Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

甘薯肉桂酸-4-羟化酶基因克隆及序列分析

肉桂酸-4-羟化酶(Cinnamic acid-4-hydroxylase,C4H,EC 1.14.13.11)是苯丙烷途径中第二步反应酶,同时也是花色素苷前体生物合成途径中关键酶。该研究根据植物C4H的同源序列设计引物,通过RTPCR结合RACE的方法,在紫色甘薯中获得了与其相应的C4H基因,命名为Ib C4H(Gen Bank登录号GQ373157)。结果表明:(1)序列分析表明Ib C4H长1 668 bp,编码505个氨基酸,该氨基酸序列与其c DNA序列与Ib C4H蛋白与马铃薯C4H蛋白序列最为接近,与苹果、黑莓、大阿米芹、油菜一致性很高,均在70%以上。(2)二级结构预测表明α-螺旋和无规则卷曲是Ib C4H蛋白最大量的结构元件,而延伸链则散布于整个蛋白中。(3)三维结构建模预测,Ib C4H蛋白具备细胞色素P450氧和铁离子结合位点等典型的C4H结构。该研究结果为进一步了解花色素苷生物合成途径中的作用奠定了基础,也为花青素生物合成分子机理和代谢调控提供了靶位点和理论参考。     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

The cycling gonad: retinoic‐acid synthesis and degradation patterns during adult zebrafish Danio rerio oogenesis

The expression pattern of genes coding for enzymes of the retinoic acid (RA) synthetic and degradation pathways was characterized in adult female zebrafish Danio rerio. Females were conditioned until maturation and post‐spawn expression dynamics were determined. A striking upregulation of cyp26b1, but not cyp26a1, was observed following egg deposition, decreasing to initial levels during recovery. A similar, yet lower, fluctuation was observed for aldh1a2 and rdh10a, the enzymes participating in the two‐step RA biosynthesis cascade. The present work highlights the dynamics of the adult D. rerio oogenesis and uncovers novel, yet elusive, metabolic contributors. Possible compartmentalized roles for the different gene paralogue isoforms are discussed.     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.     

Gibberellin Biosynthetic Pathway inGibberella fujikuroi:Evidence for a Gene Cluster

Differential screening of aGibberella fujikuroicDNA library was used to successfully clone and identify genes involved in the pathway of gibberellin biosynthesis. Several cDNA clones that hybridized preferentially to a cDNA probe prepared from mycelium induced for gibberellin production were isolated and characterized. The deduced amino acid sequences of two (identical) clones contained the conserved heme-binding motif of cytochrome P450 monooxygenases (FXXGXXXCXG). One of these cDNA fragments was used as a homologous probe for the screening of a genomic library. A hybridizing 6.7-kb genomicSalI fragment was cloned into pUC19. The sequencing of this clone revealed that a second cytochrome P450 monooxygenase gene was closely linked to the first one. Since at least four cytochrome P450 monooxygenase-catalyzed steps are involved in the synthesis of gibberellins, chromosome walking was performed to find a further gene of this family or other genes involved in gibberellin pathway. Next to the two P450 monooxygenase genes, a putative geranylgeranyl diphosphate synthase gene, the copalyl diphosphate synthase gene, which is the first specific gene of the gibberellin pathway, and a third P450 monooxygenase gene were identified. These results suggest that at least some of the genes involved in the biosynthesis of gibberellins are closely linked in a gene cluster inG. fujikuroi,as has been recently found for other “dispensable” pathways in fungi.     

Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish

Teleost fish are characterized by exceptionally high levels of brain estrogen biosynthesis when compared to the brains of other vertebrates or to the ovaries of the same fish. Goldfish (Carassius auratus) and zebrafish (Danio rerio) have utility as complementary models for understanding the molecular basis and functional significance of exaggerated neural estrogen biosynthesis. Multiple cytochrome P450 aromatase (P450arom) cDNAs that derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (P450aromBA) and ovary (P450aromAB) and have a different developmental program (BA) and response to estrogen upregulation (B only). As measured by increased P450aromB mRNA, a functional estrogen response system is first detected 24–48 h post-fertilization (hpf), consistent with the onset of estrogen receptor (ER) expression (α, β, and γ). The 5′-flanking region of the cyp19b gene has a TATA box, two estrogen response elements (EREs), an ERE half-site (ERE1/2), a nerve growth factor inducible-B protein (NGFI-B)/Nur77 responsive element (NBRE) binding site, and a sequence identical to the zebrafish GATA-2 gene neural specific enhancer. The cyp19a promoter region has TATA and CAAT boxes, a steroidogenic factor-1 (SF-1) binding site, and two aryl hydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) binding motifs. Both genes have multiple potential SRY/SOX binding sites (16 and 8 in cyp19b and cyp19a, respectively). Luciferase reporters have basal promoter activity in GH3 cells, but differences (ab) are opposite to fish pituitary (ba). When microinjected into fertilized zebrafish eggs, a cyp19b promoter-driven green fluorescent protein (GFP) reporter (but not cyp19a) is expressed in neurons of 30–48 hpf embryos, most prominently in retinal ganglion cells (RGCs) and their projections to optic tectum. Further studies are required to identify functionally relevant cis-elements and cellular factors, and to determine the regulatory role of estrogen in neurodevelopment.     

PERMANENT GENETIC RESOURCES: Consensus primers of cyp73 genes discriminate willow species and hybrids (Salix, Salicaceae)

Consensus primers, based on exon sequences of the cyp73 gene family coding for cinnamate 4-hydroxylase (C4H) of the lignin biosynthesis pathway, were designed for the tetraploid willow species Salix alba and Salix fragilis. Diagnostic alleles at species level were observed among introns of three cyp73 genes and allowed unambiguous detection of the first generation and introgressed hybrids in populations. Progeny analysis of a female S. alba with a male introgressed hybrid confirmed the codominant inheritance of each intron. Sequences of the diagnostic alleles of both species were similar to those found in the hybrids.     

Indole Glucosinolate Biosynthesis Limits Phenylpropanoid Accumulation in Arabidopsis thaliana

Plants produce an array of metabolites (including lignin monomers and soluble UV-protective metabolites) from phenylalanine through the phenylpropanoid biosynthetic pathway. A subset of plants, including many related to Arabidopsis thaliana, synthesizes glucosinolates, nitrogen- and sulfur-containing secondary metabolites that serve as components of a plant defense system that deters herbivores and pathogens. Here, we report that the Arabidopsis thaliana reduced epidermal fluorescence5 (ref5-1) mutant, identified in a screen for plants with defects in soluble phenylpropanoid accumulation, has a missense mutation in CYP83B1 and displays defects in glucosinolate biosynthesis and in phenylpropanoid accumulation. CYP79B2 and CYP79B3 are responsible for the production of the CYP83B1 substrate indole-3-acetaldoxime (IAOx), and we found that the phenylpropanoid content of cyp79b2 cyp79b3 and ref5-1 cyp79b2 cyp79b3 plants is increased compared with the wild type. These data suggest that levels of IAOx or a subsequent metabolite negatively influence phenylpropanoid accumulation in ref5 and more importantly that this crosstalk is relevant in the wild type. Additional biochemical and genetic evidence indicates that this inhibition impacts the early steps of the phenylpropanoid biosynthetic pathway and restoration of phenylpropanoid accumulation in a ref5-1 med5a/b triple mutant suggests that the function of the Mediator complex is required for the crosstalk.     

The novel pathogen-responsive glycosyltransferase UGT73C7 mediates the redirection of phenylpropanoid metabolism and promotes SNC1-dependent Arabidopsis immunity

Recent studies have shown that global metabolic reprogramming is a common event in plant innate immunity; however, the relevant molecular mechanisms remain largely unknown. Here, we identified a pathogen-induced glycosyltransferase, UGT73C7, that plays a critical role in Arabidopsis disease resistance through mediating redirection of the phenylpropanoid pathway. Loss of UGT73C7 function resulted in significantly decreased resistance to Pseudomonas syringae pv. tomato DC3000, whereas constitutive overexpression of UGT73C7 led to an enhanced defense response. UGT73C7-activated immunity was demonstrated to be dependent on the upregulated expression of SNC1, a Toll/interleukin 1 receptor-type NLR gene. Furthermore, in vitro and in vivo assays indicated that UGT73C7 could glycosylate p-coumaric acid and ferulic acid, the upstream metabolites in the phenylpropanoid pathway. Mutations that lead to the loss of UGT73C7 enzyme activities resulted in the failure to induce SNC1 expression. Moreover, glycosylation activity of UGT73C7 resulted in the redirection of phenylpropanoid metabolic flux to biosynthesis of hydroxycinnamic acids and coumarins. The disruption of the phenylpropanoid pathway suppressed UGT73C7-promoted SNC1 expression and the immune response. This study not only identified UGT73C7 as an important regulator that adjusts phenylpropanoid metabolism upon pathogen challenge, but also provided a link between phenylpropanoid metabolism and an NLR gene.     

Enantioselectivity in Developmental Toxicity of rac‐metalaxyl and R‐metalaxyl in Zebrafish (Danio rerio) Embryo

Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac‐metalaxyl and R‐metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac‐metalaxyl and R‐metalaxyl, and the LC₅₀s at 96 h are 416.41 (353.91, 499.29) mg · L^‐1 and 320.650 (279.80, 363.46) mg · L^‐1, respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic–pituitary–gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic–pituitary–thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real‐time polymerase chain reaction (qRT‐PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac‐metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R‐metalaxyl. And likewise, rac‐metalaxyl only caused the upregulation of dio2, while R‐metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489–494, 2016. © 2016 Wiley Periodicals, Inc.