Glucosinolate Production Research Articles

Two-tier morpho-chemical defence tactic in Aethionema via fruit morph plasticity and glucosinolates allocation in diaspores.

Fruit dimorphism and the production of glucosinolates (GSLs) are two specific life history traits found in the members of Brassicales, which aid to optimize seed dispersal and defence against antagonists, respectively. We hypothesized that the bipartite dispersal strategy demands a tight control over the production of fruit morphs with expectedly differential allocation of defensive anticipins (GSLs). In dimorphic Aethionema, herbivory by Plutella xylostella at a young stage triggered the production of more dehiscent (seeds released from fruit) than indehiscent fruit morphs (seeds enclosed within persistent pericarp) on the same plant upon maturity. Total GSL concentrations were highest in the mature seeds of dehiscent fruits from Aethionema arabicum and Aethionema saxatile among the different ontogenetic stages of the diaspores. Multivariate analyses of GSL profiles indicated significantly higher concentrations of specific indole GSLs in the diaspores, which require optimal defence after dispersal (i.e., seeds of dehiscent and fruit/pericarp of indehiscent fruit). Bioassays with a potentially coinhabitant fungus, Aspergillus quadrilineatus, support the distinct defensive potential of the diaspores corresponding to their GSL allocation. These findings indicate a two-tier morpho-chemical defence tactic of Aethionema via better protected fruit morphs and strategic provision of GSLs that optimize protection to the progeny for survival in nature.

Biotechnological approaches in glucosinolate production

Glucosinolates (GLSs) are sulfur‐rich, amino acid‐derived defense compounds characteristic of the Brassicales order. In the past, GLSs were mostly known as anti‐nutritional factors in fodder, biopesticides in agriculture, and flavors in condiments such as mustard. However, in recent times, GLSs have received increased attention as promoters of human health. This has spurred intensive research towards generating rich sources of health‐promoting GLSs. We provide a comprehensive overview of the biotechnological approaches applied to reach this goal. This includes optimization of GLS production and composition in native, GLS‐producing plants, including hairy root and cell cultures thereof, as well as synthetic biology approaches in heterologous hosts, such as tobacco and the microbial organisms Escherichia coli and Saccharomyces cerevisiae. The progress using these different approaches is discussed.

Optimizing growth conditions for glucosinolate production in Chinese cabbage

Glucosinolates are well known functional food components that were discovered in Cruciferae and have been the object of study from diverse disciplinary perspectives, including metabolism, quantitative analysis, and breeding. However, the effects of the growth environment and post-harvest processes on glucosinolate production in Chinese cabbage are not well studied. Hence, this study aimed to identify the major factors that affect glucosinolate accumulation in Chinese cabbage during growth, and to optimize growth conditions in order to maximize glucosinolate content. Therefore, we measured glucosinolate content in cabbage grown in five environmental conditions that altered electrical conductivity, pH, cultivation time, temperature, and relative humidity. We used principal component analysis (PCA) to identify the variables primarily affecting growth, followed by response surface methodology (RSM) to determine optimal growth conditions that maximize glucosinolate content in Chinese cabbage. Results from PCA indicated that cultivation time, temperature, and relative humidity were the principal components that explained 85.8% of the total variance, suggesting these are the most significant environmental factors that affect glucosinolate accumulation. Moreover, RSM indicated that the total model for evaluating glucosinolate content was significant (R2 = 0.934) and showed that the optimal temperature and relative humidity for maximizing glucosinolate content are 28 °C, and 66%, respectively. This study provided practical information of optimal condition for producing functional Chinese cabbage intensifying glucosinolate.

Identification and expression pattern analysis of BoMYB51 involved in indolic glucosinolate biosynthesis from broccoli (Brassica oleracea var. italica)

Glucosinolates are a class of amino acid-derived specialized metabolites characteristic of the Brassicales order. Trp derived indolic glucosinolates are essential for the effective plant defense responses to a wide range of pathogens and herbivores. In Arabidopsis, MYB51 is the key transcription factor positively regulates indolic glucosinolate production by activating certain biosynthetic genes. In this study, we report the isolation and identification of a MYB51 from broccoli designated as BoMYB51. Overexpression of BoMYB51 in Arabidopsis increased indolic glucosinolate production by upregulating biosynthetic genes and resulted in enhanced flagellin22 (Flg22) induced callose deposition. The spatial expression pattern and responsive expression of BoMYB51 to several hormones and stress treatments were investigated by expressing the β-glucuronidase (GUS) reporter gene driven by BoMYB51 promotor in Arabidopsis and quantitative real-time PCR analysis in broccoli. Our study provides information on molecular characteristics of BoMYB51 and possible physiological process BoMYB51 may involve.

Cross‐talk among biochemical defense pathways in Arabidopsis thaliana

Cellular metabolism is a complex set of interwoven interactions. That complexity challenges the prediction of how metabolism responds to change. To try to increase our ability to predict how single‐gene changes result in global metabolic shifts, we studied how changing expression in the indolic glucosinolate chemical defense pathway in Arabidopsis thaliana influences production in the related aliphatic glucosinolate pathway.Aliphatic glucosinolates are synthesized in a pathway parallel to the indolic pathway and share a number of enzymes (SUR1, GGP1). Research on the aliphatic pathway has shown aliphaticspecific and shared gene knockdowns have no control over the indolic pathway. However, by knocking down activity of each step in the indolic pathway and measuring the production of all glucosinolates via High Performance Liquid Chromatography (HPLC), our results suggest crosstalk between these pathways.Preliminary results reveal that when the gene GTSF9 of the indolic pathway is knocked down, the production of aliphatic glucosinolates is reduced, but the production of indolic glucosinolates is not affected. These results are puzzling given that GSTF9 is not thought to be shared between the pathways like the enzymes SUR1 and GGP1. Indeed, previous research suggests GSTF9 is predicted to act in the aliphatic pathway exclusively. To further study this pathway cross‐talk, we will extract and sequence RNA from single‐gene knockouts of the genes in the indolic pathway to track how changes in gene expression of individual enzymes change global patterns of expression. These results will allow us to begin to understand the genetic mechanisms that mediate cross‐talk between these pathways.Support or Funding InformationSD BRIN P20GM103443 SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pleiotropy and Flux Control in the Indolic Biochemical Pathways in Arabidopsis thaliana

Metabolic pathways are a series of highly interconnected networks. Predicting how changes in one pathway influence the rest of metabolism is a challenge. Small changes in the expression of one gene in a single pathway may have far reaching pleiotropic effects across the genome or, alternatively, large changes in expression of one gene may be buffered and have only local effects. However, taking into account pathway properties of an enzyme (including the connectedness to other parts of metabolism or the amount of control an enzyme has over pathway production) may lead to predictable impacts on gene expression across the genome (e.g. perturbations in highly connected enzymes to influence a wide range of pathways). To address this question, we are studying the indolic glucosinolate pathway in Arabidopsis thaliana. Glucosinolates are herbivore defense compounds produced by relatives of Arabidopsis and protect the plant against generalist herbivores. The indolic glucosinolate pathway is also connected to the incredibly important plant hormone pathway, auxin, in that the pathways share a product (indole‐3‐acetaldoxime). Auxin is a growth hormone involved in nearly every step of plant development, thus we predict that some changes to the indolic pathway will produce only local effects while some changes may produce wide‐ranging effects. To investigate the influence of pathway position and connectedness on global gene expression and glucosinolate production we created gene expression knockdown lines in every gene in the indolic glucosinolate pathway. By monitoring indole glucosinolate pathway output with High Performance Liquid Chromatography (HPLC) and measuring global gene expression with RNAseq, we can study how changing gene expression within a pathway influences the output of that pathway and gene expression across the genome.Support or Funding InformationSD BRIN P20GM103443SD EPSCOR IIA‐1355423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Crucifer glucosinolate production in legume-crucifer cover crop mixtures

Cover crops grown in rotation with cash crops provide ecosystem services by reducing water pollution and anthropogenic inputs. Bispecific crucifer–legume cover crop mixtures are seen as a solution to increase biodiversity and to combine ecosystem services of both species. Legumes fix nitrogen while crucifers have the capacity to suppress pathogens due to the biocidal hydrolysis products of the endogenous secondary metabolites called glucosinolates (GSL). However there is a lack of information on the impact of plant–plant interactions in the crucifer-legume mixtures on crucifer GSL production compared to sole crop. The aim of our study was to assess GSL production of a wide range of bispecific crucifer-legume mixtures in comparison to sole crops. Experiments were conducted at two sites (near Toulouse and Orleans, France) over two years. Various cultivars from eight crucifer (Brassicaceae) species (rape, white mustard, Indian mustard, Ethiopian mustard, turnip, turnip rape, radish and rocket) and nine legume (Fabaceae) species (Egyptian clover, crimson clover, common vetch, purple vetch, hairy vetch, pea, soya bean, faba bean, and white lupin) were tested in sole crop and bispecific mixtures (substitutive design of 50%–50% sole crops). We show that for a wide range of species, crucifers in cover crop mixtures had the same GSL types, concentrations and proportions (aliphatic, aromatic and indole) as in sole crops. Crucifers in mixtures tended to have more biomass per plant, both for shoots and roots, than in sole crops so that the total GSL production per plant generally increased in the mixture compared to the sole crop. Thus despite halving the crucifer density in the mixtures the GSL production on an area basis declined by only 20%. These results have been validated for a wide range of crucifer species and provide support for crucifer-legume mixtures to produce nutrient-related services while potentially maintaining high GSL production. Specific experiments are needed to evaluate whether this double effect of unchanged GSL concentration and small reduction in GSL production has an impact on the biofumigation potential of cover crop mixtures compared to sole crucifers.

Impact of plant biostimulants on the production of glucosinolates in broccoli

Impact of plant biostimulants on the production of glucosinolates in broccoli

Enteric methane production and ruminal fermentation of forage brassica diets fed in continuous culture.

The aim of the current study was to determine nutrient digestibility, VFA production, N metabolism, and CH4 production of canola (Brassica napus L.), rapeseed (B. napus L.), turnip (Brassica rapa L.), and annual ryegrass (Lolium multiflorum Lam.) fed with orchardgrass (Dactylis glomerata L.) in continuous culture. Diets were randomly assigned to fermentors in a 4 × 4 Latin square design using 7 d for adaptation and 3 d for collection. Diets were: 1) 50% orchardgrass + 50% annual ryegrass (ARG); 2) 50% orchardgrass + 50% canola (CAN); 3) 50% orchardgrass + 50% rapeseed (RAP); and 4) 50% orchardgrass + 50% turnip (TUR). Feedings (82 g DM/d) occurred four times daily throughout 4, 10-d periods at 730, 1030, 1400, and 1900 h. Methane samples were collected every 10 min using a photoacoustic gas analyzer (LumaSense Technologies, Inc.; Santa Clara, CA) during the last 3 d of the experiment. Effluent samples were collected on d 8, 9, and 10, composited by fermentor, and analyzed for VFA and pH as well as DM, OM, CP, and fiber fractions for determination of nutrient digestibility. Forage samples were analyzed for CP, NDF, ADF, minerals, and glucosinolate (GLS) concentrations. Data were analyzed using the GLIMMIX procedure of SAS. Apparent DM, OM, and NDF digestibilities and true DM and OM digestibilities were similar (P > 0.28) among diets (45.1, 63.2, 44.1, 67.1, and 87.2%, respectively). Total VFA (87.2 mol/100 mol), pH (6.47), and acetate (A: 44.6 mol/100 mol) were also not different (P > 0.20) among diets. The A:P (P = propionate) ratio was greater (P < 0.01) in ARG and CAN than RAP and TUR. Daily CH4 production was greater (P < 0.01) in ARG than all other diets (68.9 vs. 11.2 mg/d). Methane, whether expressed as g per g of OM, NDF, digestible OM, or digestible NDF fed was greatest (P < 0.01) in ARG but similar (P > 0.18) among brassica diets. A significant negative correlation was observed between total GLS and CH4 production. However, when multiple regression analysis on CH4 production was completed, neither total GLS nor individual GLS were a significant component of the model. Addition of brassicas provided similar nutrient digestibility to ARG while reducing daily CH4 production, potentially making brassicas an alternative for ARG in pasture-based ruminant diets.

Influence of nutrient supply and elicitors on glucosinolate production in E. sativa hairy root cultures

Glucosinolates (GS) are secondary plant metabolites comprising different subgroups with opposing effects on human health. Hairy root cultures (HRC) are potent biotechnological tools allowing the biosynthesis of special substances under defined conditions. HRC of Eruca sativa, a brassicaceaous plant, were used to test different strategies to enhance GS levels and to alter the profile. Additional sulphur supply in the nutrient medium increased especially aliphatic GS by 2.7-fold, but also enhanced indole GS by 1.8-fold. Ethephon as well as jasmonic acid as chemical elicitors enhanced only indole GS levels, whereby especially 4-methoxyindol-3-ylmethyl or 1-methoxyindol-3-ylmethyl GS accumulated. Jasmonic acid was used in combination with pulsed electric field treatment as physical elicitor. Already within 24 h, GS levels doubled in treated HRC compared to the control. For estimation of production potency, the GS levels of HRC were compared to contents of aerial and root parts of E. sativa sprouts. HRC showed a distinct GS profile compared to the parent plant with a higher content of indole GS when compared to sprout roots, but overall lower total GS levels. Furthermore, HRC released GS into the culture medium, which could be enhanced by jasmonic acid and pulsed electric field treatment. This could comprise an efficient strategy for a continuous GS production and mining without solvent extraction.

Reconstructing Biosynthetic Pathway of the Plant-Derived Cancer Chemopreventive-Precursor Glucoraphanin in Escherichia coli.

Epidemiological data confirmed a strong correlation between regular consumption of cruciferous vegetables and lower cancer risk. This cancer preventive property is mainly attributed to the glucosinolate products, such as glucoraphanin found in broccoli that is derived from methionine. Here we report the first successful reconstruction of the complete biosynthetic pathway of glucoraphanin from methionine in Escherichia coli via gene selection, pathway design, and protein engineering. We used branched-chain amino transferase 3 to catalyze two transamination steps to ensure the purity of precursor molecules and used cysteine as a sulfur donor to simplify the synthesis pathway. Two chimeric cytochrome P450 enzymes were engineered and expressed in E.coli functionally. The original plant C-S lyase was replaced by the Neurospora crassa hercynylcysteine sulfoxide lyase. Other pathway enzymes were successfully mined from Arabidopsis thaliana, Brassica rapa, and Brassica oleracea. Biosynthesis of glucoraphanin upon coexpression of the optimized enzymes in vivo was confirmed by liquid chromatography-tandem mass spectrometry analysis. No other glucosinolate analogues (except for glucoiberin) were identified that could facilitate the downstream purification processes. Production of glucoraphanin in this study laid the foundation for microbial production of such health-beneficial glucosinolates in a large-scale.

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.)

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed.

Leptosphaeria maculans Alters Glucosinolate Profiles in Blackleg Disease-Resistant and -Susceptible Cabbage Lines.

Blackleg, a fungal disease caused by Leptosphaeria maculans, is one of the most devastating diseases of Brassica crops worldwide. Despite notable progress elucidating the roles of glucosinolates in pathogen defense, the complex interaction between B. oleracea (cabbage) and L. maculans infection that leads to the selective induction of genes involved in glucosinolate production and subsequent modulation of glucosinolate profiles remains to be fully understood. The current study was designed to identify glucosinolate-biosynthesis genes induced by L. maculans and any associated alterations in glucosinolate profiles to explore their roles in blackleg resistance in 3-month-old cabbage plants. The defense responses of four cabbage lines, two resistant and two susceptible, were investigated using two L. maculans isolates, 03–02 s and 00–100 s. A simultaneous increase in the aliphatic glucosinolates glucoiberverin (GIV) and glucoerucin (GER) and the indolic glucosinolates glucobrassicin (GBS) and neoglucobrassicin (NGBS) was associated with complete resistance. An increase in either aliphatic (GIV) or indolic (GBS and MGBS) glucosinolates was associated with moderate resistance. Indolic glucobrassicin (GBS) and neoglucobrassicin (NGBS) were increased in both resistant and susceptible interactions. Pearson correlation showed positive association between GER content with GSL-OH (Bol033373) expression. Expressions of MYB34 (Bol007760), ST5a (Bol026200), and CYP81F2 (Bol026044) were positively correlated with the contents of both GBS and MGBS. Our results confirm that L. maculans infection induces glucosinolate-biosynthesis genes in cabbage, with concomitant changes in individual glucosinolate contents. In resistant lines, both aliphatic and indolic glucosinolates are associated with resistance, with aliphatic GIV and GER and indolic MGBS glucosinolates particularly important. The association between the genes, the corresponding glucosinolates, and plant resistance broaden our molecular understanding of glucosinolate mediated defense against L. maculans in cabbage.

Molecular constraints on resistance-tolerance trade-offs.

Plants have numerous mechanisms to cope with the negative effects of herbivory, including plant resistance, structural and chemical traits that reduce damage, and plant tolerance, the ability to compensate for tissues lost. It has been argued that resistance and tolerance represent alternate strategies and thus there should be a trade-off between resistance and tolerance. However, resistance and tolerance are controlled via the same molecular pathway, the oxidative pentose phosphate pathway and the process of endoreduplication. Endoreduplication is the replication of the genome without mitosis, which leads to an increase in cellular chromosome number. Increasing chromosome number and therefore gene copy number provides a means of increasing gene expression that has been shown to enhance compensation following herbivory. By measuring glucosinolate levels and seed production following the removal of apical dominance in genotypes of Arabidopsis thaliana we show that there is a positive association between tolerance and induced chemical defense. Similarly, the direct association between tolerance and resistance is demonstrated by genetically manipulating the endoreduplication pathway. By overexpressing ILP1, a positive regulator of endoreduplication, and thus compensation, we experimentally increased glucosinolate production and tolerance in the Col-0 genotype. We suggest that many herbaceous plants that endoreduplicate (~90%) would show a positive relationship between compensation and chemical defense, given that the molecular pathways are shared in common. We discuss these findings in light of contrasting results on measures of tolerance and resistance, given that the true relationship can be masked by ignoring genetic variation in endoreduplication and the timing of chemical measurement.

A novel Filamentous Flower mutant suppresses brevipedicellus developmental defects and modulates glucosinolate and auxin levels.

BREVIPEDICELLUS (BP) encodes a class-I KNOTTED1-like homeobox (KNOX) transcription factor that plays a critical role in conditioning a replication competent state in the apical meristem, and it also governs growth and cellular differentiation in internodes and pedicels. To search for factors that modify BP signaling, we conducted a suppressor screen on bp er (erecta) plants and identified a mutant that ameliorates many of the pleiotropic defects of the parent line. Map based cloning and complementation studies revealed that the defect lies in the FILAMENTOUS FLOWER (FIL) gene, a member of the YABBY family of transcriptional regulators that contribute to meristem organization and function, phyllotaxy, leaf and floral organ growth and polarity, and are also known to repress KNOX gene expression. Genetic and cytological analyses of the fil-10 suppressor line indicate that the role of FIL in promoting growth is independent of its previously characterized influences on meristem identity and lateral organ polarity, and likely occurs non-cell-autonomously from superior floral organs. Transcription profiling of inflorescences revealed that FIL downregulates numerous transcription factors which in turn may subordinately regulate inflorescence architecture. In addition, FIL, directly or indirectly, activates over a dozen genes involved in glucosinolate production in part by activating MYB28, a known activator of many aliphatic glucosinolate biosynthesis genes. In the bp er fil-10 suppressor mutant background, enhanced expression of CYP71A13, AMIDASE1 (AMI) and NITRILASE genes suggest that auxin levels can be modulated by shunting glucosinolate metabolites into the IAA biosynthetic pathway, and increased IAA levels in the bp er fil-10 suppressor accompany enhanced internode and pedicel elongation. We propose that FIL acts to oppose KNOX1 gene function through a complex regulatory network that involves changes in secondary metabolites and auxin.

Biosynthesis and bioactivity of glucosinolates and their production in plant in vitro cultures.

Glucosinolates are biologically active compounds which are involved in plant defense reaction. The use of plant in vitro cultures and genetic engineering is a promising strategy for their sustainable production. Glucosinolates are a class of secondary metabolites found mainly in Brassicaceae, which contain nitrogen and sulfur in their structures. Glucosinolates are divided into three groups depending on the amino acid from which they are biosynthesized. Aliphatic glucosinolates are generally derived from leucine, valine, methionine, isoleucine and alanine while indole and aromatic glucosinolates are derived from tryptophan and phenylalanine or tyrosine, respectively. These compounds are hydrolyzed by the enzyme myrosinase when plants are stressed by biotic and abiotic factors, obtaining different degradation products. Glucosinolates and their hydrolysis products play an important role in plant defense responses against different types of stresses. In addition, these compounds have beneficial effect on human health because they are strong antioxidants and they have potent cardiovascular, antidiabetic, antimicrobial and antitumoral activities. Due to all the properties described above, the demand for glucosinolates and their hydrolysis products has enormously increased, and therefore, new strategies that allow the production of these compounds to be improved are needed. The use of plant in vitro cultures is emerging as a biotechnological strategy to obtain glucosinolates and their derivatives. This work is focused on the biosynthesis of glucosinolates and the bioactivity of these compounds in plants. In addition, a detailed study on the strategies used to increase the production of several glucosinolates, in particular those synthesized in Brassicaceae, using in vitro plant cultures has been made. Special attention has been paid for increasing the production of glucosinolates and their derivatives using metabolic engineering.

Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1.

BackgroundModulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many – if not all – molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase.ResultsHere we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates.ConclusionThe here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0900-7) contains supplementary material, which is available to authorized users.

Production of glucosinolates, phenolic compounds and associated gene expression profiles of hairy root cultures in turnip (Brassica rapa ssp. rapa).

Turnip (Brassica rapa ssp. rapa) is an important vegetable crop producing glucosinolates (GSLs) and phenolic compounds. The GSLs, phenolic compound contents and transcript levels in hairy root cultures, as well as their antioxidant, antimicrobial and anticancer activity were studied in turnip. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR. GSLs levels (glucoallysin, glucobrassicanapin, gluconasturtiin, glucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin and 4-hydroxyglucobrassicin) and their gene expression levels (BrMYB28, BrMYB29, BrMYB34, BrMYB51, BrMYB122, CYP79 and CYP83) significantly increased in hairy roots compared with that in non-transformed roots. Furthermore, hairy roots efficiently produced several important individual phenolic compounds (flavonols, hydroxybenzoic and hydroxycinnamic acids). Colorimetric analysis revealed that the highest levels of total phenol, flavonoid contents, and their gene expression levels (PAL, CHI and FLS) in hairy roots than non-transformed roots. Our study provides beneficial information on the molecular and physiological active processes that are associated with the phytochemical content and biosynthetic gene expression in turnip. Moreover, antioxidant activity, as measured by DPPH scavenging activity, reducing potential, phosphomolybdenum and ferrous ion chelating ability assays was significantly higher in hairy roots. Hairy root extracts exhibited higher antimicrobial activity against bacterial and fungal species. The extract of hairy roots showed inhibition of human breast and colon cancer cell lines.

Genome-wide transcriptomic analyses provide insights into the lifestyle transition and effector repertoire of Leptosphaeria maculans during the colonization of Brassica napus seedlings.

Molecular interaction between the causal agent of blackleg disease, Leptosphaeria maculans (Lm), and its host, Brassica napus, is largely unknown. We applied a deep RNA-sequencing approach to gain insight into the pathogenicity mechanisms of Lm and the defence response of B. napus. RNA from the infected susceptible B. napus cultivar Topas DH16516, sampled at 2-day intervals (0-8 days), was sequenced and used for gene expression profiling. Patterns of gene expression regulation in B. napus showed multifaceted defence responses evident by the differential expression of genes encoding the pattern recognition receptor CERK1 (chitin elicitor receptor kinase 1), receptor like proteins and WRKY transcription factors. The up-regulation of genes related to salicylic acid and jasmonic acid at the initial and late stages of infection, respectively, provided evidence for the biotrophic and necrotrophic life stages of Lm during the infection of B. napus cotyledons. Lm transition from biotrophy to necrotropy was also supported by the expression function of Lm necrosis and ethylene-inducing (Nep-1)-like peptide. Genes encoding polyketide synthases and non-ribosomal peptide synthetases, with potential roles in pathogenicity, were up-regulated at 6-8 days after inoculation. Among other plant defence-related genes differentially regulated in response to Lm infection were genes involved in the reinforcement of the cell wall and the production of glucosinolates. Dual RNA-sequencing allowed us to define the Lm candidate effectors expressed during the infection of B. napus. Several candidate effectors suppressed Bax-induced cell death when transiently expressed in Nicotiana benthamaina leaves.

Augmenting Sulfur Metabolism and Herbivore Defense in Arabidopsis by Bacterial Volatile Signaling.

Sulfur is an element necessary for the life cycle of higher plants. Its assimilation and reduction into essential biomolecules are pivotal factors determining a plant’s growth and vigor as well as resistance to environmental stress. While certain soil microbes can enhance ion solubility via chelating agents or oxidation, microbial regulation of plant-sulfur assimilation has not been reported. With an increasing understanding that soil microbes can activate growth and stress tolerance in plants via chemical signaling, the question arises as to whether such beneficial bacteria also regulate sulfur assimilation. Here we report a previously unidentified mechanism by which the growth-promoting rhizobacterium Bacillus amyloliquefaciens (GB03) transcriptionally activates genes responsible for sulfur assimilation, increasing sulfur uptake and accumulation in Arabidopsis. Transcripts encoding for sulfur-rich aliphatic and indolic glucosinolates are also GB03 induced. As a result, GB03-exposed plants with elevated glucosinolates exhibit greater protection against the generalist herbivore, Spodoptera exigua (beet armyworm, BAW). In contrast, a previously characterized glucosinolate mutant compromised in the production of both aliphatic and indolic glucosinolates is also compromised in terms of GB03-induced protection against insect herbivory. As with in vitro studies, soil-grown plants show enhanced glucosinolate accumulation and protection against BAW feeding with GB03 exposure. These results demonstrate the potential of microbes to enhance plant sulfur assimilation and emphasize the sophisticated integration of microbial signaling in plant defense.