Ralph Kissen

Phytoalexins in defense against pathogens.

Plants use an intricate defense system against pests and pathogens, including the production of low molecular mass secondary metabolites with antimicrobial activity, which are synthesized de novo after stress and are collectively known as phytoalexins. In this review, we focus on the biosynthesis and regulation of camalexin, and its role in plant defense. In addition, we detail some of the phytoalexins produced by a range of crop plants from Brassicaceae, Fabaceae, Solanaceae, Vitaceae and Poaceae. This includes the very recently identified kauralexins and zealexins produced by maize, and the biosynthesis and regulation of phytoalexins produced by rice. Molecular approaches are helping to unravel some of the mechanisms and reveal the complexity of these bioactive compounds, including phytoalexin action and metabolism.

The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system

Glucosinolates are plant secondary metabolites that are hydrolysed by the action of myrosinases into various products (isothiocyanates, thiocyanates, epithionitriles, nitriles, oxazolidines). Massive hydrolysis of glucosinolates occurs only upon tissue damage but there is also evidence indicating metabolism of glucosinolates in intact plant tissues. It was originally believed that the glucosinolate–myrosinase system in intact plants was stable due to a spatial separation of the components. This has been coined as the ‘mustard oil bomb’ theory. Proteins that form complexes with myrosinases have been described: myrosinase-binding proteins (MBPs) and myrosinase-associated proteins (MyAPs/ESM). The roles of these proteins and their biological relevance are not yet completely known. Other proteins of the myrosinase enzyme system are the epithiospecifier protein (ESP) and the thiocyanate-forming protein (TFP) that divert the glucosinolate hydrolysis from isothiocyanate production to nitrile/epithionitrile or thiocyanate production. Some glucosinolate hydrolysis products act as plant defence compounds against insects and pathogens or have beneficial health effects on humans. In this review, we survey and critically assess the available information concerning the localization, both at the tissular/cellular and subcellular level, of the different components of the myrosinase enzyme system. Data from the model plant Arabidopsis thaliana is compared to that from other glucosinolate-producing Brassicaceae in order to show common as well as divergent features of the ‘mustard oil bomb’ among these species.

Nitrile-specifier proteins involved in glucosinolate hydrolysis in Arabidopsis thaliana.

Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30–45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.

Comparative innate responses of the aphid parasitoid diaeretiella rapae to Alkenyl Glucosinolate derived isothiocyanates, Nitriles, and Epithionitriles

Cruciferous plants (Brassicaceae) are characterized by the accumulation of a group of secondary metabolites known as glucosinolates that, following attack by pathogens or herbivores, may be hydrolyzed to one of a number of products including isothiocyanates and nitriles. Despite the range of hydrolysis products that may be produced, the toxicity of glucosinolates to pathogens and herbivores may be explained largely by the production of isothiocyanates. Isothiocyanates are also known to provide an indirect defense by acting as host finding cues for parasitoids of insect herbivores that attack crucifers. It has been speculated that nitriles may provide a similar indirect defense. Here, we investigate the olfactory perception and orientation behavior of the aphid parasitoid Diaeretiella rapae, to a range of alkenylglucosinolate hydrolysis products, including isothiocyanates, nitriles, and epithionitriles. Electroantennogram responses indicated peripheral odor perception in D. rapae females to all 3-butenylglucosinolate hydrolysis products tested. By contrast, of the 2-propenylglucosinolate hydrolysis products tested, only the isothiocyanate elicited significant responses. Despite showing peripheral olfactory detection of a range of 3-butenylglucosinolate hydrolysis products, naïve females oriented only to the isothiocyanate. Similarly, parasitoids oriented to 3-isothiocyanatoprop-1-ene, but not to the corresponding nitrile or epithionitrile. However, by rearing D. rapae either on Brassica nigra, characterized by the accumulation of 2-propenylglucosinolate, or Brassica rapa var rapifera, characterized by the accumulation of 3-butenylglucosinolate, altered the innate response of parasitoids to 3-isothiocyanatoprop-1-ene and 4-isothiocyanatobut-1-ene. These results are discussed in relation to the defensive roles of glucosinolate hydrolysis products and the influence of the host plant on aphid parasitoid behavior.

Transcriptional profiling of an Fd-GOGAT1/GLU1 mutant in Arabidopsis thaliana reveals a multiple stress response and extensive reprogramming of the transcriptome

BackgroundGlutamate plays a central position in the synthesis of a variety of organic molecules in plants and is synthesised from nitrate through a series of enzymatic reactions. Glutamate synthases catalyse the last step in this pathway and two types are present in plants: NADH- or ferredoxin-dependent. Here we report a genome wide microarray analysis of the transcriptional reprogramming that occurs in leaves and roots of the A. thaliana mutant glu1-2 knocked-down in the expression of Fd-GOGAT1 (GLU1; At5g04140), one of the two genes of A. thaliana encoding ferredoxin-dependent glutamate synthase.ResultsTranscriptional profiling of glu1-2 revealed extensive changes with the expression of more than 5500 genes significantly affected in leaves and nearly 700 in roots. Both genes involved in glutamate biosynthesis and transformation are affected, leading to changes in amino acid compositions as revealed by NMR metabolome analysis. An elevated glutamine level in the glu1-2 mutant was the most prominent of these changes. An unbiased analysis of the gene expression datasets allowed us to identify the pathways that constitute the secondary response of an FdGOGAT1/GLU1 knock-down. Among the most significantly affected pathways, photosynthesis, photorespiratory cycle and chlorophyll biosynthesis show an overall downregulation in glu1-2 leaves. This is in accordance with their slight chlorotic phenotype. Another characteristic of the glu1-2 transcriptional profile is the activation of multiple stress responses, mimicking cold, heat, drought and oxidative stress. The change in expression of genes involved in flavonoid biosynthesis is also revealed. The expression of a substantial number of genes encoding stress-related transcription factors, cytochrome P450 monooxygenases, glutathione S-transferases and UDP-glycosyltransferases is affected in the glu1-2 mutant. This may indicate an induction of the detoxification of secondary metabolites in the mutant.ConclusionsAnalysis of the glu1-2 transcriptome reveals extensive changes in gene expression profiles revealing the importance of Fd-GOGAT1, and indirectly the central role of glutamate, in plant development. Besides the effect on genes involved in glutamate synthesis and transformation, the glu1-2 mutant transcriptome was characterised by an extensive secondary response including the downregulation of photosynthesis-related pathways and the induction of genes and pathways involved in the plant response to a multitude of stresses.

Characterization of recombinant nitrile-specifier proteins (NSPs) of Arabidopsis thaliana: dependency on Fe(II) ions and the effect of glucosinolate substrate and reaction conditions.

Glucosinolates are plant secondary metabolites that are part of a plant defence system against pathogens and pests, the myrosinase-glucosinolate system, in which glucosinolates get activated by enzymic degradation through thioglucoside glucohydrolases called myrosinases. Epithiospecifier protein (ESP) and nitrile-specifier proteins (NSPs) divert myrosinase-catalyzed hydrolysis of a given glucosinolate from the formation of isothiocyanate to that of epithionitrile and/or nitrile. As the biological activity of glucosinolate hydrolysis products varies considerably, a detailed characterization of these specifier proteins is of utmost importance to understand their biological role. Therefore, the Arabidopsis thaliana AtNSP1, AtNSP2 and AtNSP5 and a supposed ancestor protein AtNSP-like1 were expressed in Escherichia coli and the activity of the purified recombinant proteins was tested in vitro on three highly different glucosinolates and compared to that of purified AtESP. As previously reported, only AtESP showed epithiospecifier activity on 2-propenylglucosinolate. We further confirmed that purified AtNSP1, AtNSP2 and AtNSP5, but not the ancestor AtNSP-like1 protein, show nitrile-specifier activity on 2-propenylglucosinolate and benzylglucosinolate. We now show for the first time that in vitro AtNSP1, AtNSP2 and AtNSP5 are able to generate nitrile from indol-3-ylmethylglucosinolate. We also tested the effect of different Fe(II) ion concentrations on the nitrile-specifier activity of purified AtNSP1, AtNSP2 and AtNSP5 on 2-propenylglucosinolate and benzylglucosinolate. AtNSP-related nitrile production was highly dependent on the presence of Fe(II) ions in the reaction assay. In the absence of added Fe(II) ions nitriles were only detected when benzylglucosinolate was incubated with AtNSP1. While AtNSP1 also exhibited overall higher nitrile-specifier activity than AtNSP2 and AtNSP5 at a given Fe(II) ion concentration, the pattern of nitrile formation in relation to Fe(II) ion concentrations depended on the AtNSP and the glucosinolate substrate. The pH of the solution also affected the reaction outcome, with a higher proportion of nitrile being produced at the higher pH for AtNSP2 and AtNSP5.

Modifying the Alkylglucosinolate Profile in Arabidopsis thaliana Alters the Tritrophic Interaction with the Herbivore Brevicoryne brassicae and Parasitoid Diaeretiella rapae

Arabidopsis thaliana was used as an experimental model plant to investigate a tritrophic interaction between the plant, a specialist aphid herbivore, Brevicoryne brassicae, and its natural enemy, the parasitoid Diaeretiella rapae. The A. thaliana ecotype Col-5 was transformed with a functional 2-oxoglutarate dependent dioxygenase (BniGSL-ALK) that converts 3-methylsulfinylpropylglucosinolate and 4-methylsulfinylbutylglucosinolate to 2-propenylglucosinolate and 3-butenylglucosinolate, respectively. This transformation results in a change in the glucosinolate hydrolysis profile where 3-butenylisothiocyanate, 2-propenylisothiocyanate and 5-vinyloxazolidine-2-thione are produced in contrast to the wild-type plant where 4-methylsulfinylbutylisothiocyanate is the main product. Performance of B. brassicae was affected negatively by transforming Col-5 with BniGSL-ALK in terms of mean relative growth rates. In a series of behavioral bioassays, naïve D. rapae females were able to discriminate between B. brassicae infested and uninfested Col-5 plants transformed with BniGSL-ALK, with parasitoids showing a preference for B. brassicae infested plants. By contrast, naïve D. rapae females were unable to discriminate between aphid infested and uninfested Col-5 plants. Subsequent air entrainments of B. brassicae infested Col-5 plants transformed with BniGSL-ALK further confirmed the presence of 3-butenylisothiocyanate in the headspace. By contrast, no glucosinolate hydrolysis products were recorded from similarly infested Col-5 plants.

Ecotype dependent expression and alternative splicing of epithiospecifier protein (ESP) in Arabidopsis thaliana

Epithiospecifier protein (ESP) is responsible for diverting glucosinolate hydrolysis from the generation of isothiocyanates to that of epithionitriles or nitriles, and thereby negatively affects the ability of the plant to defend itself against certain insects. Despite this important role of ESP, little is known about its expression in plant tissues and the regulation thereof. We therefore investigated ESP expression by qPCR and Western blot in different organs during the growth cycle of the two Arabidopsis thaliana ecotypes Col-0 and Mt-0. Besides the fact that ESP transcript and protein levels were revealed to be much higher in Mt-0 than in Col-0 in all cases, our qPCR results also indicated that ESP expression is regulated differently in the two A. thaliana ecotypes. No ESP protein was detected by Western blot in any organ or developmental stage for Col-0. During the assays an alternative splice variant of ESP was identified in Col-0, but not Mt-0, leading to a mis-spliced transcript which could explain the low expression levels of ESP in the former ecotype. Analysis of genomic sequences containing the ESP splice sites, of ESP protein level and ESP activity from seven A. thaliana ecotypes showed a positive correlation between the presence of a non-canonical 5′ splice site for ESP and the absence of detectable ESP protein levels and ESP activity. When analysing the expression of both transcript variants in Col-0 after treatment with methyl jasmonate, a condition known to “induce ESP”, it was indeed the alternative splice variant that was preferentially induced.

Allyl-isothiocyanate treatment induces a complex transcriptional reprogramming including heat stress, oxidative stress and plant defence responses in Arabidopsis thaliana

BackgroundIsothiocyanates (ITCs) are degradation products of the plant secondary metabolites glucosinolates (GSLs) and are known to affect human health as well as plant herbivores and pathogens. To investigate the processes engaged in plants upon exposure to isothiocyanate we performed a genome scale transcriptional profiling of Arabidopsis thaliana at different time points in response to an exogenous treatment with allyl-isothiocyanate.ResultsThe treatment triggered a substantial response with the expression of 431 genes affected (P < 0.05 and log2 ≥ 1 or ≤ -1) already after 30 min and that of 3915 genes affected after 9 h of exposure, most of the affected genes being upregulated. These are involved in a considerable number of different biological processes, some of which are described in detail: glucosinolate metabolism, sulphate uptake and assimilation, heat stress response, oxidative stress response, elicitor perception, plant defence and cell death mechanisms.ConclusionExposure of Arabidopsis thaliana to vapours of allyl-isothiocyanate triggered a rapid and substantial transcriptional response affecting numerous biological processes. These include multiple stress stimuli such as heat stress response and oxidative stress response, cell death and sulphur secondary defence metabolism. Hence, effects of isothiocyanates on plants previously reported in the literature were found to be regulated at the gene expression level. This opens some avenues for further investigations to decipher the molecular mechanisms underlying the effects of isothiocyanates on plants.

Heavy metal accumulation by Saccharomyces cerevisiae cells armed with metal binding hexapeptides targeted to the inner face of the plasma membrane

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.