA was mainly abundant in young organs and decreased as organs matured with the exception of fruits that presented a biphasic accumulation pattern. In this latter case in situ hybridization studies have shown that FPS mRNA is similarly abundant in all tissues of young fruit. Taken together our results suggest that several FPS isoforms are involved in tomato farnesyl pyrophosphate metabolism and that FPS genes are mostly expressed in relation to cell division and enlargement.     

Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H₂O₂ accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.