Loss of CpFTSY Reduces Photosynthetic Performance and Affects Insertion of PsaC of PSI in Diatoms.

The chloroplast signal recognition particle (CpSRP) receptor (CpFTSY) is a component of the CpSRP pathway that post-translationally targets light-harvesting complex proteins (LHCPs) to the thylakoid membranes in plants and green algae containing chloroplasts derived from primary endosymbiosis. In plants, CpFTSY also plays a major role in the co-translational incorporation of chloroplast-encoded subunits of photosynthetic complexes into the thylakoids. This role has not been demonstrated in green algae. So far, its function in organisms with chloroplasts derived from secondary endosymbiotic events has not been elucidated. Here, we report the generation and characterization of mutants lacking CpFTSY in the diatom Phaeodactylum tricornutum. We found that this protein is not involved in inserting LHCPs into thylakoid membranes, indicating that the post-translational part of the CpSRP pathway is not active in this group of microalgae. The lack of CpFTSY caused an increased level of photoprotection, low electron transport rates, inefficient repair of photosystem II (PSII), reduced growth, a strong decline in the PSI subunit PsaC and upregulation of proteins that might compensate for a non-functional co-translational CpSRP pathway during light stress conditions. The phenotype was highly similar to the one described for diatoms lacking another component of the co-translational CpSRP pathway, the CpSRP54 protein. However, in contrast to cpsrp54 mutants, only one thylakoid membrane protein, PetD of the Cytb6f complex, was downregulated in cpftsy. Our results point to a minor role for CpFTSY in the co-translational CpSRP pathway, suggesting that other mechanisms may partially compensate for the effect of a disrupted CpSRP pathway.

The Imaging of Guard Cells of thioglucosidase (tgg) Mutants of Arabidopsis Further Links Plant Chemical Defence Systems with Physical Defence Barriers.

The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants' physical defence barriers.

Assessment of oxidative stress response genes in Avicennia marina exposed to oil contamination - Polyphenol oxidase (PPOA) as a biomarker.

Mangrove plants, which inhabit and form sensitive ecosystems in the intertidal zones of tropical and subtropical coastlines, though vulnerable to petroleum pollution, still maintain their growth under oil contamination. To elucidate the molecular response of mangrove plants to crude oil-sediment mixture, seeds of Avicennia marina were planted and grown on 0, 2.5, 5.0, 7.5 and10 % (w/w) oil-contaminated soil. Plant biomass was highly affected from 3.05 ± 0.28 (Control) to 0.50 ± .07 (10 %) and from 3.47 ± 0.12 to 1.88 ± 0.08 in 2 and 4 months old plants respectively. The expression analysis of 11genes belonging to detoxification pathways in the roots and leaves of 2 and 4 month-old plants was evaluated by qRT-PCR. Our results showed changes in expression levels of Fe-SOD, Mn-SOD, CAT, PRX, PPOs, GSTs, and NAP2 whose products are involved in reactive oxygen species (ROS) and xenobiotic detoxification. PPOA showed the highest expression induction of 43 ± 1.15, followed by CAT (12.61 ± 3.25) and PPOB (6.38 ± 1.34) in leaves of 2 months old seedlings grown on 7.5, 10 and 7.5 % oil contaminated soil respectively. PPOA (39.23 ± 2.1), PRX (32.13 ± 1.2) as well as PPOB (26.11 ± 1.3) showed the highest expression induction in leaves of 4 months old plants grown in 2.5 % oil contaminated soil. Our data indicated that PPOA can be a good biomarker candidate gene for long term exposure to oil contamination in A. marina.

The Role of a Glucosinolate-Derived Nitrile in Plant Immune Responses.

Glucosinolates are defense-related secondary metabolites found in Brassicaceae. When Brassicaceae come under attack, glucosinolates are hydrolyzed into different forms of glucosinolate hydrolysis products (GHPs). Among the GHPs, isothiocyanates are the most comprehensively characterized defensive compounds, whereas the functional study of nitriles, another group of GHP, is still limited. Therefore, this study investigates whether 3-butenenitrile (3BN), a nitrile, can trigger the signaling pathways involved in the regulation of defense responses in Arabidopsis thaliana against biotic stresses. Briefly, the methodology is divided into three stages, (i) evaluate the physiological and biochemical effects of exogenous 3BN treatment on Arabidopsis, (ii) determine the metabolites involved in 3BN-mediated defense responses in Arabidopsis, and (iii) assess whether a 3BN treatment can enhance the disease tolerance of Arabidopsis against necrotrophic pathogens. As a result, a 2.5 mM 3BN treatment caused lesion formation in Arabidopsis Columbia (Col-0) plants, a process found to be modulated by nitric oxide (NO). Metabolite profiling revealed an increased production of soluble sugars, Krebs cycle associated carboxylic acids and amino acids in Arabidopsis upon a 2.5 mM 3BN treatment, presumably via NO action. Primary metabolites such as sugars and amino acids are known to be crucial components in modulating plant defense responses. Furthermore, exposure to 2.0 mM 3BN treatment began to increase the production of salicylic acid (SA) and jasmonic acid (JA) phytohormones in Arabidopsis Col-0 plants in the absence of lesion formation. The production of SA and JA in nitrate reductase loss-of function mutant (nia1nia2) plants was also induced by the 3BN treatments, with a greater induction for JA. The SA concentration in nia1nia2 plants was lower than in Col-0 plants, confirming the previously reported role of NO in controlling SA production in Arabidopsis. A 2.0 mM 3BN treatment prior to pathogen assays effectively alleviated the leaf lesion symptom of Arabidopsis Col-0 plants caused by Pectobacterium carotovorum ssp. carotovorum and Botrytis cinerea and reduced the pathogen growth on leaves. The findings of this study demonstrate that 3BN can elicit defense response pathways in Arabidopsis, which potentially involves a coordinated crosstalk between NO and phytohormone signaling.

The Myb-like transcription factor phosphorus starvation response (PtPSR) controls conditional P acquisition and remodelling in marine microalgae.

Phosphorus (P) is one of the limiting macronutrients for algal growth in marine environments. Microalgae have developed adaptation mechanisms to P limitation that involve remodelling of internal phosphate resources and accumulation of lipids. Here, we used in silico analyses to identify the P-stress regulator PtPSR (Phaeodactylum tricornutum phosphorus starvation response) in the diatom P. tricornutum. ptpsr mutant lines were generated using gene editing and characterised by various molecular, genetics and biochemical tools. PtPSR belongs to a clade of Myb transcription factors that are conserved in stramenopiles and distantly related to plant P-stress regulators. PtPSR bound specifically to a conserved cis-regulatory element found in the regulatory region of P-stress-induced genes. ptpsr knockout mutants showed reduction in cell growth under P limitation. P-stress responses were impaired in ptpsr mutants compared with wild-type, including reduced induction of P-stress response genes, near to complete loss of alkaline phosphatase activity and reduced phospholipid degradation. We conclude that PtPSR is a key transcription factor influencing P scavenging, phospholipid remodelling and cell growth in adaptation to P stress in diatoms.

Accumulation of Ag(I) by Saccharomyces cerevisiae Cells Expressing Plant Metallothioneins.

The various applications of Ag(I) generated the necessity to obtain Ag(I)-accumulating organisms for the removal of surplus Ag(I) from contaminated sites or for the concentration of Ag(I) from Ag(I)-poor environments. In this study we obtained Ag(I)-accumulating cells by expressing plant metallothioneins (MTs) in the model Saccharomyces cerevisiae. The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) fused to myrGFP displaying an N-terminal myristoylation sequence for plasma membrane targeting were expressed in S. cerevisiae and checked for Ag(I)-related phenotype. The transgenic yeast cells were grown in copper-deficient media to ensure the expression of the plasma membrane high-affinity Cu(I) transporter Ctr1, and also to elude the copper-related inhibition of Ag(I) transport into the cell. All plant MTs expressed in S. cerevisiae conferred Ag(I) tolerance to the yeast cells. Among them, myrGFP-NcMT3 afforded Ag(I) accumulation under high concentration (10⁻50 µM), while myrGFP-AtMT1a conferred increased accumulation capacity under low (1 µM) or even trace Ag(I) (0.02⁻0.05 µM). The ability to tolerate high concentrations of Ag(I) coupled with accumulative characteristics and robust growth showed by some of the transgenic yeasts highlighted the potential of these strains for biotechnology applications.

Benzyl Cyanide Leads to Auxin-Like Effects Through the Action of Nitrilases in Arabidopsis thaliana.

Plants within the Brassicales order generate glucosinolate hydrolysis products that can exert different biological effects on several organisms. Here, we evaluated the physiological effects of one of these compounds, benzyl cyanide (phenylacetonitrile), when exogenously applied on Arabidopsis thaliana. Treatment with benzyl cyanide led to a dose-dependent reduction of primary root length and total biomass. Further morphological changes like elongated hypocotyls, epinastic cotyledons, and increased formation of adventitious roots resembled a severe auxin-overproducer phenotype. The elevated auxin response was confirmed by histochemical staining and gene expression analysis of auxin-responsive genes. Nitriles are converted by specific enzymes, nitrilases (NIT1-3), to their corresponding carboxylic acids. The nitrilase mutants nit1 and nit2 tolerated benzyl cyanide treatments better than the wild type, with nit2 being less resistant than nit1. A NIT2RNAi line suppressing several nitrilases was resistant to all tested benzyl cyanide concentrations. When exposed to phenylacetic acid (PAA) - the corresponding carboxylic acid of benzyl cyanide - wild type and mutant seedlings were, however, equally susceptible and showed a more severe auxin phenotype than upon cyanide treatment. Here, we demonstrate that the auxin-like effects triggered by benzyl cyanide on Arabidopsis are due to its nitrilase-mediated conversion to the natural auxin PAA.

Arabidopsis mutants impaired in glutathione biosynthesis exhibit higher sensitivity towards the glucosinolate hydrolysis product allyl-isothiocyanate.

Upon tissue damage the plant secondary metabolites glucosinolates can generate various hydrolysis products, including isothiocyanates (ITCs). Their role in plant defence against insects and pest and their potential health benefits have been well documented, but our knowledge regarding the endogenous molecular mechanisms of their effect in plants is limited. Here we investigated the effect of allyl-isothiocyanate (AITC) on Arabidopsis thaliana mutants impaired in homeostasis of the low-molecular weight thiol glutathione. We show that glutathione is important for the AITC-induced physiological responses, since mutants deficient in glutathione biosynthesis displayed a lower biomass and higher root growth inhibition than WT seedlings. These mutants were also more susceptible than WT to another ITC, sulforaphane. Sulforaphane was however more potent in inhibiting root growth than AITC. Combining AITC with the glutathione biosynthesis inhibitor L-buthionine-sulfoximine (BSO) led to an even stronger phenotype than observed for the single treatments. Furthermore, transgenic plants expressing the redox-sensitive fluorescent biomarker roGFP2 indicated more oxidative conditions during AITC treatment. Taken together, we provide genetic evidence that glutathione plays an important role in AITC-induced growth inhibition, although further studies need to be conducted to reveal the underlying mechanisms.

Glucosinolate-Derived Isothiocyanates Inhibit Arabidopsis Growth and the Potency Depends on Their Side Chain Structure.

Isothiocyanates (ITCs), the biologically important glucosinolate breakdown products, can present health-promoting effects, play an important role in plant defense and affect plant cellular mechanisms. Here, we evaluated the biological effects of ITCs on Arabidopsis thaliana by assessing growth parameters after long-term exposure to low concentrations of aliphatic and aromatic ITCs, ranging from 1 to 1000 µM. Treatment with the aliphatic allylisothiocyanate (allyl-ITC) led to a significant reduction of root length and fresh weight in a dose-dependent manner and affected the formation of lateral roots. To assess the importance of a hormonal crosstalk in the allyl-ITC-mediated growth reduction, the response of auxin and ethylene mutants was investigated, but our results did not allow us to confirm a role for these hormones. Aromatic ITCs generally led to a more severe growth inhibition than the aliphatic allyl-ITC. Interestingly, we observed a correlation between the length of their side chain and the effect these aromatic ITCs caused on Arabidopsis thaliana, with the greatest inhibitory effect seen for 2-phenylethyl-ITC. Root growth recovered when seedlings were removed from exposure to ITCs.

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.

Anchoring plant metallothioneins to the inner face of the plasma membrane of Saccharomyces cerevisiae cells leads to heavy metal accumulation.

In this study we engineered yeast cells armed for heavy metal accumulation by targeting plant metallothioneins to the inner face of the yeast plasma membrane. Metallothioneins (MTs) are cysteine-rich proteins involved in the buffering of excess metal ions, especially Cu(I), Zn(II) or Cd(II). The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) were each translationally fused to the C-terminus of a myristoylation green fluorescent protein variant (myrGFP) and expressed in Saccharomyces cerevisiae cells. The myrGFP cassette introduced a yeast myristoylation sequence which allowed directional targeting to the cytosolic face of the plasma membrane along with direct monitoring of the intracellular localization of the recombinant protein by fluorescence microscopy. The yeast strains expressing plant MTs were investigated against an array of heavy metals in order to identify strains which exhibit the (hyper)accumulation phenotype without developing toxicity symptoms. Among the transgenic strains which could accumulate Cu(II), Zn(II) or Cd(II), but also non-canonical metal ions, such as Co(II), Mn(II) or Ni(II), myrGFP-NcMT3 qualified as the best candidate for bioremediation applications, thanks to the robust growth accompanied by significant accumulative capacity.

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

BACKGROUND: Isothiocyanates (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. RESULTS: The 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. CONCLUSION: Exposure 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.

Effect of growth temperature on glucosinolate profiles in Arabidopsis thaliana accessions.

Glucosinolates are plant secondary metabolites with important roles in plant defence against pathogens and pests and are also known for their health benefits. Understanding how environmental factors affect the level and composition of glucosinolates is therefore of importance in the perspective of climate change. In this study we analysed glucosinolates in Arabidopsis thaliana accessions when grown at constant standard (21 °C), moderate (15 °C) and low (9 °C) temperatures during three generations. In most of the tested accessions moderate and pronounced chilling temperatures led to higher levels of glucosinolates, especially aliphatic glucosinolates. Which temperature yielded the highest glucosinolate levels was accession-dependent. Transcriptional profiling revealed also accession-specific gene responses, but only a limited correlation between changes in glucosinolate-related gene expression and glucosinolate levels. Different growth temperatures in one generation did not consistently affect glucosinolate composition in subsequent generations, hence a clear transgenerational effect of temperature on glucosinolates was not observed.

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.

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.

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

BACKGROUND: Glutamate 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. RESULTS: Transcriptional 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. CONCLUSIONS: Analysis 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.

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.

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.