Novel chemically synthesized hydroxyl-containing jasmonates as powerful inducing signals for plant secondary metabolism

Novel hydroxyl-containing jasmonate derivatives were chemically synthesized and evaluated by bioassay as potential elicitors for stimulating the biosynthesis of plant secondary metabolites. A suspension culture of Taxus chinensis, which produces a bioactive taxoid, taxuyunnanine C (Tc), was taken as a model plant cell system. Experiments on the timing of addition of jasmonates and dose response indicated that day 7 and 100 microM was the optimal elicitation time and concentration, respectively, for both cell growth and Tc accumulation. Tc accumulation was increased more in the presence of novel hydroxyl-containing jasmonates compared to that with methyljasmonate (MJA) addition. For example, addition of 100 microM 2,3-dihydroxypropyl jasmonate on day 7 led to a very high Tc content of 47.2 +/- 0.5 mg/g (at day 21), whereas the Tc content was 29.2 +/- 0.6 mg/g (on the same day) with addition of 100 microM MJA. Quantitative structure-activity analysis of various jasmonates suggests that the optimal lipophilicity and the number of hydroxyl groups may be two important factors affecting their elicitation activity. In addition, the jasmonate elicitors were found to induce plant defense responses, including oxidative burst and activation of L-phenylalanine ammonia lyase (PAL). Interestingly, a higher level of H(2)O(2) production and PAL activity was detected with elicitation by the synthesized jasmonates compared with that by MJA, which corresponded well to the superior stimulating activity in the former. This work indicates that the newly synthesized hydroxyl-containing jasmonates can act as powerful inducing signals for secondary metabolite biosynthesis in plant cell cultures.     

中国红豆杉细胞经重复诱导和蔗糖饲喂后云南紫杉烷C生产的相应基因表达变化

红豆杉悬浮培养细胞具有可持续生产抗癌药物紫杉醇及其他紫杉烷的潜力。在中国红豆杉悬浮培养细胞中,云南紫杉烷 C(Tc) 是主要的次生代谢产物。为促使代谢前体由生成其他紫杉烷的代谢支路转到生产紫杉醇,实验采用实时定量PCR技术 (RQ-PCR) 揭示细胞培养过程中紫杉醇及紫杉烷合成关键基因的动态变化。在细胞培养的第7天和第12天,以100 μmol/L 2,3-二羟丙基茉莉酸 (DHPJA) 进行诱导,同时在第7天饲喂20 g/L的蔗糖,在此过程中考察6个关键基因 (TASY,TDAT,T5αH,TαH,T10βH和T14βH) 的表达变化。上述联合调控手段使得Tc产量在第1次诱导8 d后达 (554.46±21.28) mg/L,第2次诱导9 d后高达 (997.72±1.51) mg/L。代谢早期基因TASY和TDAT在第1次诱导后表达量分别提高了182和98倍,在第2次诱导后表达量分别提高了208和131倍。在每次诱导后基因表达量提高约持续24 h,之后下降。其他4个基因 (T5αH、TαH、T10βH和T14βH) 的情况有所不同。基因TαH在2次诱导后表达量分别提高了3 061和1 016倍。其他3个基因T5αH、T10βH、T14βH在第1次诱导后表达量分别提高13、38、20倍,在第2次诱导后分别提高7、16、6倍。RQ-PCR结果表明基因表达和Tc积累之间存在紧密相关性：基因表达的变化与Tc产量的变化相一致,诱导可提高6个基因的表达量。基因的高表达随着培养过程逐渐衰减,再次诱导可再次促使基因的高表达。     

Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.     

原位吸附和茉莉酸甲酯联合使用显著提高中国红豆杉细胞云南紫杉烷c的生产

本实验所用的中国红豆杉细胞悬浮培养体系中,云南紫杉烷c(Tc)是主要的次生代谢产物,该化合物有类神经生长因子活性,提高其产量是进一步规模化生产的前提。本研究考察了原位吸附和茉莉酸甲酯(MJA)联合调控提高Tc产量的可能性。在培养的第7天加入浓度为100μmol/L的MJA虽然会使细胞的生物量下降10%～30%,但是单位细胞内Tc含量和Tc产量均有显著提高,分别是对照的3.6和3.3倍。吸附剂XAD-7在不同时间加入对Tc的合成影响显著。在培养的第7天同时加入100μmol/L的MJA和100g/L的XAD-7会使细胞生物量增加,Tc产量显著提高。培养到第21天,Tc产量达477.4mg/L,为对照的6.3倍,为只加MJA的1.9倍,其中94%的Tc被树脂吸附。实验结果表明,在MJA诱导高表达的过程中,吸附剂XAD-7的加入使细胞内代谢产物外泌,浓度降低,减轻产物反馈抑制现象,从而大幅度提高代谢物产量,有较好的生产前景。     

Novel synthetic jasmonates as highly efficient elicitors for taxoid production by suspension cultures of Taxus chinensis

Suspension cultures of Taxus chinensis were used as a model plant cell system to evaluate novel synthetic jasmonates as elicitors for stimulating the biosynthesis of secondary metabolites. Significant increases in accumulation of taxuyunnanine C (Tc) were observed in the presence of newly synthesized 2-hydroxyethyl jasmonate (HEJA) and trifluoroethyl jasmonate (TFEJA) without their inhibition on cell growth. Addition of 100 microM HEJA or TFEJA on day 7 led to a high Tc content of 44.3 +/- 1.1mg/g or 39.7 +/- 1.1 mg/g (at day 21), while the Tc content was 14.0 +/- 0.1 mg/g and 32.4 +/- 1.6 mg/g for the control and that with addition of 100 microM methyl jasmonate (MJA), respectively. The superior stimulating ability of HEJA and TFEJA over MJA, which was generally considered as the best chemical for eliciting taxoid biosynthesis, suggests that the novel jasmonate analogues may have great potential in application to other cell culture systems for effcient elicitation of plant secondary metabolites.     

Gibberellin and Jasmonate Crosstalk during Stamen Development

Gibberellin (GA) and jasmonate (JA) are two types of phytohormones that play important roles during stamen development. For example, Arabidopsis plants deficient in either of GA or JA develop short stamens. An apparent question to ask is whether GA action and JA action during stamen filament development are independent of each other or are in a hierarchy. Recent studies showed that GA modulates the expression of genes essential for JA biosynthesis to promote JA production and high levels of JA will induce the expression of three MYB genes MYB21, MYB24 and MYB57. These three MYB genes are crucial factors for the normal development of stamen filament in Arabidopsis.     

Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites.     

Coordinated activation of metabolic pathways for antioxidants and defence compounds by jasmonates and their roles in stress tolerance in Arabidopsis

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively termed jasmonates, are ubiquitous plant signalling compounds. Several types of stress conditions, such as wounding and pathogen infection, cause endogenous JA accumulation and the expression of jasmonate-responsive genes. Although jasmonates are important signalling components for the stress response in plants, the mechanism by which jasmonate signalling contributes to stress tolerance has not been clearly defined. A comprehensive analysis of jasmonate-regulated metabolic pathways in Arabidopsis was performed using cDNA macroarrays containing 13516 expressed sequence tags (ESTs) covering 8384 loci. The results showed that jasmonates activate the coordinated gene expression of factors involved in nine metabolic pathways belonging to two functionally related groups: (i) ascorbate and glutathione metabolic pathways, which are important in defence responses to oxidative stress, and (ii) biosynthesis of indole glucosinolate, which is a defence compound occurring in the Brassicaceae family. We confirmed that JA induces the accumulation of ascorbate, glutathione and cysteine and increases the activity of dehydroascorbate reductase, an enzyme in the ascorbate recycling pathway. These antioxidant metabolic pathways are known to be activated under oxidative stress conditions. Ozone (O3) exposure, a representative oxidative stress, is known to cause activation of antioxidant metabolism. We showed that O3 exposure caused the induction of several genes involved in antioxidant metabolism in the wild type. However, in jasmonate-deficient Arabidopsis 12-oxophytodienoate reductase 3 (opr3) mutants, the induction of antioxidant genes was abolished. Compared with the wild type, opr3 mutants were more sensitive to O3 exposure. These results suggest that the coordinated activation of the metabolic pathways mediated by jasmonates provides resistance to environmental stresses.     

Jasmonate Signal Pathway in Arabidopsis

Jasmonates （JAs）, which include jasmonic acid and its cyclopentane derivatives are synthesized from the octadecanoid pathway and widely distributed throughout the plant kingdom. JAs modulate the expression of numerous genes and mediate responses to stress, wounding, insect attack, pathogen infection, and UV damage. They also affect a variety of processes in many plant developmental processes. The JA signal pathway involves two important events： the biosynthesis of JA and the transduction of JA signal. Several important Arabidopsis mutants in jasmonate signal pathway were described in this review.     

The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.     

Monitoring of methyl jasmonate-responsive genes in Arabidopsis by cDNA macroarray: self-activation of jasmonic acid biosynthesis and crosstalk with other phytohormone signaling pathways.

Jasmonates mediate various physiological events in plant cells such as defense responses, flowering, and senescence through intracellular and intercellular signaling pathways, and the expression of a large number of genes appears to be regulated by jasmonates. In order to obtain information on the regulatory network of jasmonate-responsive genes (JRGs) in Arabidopsis thaliana (Arabidopsis), we screened 2880 cDNA clones for jasmonate responsiveness by a cDNA macroarray procedure. Since many of the JRGs reported so far have been identified in leaf tissues, the cDNA clones used were chosen from a non-redundant EST library that was prepared from above-ground organs. Hybridization to the filters was achieved using alpha-33P-labeled single-strand DNAs synthesized from mRNAs obtained from methyl jasmonate (MeJA)-treated and untreated Arabidopsis seedlings. Data analysis identified 41 JRGs whose mRNA levels were changed by more than three fold in response to MeJA. This was confirmed by Northern blot analysis by using eight representatives. Among the 41 JRGs identified, 5 genes were JA biosynthesis genes and 3 genes were involved in other signaling pathways (ethylene, auxin, and salicylic acid). These results suggest the existence of a positive feedback regulatory system for JA biosynthesis and the possibility of crosstalk between JA signaling and other signaling pathways.     

A gain-of-function allele of TPC1 activates oxylipin biogenesis after leaf wounding in Arabidopsis

Jasmonates, potent lipid mediators of defense gene expression in plants, are rapidly synthesized in response to wounding. These lipid mediators also stimulate their own production via a positive feedback circuit, which depends on both JA synthesis and JA signaling. To date, molecular components regulating the activation of jasmonate biogenesis and its feedback loop have been poorly characterized. We employed a genetic screen capable of detecting the misregulated activity of 13-lipoxygenase, which operates at the entry point of the jasmonate biosynthesis pathway. Leaf extracts from the Arabidopsis fou2 (fatty acid oxygenation upregulated 2) mutant displayed an increased capacity to catalyze the synthesis of lipoxygenase (LOX) metabolites. Quantitative oxylipin analysis identified less than twofold increased jasmonate levels in healthy fou2 leaves compared to wild-type; however, wounded fou2 leaves strongly increased jasmonate biogenesis compared to wounded wild-type. Furthermore, the plants displayed enhanced resistance to the fungus Botrytis cinerea. Higher than wild-type LOX activity and enhanced resistance in the fou2 mutant depend fully on a functional jasmonate response pathway. The fou2 mutant carries a missense mutation in the putative voltage sensor of the Two Pore Channel 1 gene (TPC1), which encodes a Ca(2+)-permeant non-selective cation channel. Patch-clamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. The results indicate that cation fluxes exert strong control over the positive feedback loop whereby JA stimulates its own synthesis.     

Ectopic expression of AtJMT in Nicotiana attenuata: creating a metabolic sink has tissue-specific consequences for the jasmonate metabolic network and silences downstream gene expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.     

荣莉酸甲酯对丹参培养细胞中迷迭香酸生物合成及相关酶活性的影响

茉莉酸甲酯是植物细胞响应外界刺激产生的重要信号分子,与植物次生代谢物的生物合成有关。本研究考察了茉莉酸甲酯（methyl jasmonate,MeJA）对丹参培养细胞中迷迭香酸（rosmarinic acid,RA）生物合成的影响。结果显示,诱导24h后可显著提高丹参愈伤细胞中RA的积累量及其相关酶（PAL、TAT）的活性,在48h时RA积累量和酶活性达到最大。布洛芬（IBu）处理可抑制MeJA对RA积累量和相关酶活性的促进作用,外源施加MeJA可部分解除IBU对RA合成及其相关酶活性的抑制作用。说明MeJA可以显著促进丹参培养细胞中RA的生物合成,IBU抑制了MeJA合成、PAL和TAT活性,从而导致了RA合成受阻。     

Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.     

外源茉莉酸和茉莉酸甲酯诱导植物抗虫作用及其机理

综述了茉莉酸(jasmonic acid, JA)和茉莉酸甲酯(methyl jasmo nate, MJA)的分子结构和应用其诱导的植物抗虫作用及其机制。植物受外源茉莉酸或茉莉酸甲酯刺激后,一条反应途径是由硬脂酸途径激活防御基因,另一条途径是直接激活防御基因。防御基因激活后导致代谢途径重新配置,并可能诱导植物产生下列4种效应：（1）直接防御,即植物产生对害虫有毒的物质、抗营养和抗消化的酶类,或具驱避性和妨碍行为作用的化合物;（2）间接防御,即产生吸引天敌的挥发物;（3）不防御,即无防御反应;（4）负防御,即产生吸引害虫的挥发物。