Metabolite profiling of Arabidopsis seedlings in response to exogenous sinalbin and sulfur deficiency

In order to determine how plant uptake of a sulfur-rich secondary metabolite, sinalbin, affects the metabolic profile of sulfur-deficient plants, gas chromatography time-of-flight mass spectrometry (GC-TOF-MS), in combination with liquid chromatography-mass spectrometry (LC-MS), was used to survey the metabolome of Arabidopsis seedlings grown in nutrient media under different sulfur conditions. The growth media had either sufficient inorganic sulfur for normal plant growth or insufficient inorganic sulfur in the presence or absence of supplementation with organic sulfur in the form of sinalbin (p-hydroxybenzylglucosinolate). A total of 90 metabolites were identified by GC-TOF-MS and their levels were compared across the three treatments. Of the identified compounds, 21 showed similar responses in plants that were either sulfur deficient or sinalbin supplemented compared to sulfur-sufficient plants, while 12 metabolites differed in abundance only in sulfur-deficient plants. Twelve metabolites accumulated to higher levels in sinalbin-supplemented than in the sulfur-sufficient plants. Secondary metabolites such as flavonol conjugates, sinapinic acid esters and glucosinolates, were identified by LC-MS and their corresponding mass fragmentation patterns were determined. Under sinalbin-supplemented conditions, sinalbin was taken up by Arabidopsis and contributed to the endogenous formation of glucosinolates. Additionally, levels of flavonol glycosides and sinapinic acid esters increased while levels of flavonol diglycosides with glucose attached to the 3-position were reduced. The exogenously administered sinalbin resulted in inhibition of root and hypocotyl growth and markedly influenced metabolite profiles, compared to control and sulfur-deficient plants. These results indicate that, under sulfur deficient conditions, glucosinolates can be a sulfur source for plants. This investigation defines an opportunity to elucidate the mechanism of glucosinolate degradation in vivo.     

拟南芥莲座叶芥子油苷含量对水分胁迫的响应

芥子油苷(glucosinolates)是十字花科植物中一类含氮、含硫的次生代谢产物,与其水解产物在植物防御功能中有重要意义且与环境因子关系密切.通过控制供水的方式对营养生长时期的拟南芥幼苗进行水分胁迫,观察了土壤自然干旱对营养生长时期拟南芥莲座叶芥子油苷含量及组成的影响.结果表明,土壤自然干旱处理下,拟南芥莲座叶的芥子油苷总量从处理3 d起低于对照,且随着处理天数的增加与对照组的差异逐渐增大,脂肪族芥子油苷的响应均比较明显,与芥子油苷总量的变化趋势基本一致,而吲哚族芥子油苷对水分胁迫则不敏感.脂肪族中的4-甲基亚磺酰丁基芥子油苷(4-methylsulphinylbutyl GS,4MSOB)占脂肪族芥子油苷的比例最大,它的含量变化成为影响莲座叶中芥子油苷组合模式的主导因素.     

Metabolic engineering of valine- and isoleucine-derived glucosinolates in Arabidopsis expressing CYP79D2 from Cassava

Glucosinolates are amino acid-derived natural products that, upon hydrolysis, typically release isothiocyanates with a wide range of biological activities. Glucosinolates play a role in plant defense as attractants and deterrents against herbivores and pathogens. A key step in glucosinolate biosynthesis is the conversion of amino acids to the corresponding aldoximes, which is catalyzed by cytochromes P450 belonging to the CYP79 family. Expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in Arabidopsis resulted in the production of valine (Val)- and isoleucine-derived glucosinolates not normally found in this ecotype. The transgenic lines showed no morphological phenotype, and the level of endogenous glucosinolates was not affected. The novel glucosinolates were shown to constitute up to 35% of the total glucosinolate content in mature rosette leaves and up to 48% in old leaves. Furthermore, at increased concentrations of these glucosinolates, the proportion of Val-derived glucosinolates decreased. As the isothiocyanates produced from the Val- and isoleucine-derived glucosinolates are volatile, metabolically engineered plants producing these glucosinolates have acquired novel properties with great potential for improvement of resistance to herbivorous insects and for biofumigation.     

Localization of the glucosinolate biosynthetic enzymes reveals distinct spatial patterns for the biosynthesis of indole and aliphatic glucosinolates

Glucosinolates constitute the primary defense metabolites in Arabidopsis thaliana (Arabidopsis). Indole and aliphatic glucosinolates, biosynthesized from tryptophan and methionine, respectively, are known to serve distinct biological functions. Although all genes in the biosynthetic pathways are identified, and it is known where glucosinolates are stored, it has remained elusive where glucosinolates are produced at the cellular and tissue level. To understand how the spatial organization of the different glucosinolate biosynthetic pathways contributes to their distinct biological functions, we investigated the localization of enzymes of the pathways under constitutive conditions and, for indole glucosinolates, also under induced conditions, by analyzing the spatial distribution of several fluorophore‐tagged enzymes at the whole plant and the cellular level. We show that key steps in the biosynthesis of the different types of glucosinolates are localized in distinct cells in separate as well as overlapping vascular tissues. The presence of glucosinolate biosynthetic enzymes in parenchyma cells of the vasculature may assign new defense‐related functions to these cell types. The knowledge gained in this study is an important prerequisite for understanding the orchestration of chemical defenses from site of synthesis to site of storage and potential (re)mobilization upon attack.     

Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis by defense signaling pathways

Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3- to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 and CYP79B3 were both highly induced. Specifically, the indole glucosinolate N-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutant coi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducing mpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.     

Genetic control of natural variation in Arabidopsis glucosinolate accumulation

Glucosinolates are biologically active secondary metabolites of the Brassicaceae and related plant families that influence plant/insect interactions. Specific glucosinolates can act as feeding deterrents or stimulants, depending upon the insect species. Hence, natural selection might favor the presence of diverse glucosinolate profiles within a given species. We determined quantitative and qualitative variation in glucosinolates in the leaves and seeds of 39 Arabidopsis ecotypes. We identified 34 different glucosinolates, of which the majority are chain-elongated compounds derived from methionine. Polymorphism at only five loci was sufficient to generate 14 qualitatitvely different leaf glucosinolate profiles. Thus, there appears to be a modular genetic system regulating glucosinolate profiles in Arabidopsis. This system allows the rapid generation of new glucosinolate combinations in response to changing herbivory or other selective pressures. In addition to the qualitative variation in glucosinolate profiles, we found a nearly 20-fold difference in the quantity of total aliphatic glucosinolates and were able to identify a single locus that controls nearly three-quarters of this variation.     

Glucosinolate biochemical diversity and innovation in the Brassicales

Glucosinolates were analysed from herbarium specimens and living tissues from representative of all families of the Brassicales, following the phylogenetic schemes of Rodman et al. (1998) and Hall et al. (2002, 2004), including specimens of Akania, Setchellanthus, Emblingia, Stixis, Forchhammeria and members of the Capparaceae for which glucosinolate content had not previously been reported. The results are reviewed along with additional published data on glucosinolate content of members of the Brassicales. In addition to providing an overview of the evolution of glucosinolate biochemical diversity within the core Brassicales, there were three main findings. Firstly, the glucosinolate content of some 'orphan' taxa of the Brassicales, such as Setchellanthus and Emblingia were consistent with recent phylogentic analyses based upon DNA sequence comparisons, while further analyses of Tirania and Stixis is required. Secondly, methyl glucosinolate is found within the Capparaceae and Cleomaceae, but also, unexpectedly, within Forchhammeria, with implications for the biochemical and evolutionary origin of methyl glucosinolate and the phylogenetic relationships of Forchhammeria. Thirdly, whereas Old World Capparaceae contain methyl glucosinolate, New World Capparaceae, including New World Capparis, either contain methyl glucosinolates or glucosinolates of complex and unresolved structures, indicative of continued innovation in glucosinolate biosynthesis. These taxa may be productive sources of glucosinolate biosynthetic genes and alleles that are not found in the model plant Arabidopsis thaliana.     

Class targeted metabolomics: ESI ion trap screening methods for glucosinolates based on MSn fragmentation

Glucosinolates are naturally occurring anionic secondary plant metabolites incorporating a thioglucosidic link to the carbon of a sulphonated oxime. There are a large number of naturally occurring glucosinolates and they are found in relatively large quantities in many plant species within the family Crucifereae. These metabolites are of interest for both their anticancer and flavour properties and in the study of nitrogen and sulphur metabolism in model plants such as Arabidopsis. Parent ion mapping is an analytical mass spectrometry approach that allows rapid assessment of glucosinolate content. Ion mapping proved to be highly sensitive and the glucosinolate sinigrin could be detected at three parts per trillion. This method takes advantage of the glucosinolate anion fragmentation which consistently produces a sulphonate ring-opened glucose moiety in the ion trap mass spectrometer, m/z 259. An intramolecular transfer mechanism for this fragmentation is presented here for the first time. This fragmentation can be exploited as a general identifier of the glucosinolate class of metabolites in plant extracts and in LCMSⁿ can be employed provide positive identification and quantification of individual glucosinolates. Such approaches offer sensitive tools for focused metabolomics analysis and screening of plant breeding lines.     

ESP and ESM1 mediate indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate in Arabidopsis

Glucosinolates are plant secondary metabolites that act as direct defenses against insect herbivores and various pathogens. Recent analysis has shown that methionine-derived glucosinolates are hydrolyzed/activated into either nitriles or isothiocyanates depending upon the plants genotype at multiple loci. While it has been hypothesized that tryptophan-derived glucosinolates can be a source of indole-acetonitriles, it has not been explicitly shown if the same proteins control nitrile production from tryptophan-derived glucosinolates as from methionine-derived glucosinolates. In this report, we formally test if the proteins involved in controlling aliphatic glucosinolate hydrolysis during tissue disruption can control production of nitriles during indolic glucosinolate hydrolysis. We show that myrosinase is not sufficient for indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate and requires the presence of functional epithospecifier protein in planta and in vitro to produce significant levels of indol-3-acetonitrile. This reaction is also controlled by the Epithiospecifier modifier 1 gene. Thus, like formation of nitriles from aliphatic glucosinolates, indol-3-acetonitrile production following tissue disruption is controlled by multiple loci raising the potential for complex regulation and fine tuning of indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate.     

Tipping the scales--specifier proteins in glucosinolate hydrolysis

Glucosinolates are a group of secondary plant metabolites found in the Brassicales order that are beneficial components of our diet, determine the flavor of a number of vegetables and spices and have been implicated in pest management strategies. These properties, most of the biological activities and the pungent odor and taste associated with glucosinolate-containing plants are due to the products formed from glucosinolates by their hydrolytic enzymes, myrosinases, upon tissue disruption. Specifier proteins impact the outcome of glucosinolate hydrolysis without having hydrolytic activity on glucosinolates themselves. In the presence of specifier proteins, glucosinolate hydrolysis results in nitriles, epithionitriles and organic thiocyanates whose biological functions are currently unknown. In contrast, isothiocyanates formed in the absence of specifier proteins have been demonstrated to possess a variety of biological activities and are thought to protect plants from herbivore and pathogen attack. This review discusses the current knowledge on plant and insect specifier proteins with special emphasis on their biochemical properties and possible mechanisms of action.     

Insect Attraction versus Plant Defense: Young Leaves High in Glucosinolates Stimulate Oviposition by a Specialist Herbivore despite Poor Larval Survival due to High Saponin Content

Glucosinolates are plant secondary metabolites used in plant defense. For insects specialized on Brassicaceae, such as the diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), glucosinolates act as “fingerprints” that are essential in host plant recognition. Some plants in the genus Barbarea (Brassicaceae) contain, besides glucosinolates, saponins that act as feeding deterrents for P. xylostella larvae, preventing their survival on the plant. Two-choice oviposition tests were conducted to study the preference of P. xylostella among Barbarea leaves of different size within the same plant. P. xylostella laid more eggs per leaf area on younger leaves compared to older ones. Higher concentrations of glucosinolates and saponins were found in younger leaves than in older ones. In 4-week-old plants, saponins were present in true leaves, while cotyledons contained little or no saponins. When analyzing the whole foliage of the plant, the content of glucosinolates and saponins also varied significantly in comparisons among plants that were 4, 8, and 12 weeks old. In Barbarea plants and leaves of different ages, there was a positive correlation between glucosinolate and saponin levels. This research shows that, in Barbarea plants, ontogenetical changes in glucosinolate and saponin content affect both attraction and resistance to P. xylostella. Co-occurrence of a high content of glucosinolates and saponins in the Barbarea leaves that are most valuable for the plant, but are also the most attractive to P. xylostella, provides protection against this specialist herbivore, which oviposition behavior on Barbarea seems to be an evolutionary mistake.     

Insect herbivore counteradaptations to the plant glucosinolate-myrosinase system

The glucosinolate-myrosinase system found in plants of the Brassicales order is one of the best studied plant chemical defenses. Glucosinolates and their hydrolytic enzymes, myrosinases, are stored in separate compartments in the intact plant tissue. Upon tissue disruption, bioactivation of glucosinolates is initiated, i.e. myrosinases get access to their glucosinolate substrates, and glucosinolate hydrolysis results in the formation of toxic isothiocyanates and other biologically active products. The defensive function of the glucosinolate-myrosinase system has been demonstrated in a variety of studies with different insect herbivores. However, a number of generalist as well as specialist herbivores uses glucosinolate-containing plants as hosts causing large agronomical losses in oil seed rape and other crops of the Brassicaceae. While our knowledge of counteradaptations in generalist insect herbivores is still very limited, considerable progress has been made in understanding how specialist insect herbivores overcome the glucosinolate-myrosinase system and even exploit it for their own defense. All mechanisms of counteradaptation identified to date in insect herbivores specialized on glucosinolate-containing plants ensure that glucosinolate breakdown to toxic isothiocyanates is avoided. This is accomplished in many different ways including avoidance of cell disruption, rapid absorption of intact glucosinolates, rapid metabolic conversion of glucosinolates to harmless compounds that are not substrates for myrosinases, and diversion of plant myrosinase-catalyzed glucosinolate hydrolysis. One of these counteradaptations, the nitrile-specifier protein identified in Pierid species, has been used to demonstrate mechanisms of coevolution of plants and their insect herbivores.     

Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross-react with other metabolites. These methylsulfinylalkyl glucosinolate specific antibodies have prospective use in multiple applications such as ELISA, co-immunoprecipitation and immunolocalization of glucosinolates.     

Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana

Glucosinolates are secondary metabolites involved in pathogen and insect defense of cruciferous plants. Although seeds and vegetative tissue often have very different glucosinolate profiles, few genetic factors that determine seed glucosinolate accumulation have been identified. An HPLC-based screen of 5500 mutagenized Arabidopsis thaliana lines produced 33 glucosinolate mutants, of which 21 have seed-specific changes. Five of these mutant lines, representing three genetic loci, are compromised in the biosynthesis of benzoyloxyglucosinolates, which are only found in seeds and young seedlings of A. thaliana. Genetic mapping and analysis of T-DNA insertions in candidate genes identified BZO1 (At1g65880), which encodes an enzyme with benzoyl-CoA ligase activity, as being required for the accumulation of benzoyloxyglucosinolates. Long-chain aliphatic glucosinolates are elevated in bzo1 mutants, suggesting substrate competition for the common short-chain aliphatic glucosinolate precursors. Whereas bzo1 mutations have seed-specific effects on benzoyloxyglucosinolate accumulation, the relative abundance of 3-benzoyloxypropyl- and 4-benzoyloxybutylglucosinolates depends on the maternal genotype.     

Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification

Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxy-indol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.     

Uptake and distribution of sinigrin in microspore derived embryos of Brassica napus L

In Brassica napus, glucosinolates are transported from all parts of the plant into the embryo during seed development. In this study we describe the uptake of the alkenyl glucosinolate sinigrin by microspore derived embryos from high and low glucosinolate genotypes. Microspore derived embryos develop completely isolated from maternal tissues unlike zygotic embryos, which contains glucosinolates transported into the embryo synthesised in the vegetative tissues. The sinigrin in the culture medium was almost completely absorbed by the embryos after three days of culture. The embryos of high and low glucosinolate genotypes were equally capable of absorbing sinigrin from the medium. A significant increase in different alkenyl glucosinolates following feeding of sinigrin suggests induction of biosynthetic enzymes in the embryos. Following excess feeding of sinigrin, we found a strong uptake against a concentration gradient and stable accumulation by the embryos. The glucosinolate was detected in single dissected cotyledons by a photometric test and by HPLC. This test could potentially be useful for screening mutants defective in glucosinolate uptake into the embryo.     

Involvement of a glucosinolate (sinigrin) in the regulation of water transport in Brassica oleracea grown under salt stress

Members of the Brassicaceae are known for their contents of nutrients and health‐promoting phytochemicals, including glucosinolates. The concentrations of these chemopreventive compounds (glucosinolate‐degradation products, the bioactive isothiocyanates) may be modified under salinity. In this work, the effect of the aliphatic glucosinolate sinigrin (2‐propenyl‐glucosinolate) on plant water balance, involving aquaporins, was explored under salt stress. For this purpose, water uptake and its transport through the plasma membrane were determined in plants after NaCl addition, when sinigrin was also supplied. We found higher hydraulic conductance (L₀) and water permeability (P_f) and increased abundance of PIP2 aquaporins after the direct administration of sinigrin, showing the ability of the roots to promote cellular water transport across the plasma membrane in spite of the stress conditions imposed. The higher content of the allyl‐isothiocyanate and the absence of sinigrin in the plant tissues suggest that the isothiocyanate is related to water balance; in fact, a direct effect of this nitro‐sulphate compound on water uptake is proposed. This work provides the first evidence that the addition of a glucosinolate can regulate aquaporins and water transport: this effect and the mechanism(s) involved merit further investigation.     

flg22诱导的拟南芥转录组分析及芥子油苷代谢途径的变化

flg22是细菌鞭毛蛋白N端的一段保守性极高的区域,能够诱导植物天然的免疫反应,为全面了解植物在受到细菌性病原菌侵害后的系统响应,利用Illumina Hiseq2000对flg22处理和未处理的拟南芥幼苗进行转录组测序。对两组数据进行差异表达分析,共获得1 200个差异表达基因,包括290个下调基因和910个上调基因。对差异表达基因进行GO富集分析和KEGG pathway富集分析,结果显示,flg22处理后,拟南芥在能量代谢、氨基酸代谢及次生代谢产物的生物合成等方面产生了巨大变化。芥子油苷是一类在植物防御病原菌的天然免疫反应中起重要作用的次生代谢产物,因此对芥子油苷代谢途径的变化进行了深入分析。根据测序结果,Flg22处理后吲哚族芥子油苷合成途径的基因表达水平显著提高,而脂肪族芥子油苷代谢途径几乎没有变化,进一步对吲哚族芥子油苷合成途径的关键酶基因进行Real Time RT-PCR的分析,验证了测序结果的正确性,证明了吲哚族芥子油苷在植物抗病防御反应中的重要作用。这为深入理解病原菌诱导的植物防御性反应及吲哚族芥子油苷的抗病机制提供了大量参考数据。