Downregulation of Cinnamoyl CoA Reductase Affects Lignin and Phenolic Acids Biosynthesis in Salvia miltiorrhiza Bunge

The biosynthesis of salvianolic acid B shares the phenylpropanoid pathway with lignin, and cinnamoyl CoA reductase (CCR; EC 1.2.1.44) is a specific enzyme in the lignin pathway. In this study, a CCR gene (SmCCR1) from Salvia miltiorrhiza Bunge was cloned using DNA walking technology (GenBank ID: JF798634). The full-length SmCCR1 is 2,489?bp long and consists of four introns and five exons encoding a polypeptide of 324 amino acid residues. Sequence alignment revealed that SmCCR1 shares 83?% identity with CCR sequences reported in Camellia oleifera and other plant species. Expression pattern analysis indicated that expression of SmCCR1 can be induced by exposure to Xanthomonas campestris pv. Campestris or methyl jasmonate. To demonstrate its functioning, we selected a 296-bp fragment and established an RNA interference construct that was introduced into S. miltiorrhiza by Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants exhibited dwarfing phenotypes, and both syringyl and guaiacyl lignin monomers were decreased more than 60?%. In contrast, biosynthesis of phenolic acids??danshensu, rosmarinic acid, and salvianolic acid B??was strongly induced by 2.03-, 1.41-, and 1.45-fold, respectively, in the roots of transgenic plants from line CCR-10. Consistent with these phytochemical changes, downregulation of SmCCR1 also affected the expression of related genes in the phenolics and lignin biosynthetic pathways. Our results also provide potential opportunities for engineering danshensu and salvianolic acid B production in S. miltiorrhiza.     

Nitrogen and phosphorus requirements for raising mycorrhizal seedlings of Leucaena leucocephala in containers

A greenhouse study was undertaken to determine the nitrogen and phosphorus fertilization requirements for raising mycorrhizal seedlings in soil in containers. Seedlings of Leucaena leucocephala were grown for 40 days in dibble tubes containing fumigated or nonfumigated soil uninoculated or inoculated with Glomus aggregatum. The soil was fertilized with NH₄NO₃ solution to obtain 25–200 mg N kg^-1 soil, and with a KH₂PO₄ solution to establish target soil solution P concentrations of 0.015–0.08 mg P l^-1. At the end of 40 days, seedlings were transplanted into pots containing 5-kg portions of fumigated soil. Posttransplant vesicular arbuscular mycorrhizal fungal (VAMF) effectiveness, measured as pinnule P content, plant height, shoot dry weight and tissue N and P concentrations, was significantly increased by pretransplant VAMF colonization in both soils. The best posttransplant mycorrhizal colonization and mycorrhizal growth responses were observed if the nonfumigated pretransplant soil was amended with 50 mg N kg^-1 soil and 0.04 mg P l^-1 or if the fumigated pretransplant soil was amended with 100 mg N kg^-1 soil and 0.04 mg P 1^-1. There was no relationship between NP ratios of nutrients added to the pretransplant soil medium and shoot NP ratios observed after transplanting. Shoot NP ratio was also not correlated with root colonization level.Contribution from the Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 4025     

The effect of VA mycorrhizal fungi,phosphorus and drought stress on the growth of Acacia nilotica and Leucaena leucocephala seedlings

The effect of vesicular-arbuscular mycorrhizal (VAM) fungi on growth and drought resistance of Acacia nilotica and Leucaena leucocephala seedlings was studied in a glasshouse experiment. The experimental design was a 2·2·2 factorial: ± mycorrhizal inoculation, ± application of phosphorus fertilizer and ± repeated drought treatment. The growth promoting effect of VAM fungi equalled the effect of phosphorus fertilization after 12 weeks. The drought treatment reduced seedling biomass and nodulation. Differences between the plant species were found with respect to growth improvements due to VAM inoculation and/or phosphorus fertilization under drought stress conditions. The results are discussed in relation to plant drought resistance and reforestation in the subhumid to arid tropics.     

Sequence variation in two lignin biosynthesis genes,cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2)

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2) are genes which may influence variation in lignin content and composition within plants. Sequence variation within these genes may be responsible for changes in enzyme activity and/or specificity, which could cause variation in lignin content or composition. This study examines sequence variation within these two genes in Eucalyptus globulus, an important species used in pulp and paper-making. Twenty-one single nucleotide polymorphisms (SNPs) were identified in the exons of CCR, of which nine were neutral mutations and 12 were missense mutations. Six of the missense mutations affected highly conserved amino acids within the protein sequence of CCR. Eight SNPs were identified in the CAD2 exons, six of which were neutral mutations and two which were missense mutations. One of the missense mutations affected a highly conserved amino acid within the protein sequence. In addition, 32 SNPs were identified in the CCR introns along with four insertion/deletions and two polyA length variation regions. Polymorphism affecting highly conserved amino acids may alter enzyme function and this molecular variation may be linked to variation in lignin profiles. Selecting positive alleles which produce favourable lignin profiles would be advantageous in tree breeding programs.     

干旱胁迫对坡柳等抗旱树种幼苗膜脂过氧化及保护酶活性的影响

以金沙江干热河谷主要树种坡柳、银合欢、山毛豆实生幼苗为材料,通过盆栽苗自然干旱胁迫,同时以浇水处理为对照,研究了干旱胁迫对坡柳、银合欢、山毛豆3个树种丙二醛含量、膜相对透性及保护酶活性的影响。结果表明,干旱胁迫下3个树种幼苗的细胞膜透性、MDA及SOD, POD酶活性都发生了变化,只是变化的幅度和进程不同。干旱胁迫对银合欢膜系统损伤生成的主要降解产物不是MDA;山毛豆清除活性氧毒害作用主要不是通过SOD和POD的作用;通过叶片相对保水力测定及膜透性、MDA相对含量、酶活性变化情况的分析,3个树种中坡柳耐旱性最强,其次为银合欢,山毛豆居后。     

in Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1)

Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants.

Abbreviations

Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.     

植物肉桂酰辅酶A还原酶基因克隆研究进展

木质素单体合成的过程中涉及了许多酶的参与,而肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase,CCR)是该过程中的一个关键酶。综述了CCR基因在植物体内的克隆、基因功能及在植物组织中的表达情况,并介绍了该基因在植物的抗病虫害和抗逆性研究、饲草和能源上的应用潜力,为进一步研究CCR基因生物学功能和利用奠定了基础。     

Testa Structure and Histochemistry Related to Water Uptake in Leucaena leucocephala Lam. (De Wit)

The testa structure and histochemistry of Leucaena leucocephalaLam. (De Wit) seed were investigated by bright-field and fluorescencelight microscopy, and scanning electron microscopy. The testaconsisted of several separate layers. Externally there was anon-cellular layer made up of two parts differing in histochemicalcharacteristics: an "outer strip" rich in hydrophilic substances,and a "thicker part" showing the occurrence of phenolics, involvedin water impermeability control. A second underlying thin layerwas formed by the palisade cell caps, joined one to another.This layer, which we called the "cap film ", became metachromaticreddish-blue with Toluidine O pH 4·4 and reacted positivelywith Alcian blue pH 2·5, revealing the presence of polysaccharidehydrophilic material. The palisade cells consisted of two parts,having different structures and histochemical features. Thefurrowed upper part revealed the simultaneous presence of hydrophilicand hydrophobic substances in the furrows and in the ribs respectively.The corresponding periclinal section showed a central daisy-likepattern (made up of hydrophilic material) with a small chainof tiny spots (made up of hydrophobic material) all around it.The inner part of the palisade cells was also furrowed, butshowed only hydrophobic substances of lipidic nature, detectedby Auramine O and Phosphine 3R. The light line was found tobe rich in callose as shown by the strong fluorescence inducedby Aniline Blue. These findings add supporting evidence of thecomplex structure and composition of the Leucaena leucocephalatesta.Copyright 1994, 1999 Academic Press Leucaena leucocephala, testa, structure, histochemistry, water entry     

含羞草素及其代谢产物的降解和银合欢去毒(综述)

本文概述含羞草素及其代谢产物降解的物理、化学和生物学途径,为银合欢的去毒研究提供依据。     

Fluctuation of Endogenous Levels of Free and Conjugated Gibberellins throughout Seed Maturation in Leucaena leucocephala (Lmk) De Wit

Combined gas chromatography-selected ion monitoring (GC-SIM)analysis of a purified extract from seeds of Leucaena leucocephalashowed the presence of GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₃, GA₂₉and GA₅₃. GA₁, GA₈ and GA₂₉ were also found both as glucosylester-like and glucosyl ether-like conjugates, and GA₂₀ as aglucosyl ester-like conjugate; these conjugates were analyzedas free GAs after enzyme hydrolysis. GA₂₃ was shown to be themain free gibberellin in immature seeds (268 ng/seed), thoughits level drastically decreased during their maturation. GA₁was the main free C₁₉-gibbercllin and GA₁ glucosyl ester-likeand glucosyl ether-like conjugates were found at the highestlevels in the seeds prior to maturation. Fluctuation of endogenouslevels of gibberellins is discussed in terms of seed development. (Received May 9, 1984; Accepted August 25, 1984)     

Auxin induces mitogenic activated protein kinase (MAPK) activation in roots of Arabidopsis seedlings

Genome analyses have shown that plants contain gene families encoding various components of mitogen-activated protein kinase (MAPK) signaling pathways. Previous reports have described the involvement of MAPK pathways in stress and pathogen responses of leaves and suspension-cultured cells. Here we show that auxin treatment of Arabidopsis roots transiently induced increases in protein kinase activity with characteristics of mammalian ERK-like MAPKs. The MAPK response we monitored was the result of hormonal action of biologically active auxin, rather than a stress response provoked by auxin-like compounds. Auxin-induced MAPK pathway signaling was distinguished genetically in the Arabidopsis auxin response mutant axr4, in which MAPK activation by auxin, but not by salt stress, was significantly impaired. Perturbation of MAPK signaling in roots using inhibitors of a mammalian MAPKK blocked auxin-activated transgene expression in BA3-GUS seedlings, while potentiating higher than normal levels of MAPK activation in response to auxin. Data presented here indicate that MAPK pathway signaling is positively involved in auxin response, and further suggest that interactions among MAPK signaling pathways in plants influence plant responses to auxin.     

Strong decrease in lignin content without significant alteration of plant development is induced by simultaneous down-regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) in tobacco plants

Different transgenic tobacco lines down-regulated for either one or two enzymes of the monolignol pathway were compared for their lignin content and composition, and developmental patterns. The comparison concerned CCR and CAD down-regulated lines (homozygous or heterozygous for the transgene) and the hybrids resulting from the crossing of transgenic lines individually altered for CCR or CAD activities. Surprisingly, the crosses containing only one allele of each antisense transgene, exhibit a dramatic reduction of lignin content similar to the CCR down-regulated parent but, in contrast to this transgenic line, display a normal phenotype and only slight alterations of the shape of the vessels. Qualitatively the lignin of the double transformant displays characteristics more like the wild type control than either of the other transgenics. In the transgenics with a low lignin content, the transformations induced other biochemical changes involving polysaccharides, phenolic components of the cell wall and also soluble phenolics. These results show that the ectopic expression of a specific transgene may have a different impact depending on the genetic background and suggest that the two transgenes present in the crosses may operate synergistically to reduce the lignin content. In addition, these data confirm that plants with a severe reduction in lignin content may undergo normal development at least in controlled conditions.     

杨树木质素合成酶CCR基因的序列分析及蛋白结构预测

利用RT—PCR从欧美杨107次生木质部中克隆出-961bp的CCR基因片段。通过生物信息学软件对该序列的核苷酸序列、拟翻译的氨基酸序列的疏水性、残基带电量及表面暴露区、蛋白质二级结构、亚细胞定位及三维结构等进行了初步分析预测。结果表明该CCR基因含一个编码301个氨基酸的完整开放阅读框,其成熟蛋白为亲水性的、主要存在于细胞膜,具有大多数植物CCR蛋白普遍存在的KNWYCYGK的保守性基序,其二级结构中共包含12个α螺旋,20个β折叠,11个卷曲,并构建了其三维结构图。     

干旱胁迫下坡柳等幼苗质膜相对透性和脯氨酸含量的变化

以金沙江干热河谷主要树种坡柳、银合欢和山毛豆幼苗为材料,通过盆栽苗自然干旱胁迫,同时以浇水处理为对照,探讨了干旱胁迫下三树种幼苗叶片的相对含水量、质膜透性以及脯氨酸含量的变化情况。结果表明,干旱胁迫后三树种叶片的相对含水量、质膜相对透性以及脯氨酸含量都发生了变化,只是变化的幅度和进程不同。三项生理指标的综合分析及结合其自然表现,坡柳具有较强的抗旱能力,其次为银合欢,最后是山毛豆。     

Developmental Toxicity of the HMG‐CoA Reductase Inhibitor (PPD10558) in Rats and Rabbits

PPD10558 is an orally active, lipid‐lowering 3–hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor (statin) being developed as a treatment for hypercholesterolemia in patients who have not been able to tolerate statins because of statin‐associated myalgia. We have studied the potential developmental toxicity effects of PPD10558 in pregnant rats and rabbits given daily oral doses during the period of organogenesis. Rats were dosed with 0, 20, 80, or 320 mg/kg/day from Gestation Day (GD) 6 to 17 and rabbits received dose levels of 0, 12.5, 25, or 50 mg/kg/day from GD 6 to 18. Additional groups in both studies served as toxicokinetic animals and received the PPD10558 in the same manner as the main study groups at the same dose levels. Blood samples were collected from toxicokinetic animals at designated time points on GD 6 and 17 in rats and GD 6 and 18 in rabbits. Fetal exposure in rats was assessed on GD 20. Maternal and developmental parameters were evaluated in rats and rabbits on GD 20 and GD 29, respectively. No maternal and developmental toxicity was observed at any of the dose levels used in the rat study. Evidence of fetal exposure was determined in fetal plasma with mean fetal concentrations of PPD10558 and the metabolite (PPD11901) found to be between 1 and 6% of the mean maternal concentrations. In rabbits, marked maternal toxicity including mortality (eight deaths; 1 dose at 25 and 7 at 50 mg/kg/day), abortions (2 at 25 mg/kg/day and 6 at 50 mg/kg/day) and reduction in gestation body weight, gestation body weight changes and decreased food consumption were observed. In addition, fetal body weights of the combined sexes were significantly reduced at 50 mg/kg/day in comparison with the controls. Mean peak exposure (Cmax) and total exposure (AUC(0–24)) of PPD11901 in both rats and rabbits were higher than that of PPD10558 on GD 6 and GD 17 at each of the three dose levels.. Based on the results of these studies, the no observed adverse effect level (NOAEL) for maternal and developmental toxicity in rats was considered to be ≥320 mg/kg/day, the highest dose level used in the study. The NOAEL for maternal and developmental toxicity in rabbits was 12.5 mg/kg/day and 25 mg/kg/day, respectively.     

Zinc stress induces physiological,ultra-structural and biochemical changes in mandarin orange (Citrus reticulata Blanco) seedlings

Zinc (Zn) is an essential micronutrient for higher plants; yet, at higher concentrations it is toxic. In order to explore the effect of Zn stress on growth, biochemical, physiological and ultra-structural changes, 1 year old mandarin plants were grown under various Zn concentrations (1, 2, 3, 4, 5, 10 15 and 20 mM) for 14 weeks. The biomass of the plants increased with increasing Zn concentrations and finally declined under excess Zn concentration but the prime increase was observed at 4 and 5 mM Zn. Zn stress reduced the photosynthetic rate, stomatal conductance, and transpiration along with reduction of chlorophyll a, chlorophyll b, and carotenoids content in leaf. Superoxide anion, malondialdehyde, hydrogen peroxide and electrolyte leakage were elevated in Zn stressed plants. The activities of ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) enzymes were increased in both Zn-deficient and Zn-excess plants. Therefore it is suggested that antioxidant defense system did not sufficiently protect the plants under rigorous Zn stress which was also corroborated by the alteration in cell ultrastructure as revealed by transmission electron microscopy.