Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the β-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle's energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

Environmental pollution by heavy metals has become a serious problem in the world. Phytoextraction, which is one of the plant-based technologies, has attracted the most attention for the bioremediation of soils polluted with these contaminants. The aim of this study was to determine whether the multiple-tolerant bacterium, Brevibacterium casei MH8a isolated from the heavy metal-contaminated rhizosphere soil of Sinapis alba L., is able to promote plant growth and enhance Cd, Zn, and Cu uptake by white mustard under laboratory conditions. Additionally, the ability of the rifampicin-resistant spontaneous mutant of MH8a to colonize plant tissues and its mechanisms of plant growth promotion were also examined. In order to assess the ecological consequences of bioaugmentation on autochthonous bacteria, the phospholipid fatty acid (PLFA) analysis was used. The MH8a strain exhibited the ability to produce ammonia, 1-amino-cyclopropane-1-carboxylic acid deaminase, indole 3-acetic acid and HCN but was not able to solubilize inorganic phosphate and produce siderophores. Introduction of MH8a into soil significantly increased S. alba biomass and the accumulation of Cd (208%), Zn (86%), and Cu (39%) in plant shoots in comparison with those grown in non-inoculated soil. Introduced into the soil, MH8a was able to enter the plant and was found in the roots and leaves of inoculated plants thus indicating its endophytic features. PLFA analysis revealed that the MH8a that was introduced into soil had a temporary influence on the structure of the autochthonous bacterial communities. The plant growth-promoting features of the MH8a strain and its ability to enhance the metal uptake by white mustard and its long-term survival in soil as well as its temporary impact on autochthonous microorganisms make the strain a suitable candidate for the promotion of plant growth and the efficiency of phytoextraction.

The severity of salt stress is alarming for crop growth and production and it threatens food security. Strategies employed for the reduction in stress are not always eco-friendly or sustainable. Plant-growth-promoting rhizobacteria (PGPR) could provide an alternative sustainable stress reduction strategy owning to its role in various metabolic processes. In this study, we have used two strains of PGPR, Pseudomonas fluorescens (NAIMCC-B-00340) and Azotobacter chroococcum Beijerinck 1901 (MCC 2351), either singly or in combination, and studied their effect in the amelioration of salt toxicity in mustard cultivar Pusa Jagannath via its influence on plants' antioxidants' metabolism, photosynthesis and growth. Individually, the impact of Pseudomonas fluorescens was better in reducing stress ethylene, oxidative stress, photosynthesis and growth but maximal alleviation was observed with their combined application. MDA and H2O2 content as indicator of oxidative stress decreased by 27.86% and 45.18% and osmolytes content (proline and glycine-betaine) increased by 38.8% and 26.3%, respectively, while antioxidative enzymes (SOD, CAT, APX and GR) increased by 58.40, 25.65, 81.081 and 55.914%, respectively, over salt-treated plants through the application of Pseudomonas fluorescens. The combined application maximally resulted in more cell viability and less damage to the leaf with lesser superoxide generation due to higher antioxidative enzymes and reduced glutathione formation (GSH). Considering the obtained results, we can supplement the PGPR in combination to plants subjected to salt stress, prevent photosynthetic and growth reduction, and increase the yield of plants.

Crossing experiments indicate that hybrid sterility barriers frequently have developed within diploid, circumpolar plant species of the genus Draba. To gain insight into the rapid evolution of postzygotic reproductive isolation in this system, we augmented the linkage map of one of these species, D. nivalis, and searched for quantitative trait loci (QTLs) associated with reproductive isolation. The map adds 63 new dominant markers to a previously published dataset of 31 co-dominant microsatellites. These markers include 52 amplified fragment length polymorphisms (AFLPs) and 11 sequence-specific amplified polymorphisms (SSAPs) based on retrotransposon sequence. 22 markers displaying transmission ratio distortion were further included in the map. We resolved eight linkage groups with a total map length of 894 cM. Significant genotype-trait associations, or quantitative trait loci (QTL), were detected for reproductive phenotypes including pollen fertility (4 QTLs), seed set (3 QTLs), flowering time (3 QTLs) and number of flowers (4 QTLs). Observed patterns of inheritance were consistent with the influence of both nuclear-nuclear interactions and chromosomal changes on these traits. All seed set QTLs and one pollen fertility QTL displayed underdominant effects suggestive of the involvement of chromosomal rearrangements in hybrid sterility. Interestingly, D. nivalis is predominantly self-fertilizing, which may facilitate the establishment of underdominant loci and contribute to reproductive isolation.

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

Poly(epsilon-caprolactone) (PCL) nanocapsules have been recently developed as a modified release system for atrazine, an herbicide that can have harmful effects in the environment. Here, the post-emergence herbicidal activity of PCL nanocapsules containing atrazine was evaluated using mustard (Brassica juncea) as target plant species model. Characterization of atrazine-loaded PCL nanocapsules by nanoparticle tracking analysis indicated a concentration of 7.5 x 10(12) particles mL(-1) and an average size distribution of 240.7 nm. The treatment of mustard plants with nanocapsules carrying atrazine at 1 mg mL(-1) resulted in a decrease of net photosynthesis and PSII maximum quantum yield, and an increase of leaf lipid peroxidation, leading to shoot growth inhibition and the development of severe symptoms. Time course analysis until 72 h after treatments showed that nanoencapsulation of atrazine enhanced the herbicidal activity in comparison with a commercial atrazine formulation. In contrast to the commercial formulation, ten-fold dilution of the atrazine-containing nanocapsules did not compromise the herbicidal activity. No effects were observed when plants were treated with nanocapsules without herbicide compared to control leaves sprayed with water. Overall, these results demonstrated that atrazine-containing PCL nanocapsules provide very effective post-emergence herbicidal activity. More importantly, the use of nanoencapsulated atrazine enables the application of lower dosages of the herbicide, without any loss of efficiency, which could provide environmental benefits.

The glucosinolates which are specialized plant metabolites of Brassica vegetables are prone to hydrolysis catalyzed by an endogenous enzyme myrosinase (thioglycoside hydrolase, thioglucosidase) that exists in Brassica plant tissue causing volatile isothiocyanates release. Currently existing literature data on the inactivation of myrosinase is insufficient in particular for use in the analysis of volatile and odor compounds in vegetables rich in glucosinolates. In this study, the impact of different metal salts in effective inactivation of enzyme activity was investigated by solid-phase microextraction (SPME) and GC/MS system in aqueous samples and kohlrabi matrix. A saturated solution of calcium chloride which is commonly used to stop enzyme activity in plant tissue inactivates the myrosinase-glucosinolate system. However, even without the participation of myrosinase, it changes the reaction pathway towards nitrile formation. The model experiment shows that optimum efficiency in inhibition of the enzyme system shows iron(III) ions, silver ions, and anhydride sodium sulfate resulting in no volatile products derived from glucosinolates. However, in the kohlrabi matrix, the strongest enzyme inhibition effect was observed for silver salt resulting in no volatile products, also both anhydrous Na2SO4 and saturated CaCl2 solution seem to be useful inhibitors in flavor studies.

Angiosperms are renown for their diversity of flower colors. Often considered adaptations to pollinators, the most common underlying pigments, anthocyanins, are also involved in plants' stress response. Although the anthocyanin biosynthetic pathway is well characterized across many angiosperms and is composed of a few candidate genes, the consequences of blocking this pathway and producing white flowers has not been investigated at the transcriptome scale. We take a transcriptome-wide approach to compare expression differences between purple and white petal buds in the arctic mustard, Parrya nudicaulis, to determine which genes' expression are consistently correlated with flower color. Using mRNA-Seq and de novo transcriptome assembly, we assembled an average of 722 bp per gene (49.81% coding sequence based on the A. thaliana homolog) for 12,795 genes from the petal buds of a pair of purple and white samples. Our results correlate strongly with qRT-PCR analysis of nine candidate genes in the anthocyanin biosynthetic pathway where chalcone synthase has the greatest difference in expression between color morphs (P/W = ∼7×). Among the most consistently differentially expressed genes between purple and white samples, we found 3× more genes with higher expression in white petals than in purple petals. These include four unknown genes, two drought-response genes (CDSP32, ERD5), a cold-response gene (GR-RBP2), and a pathogen defense gene (DND1). Gene ontology analysis of the top 2% of genes with greater expression in white relative to purple petals revealed enrichment in genes associated with stress responses including cold, drought and pathogen defense. Unlike the uniform downregulation of chalcone synthase that may be directly involved in the loss of petal anthocyanins, the variable expression of several genes with greater expression in white petals suggest that the physiological and ecological consequences of having white petals may be microenvironment-dependent.

The oxylipin methyl jasmonate (MeJA) is a plant hormone active in response signalling and defence against herbivores. Although MeJA is applied experimentally to mimic herbivory and induce plant defences, its downstream effects on the plant metabolome are largely uncharacterized, especially in the context of primary growth and tissue-specificity of the response.

In Brassicaceae, tissue damage triggers the mustard oil bomb i.e., activates the degradation of glucosinolates by myrosinases leading to a rapid accumulation of isothiocyanates at the site of damage. Isothiocyanates are reactive electrophilic species (RES) known to covalently bind to thiols in proteins and glutathione, a process that is not only toxic to herbivores and microbes but can also cause cell death of healthy plant tissues. Previously, it has been shown that subtoxic isothiocyanate concentrations can induce transcriptional reprogramming in intact plant cells. Glutathione depletion by RES leading to breakdown of the redox potential has been proposed as a central and common RES signal transduction mechanism. Using transcriptome analyses, we show that after exposure of Arabidopsis seedlings (grown in liquid culture) to subtoxic concentrations of sulforaphane hundreds of genes were regulated without depletion of the cellular glutathione pool. Heat shock genes were among the most highly up-regulated genes and this response was found to be dependent on the canonical heat shock factors A1 (HSFA1). HSFA1-deficient plants were more sensitive to isothiocyanates than wild type plants. Moreover, pretreatment of Arabidopsis seedlings with subtoxic concentrations of isothiocyanates increased resistance against exposure to toxic levels of isothiocyanates and, hence, may reduce the autotoxicity of the mustard oil bomb by inducing cell protection mechanisms.

Large volumes of treated process water are required for protein extraction. Evaporation of this water contributes greatly to the energy consumed in enriching protein products. Thin stillage remaining from ethanol production is available in large volumes and may be suitable for extracting protein rich materials. In this work protein was extracted from ground defatted oriental mustard (Brassica juncea (L.) Czern.) meal using thin stillage. Protein extraction efficiency was studied at pHs between 7.6 and 10.4 and salt concentrations between 3.4 × 10-2 and 1.2 M. The optimum extraction efficiency was pH 10.0 and 1.0 M NaCl. Napin and cruciferin were the most prevalent proteins in the isolate. The isolate exhibited high in vitro digestibility (74.9 ± 0.80%) and lysine content (5.2 ± 0.2 g/100 g of protein). No differences in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability, or amino acid composition were observed between protein extracted with thin stillage and that extracted with NaCl solution. The use of thin stillage, in lieu of water, for protein extraction would decrease the energy requirements and waste disposal costs of the protein isolation and biofuel production processes.

Plants share a common history of successive whole-genome duplication (WGD) events retaining genomic patterns of duplicate gene copies (ohnologs) organized in conserved syntenic blocks. Duplication was often proposed to affect the origin of novel traits during evolution. However, genetic evidence linking WGD to pathway diversification is scarce. We show that WGD and tandem duplication (TD) accelerated genetic versatility of plant secondary metabolism, exemplified with the glucosinolate (GS) pathway in the mustard family. GS biosynthesis is a well-studied trait, employing at least 52 biosynthetic and regulatory genes in the model plant Arabidopsis. In a phylogenomics approach, we identified 67 GS loci in Aethionema arabicum of the tribe Aethionemae, sister group to all mustard family members. All but one of the Arabidopsis GS gene families evolved orthologs in Aethionema and all but one of the orthologous sequence pairs exhibit synteny. The 45% fraction of duplicates among all protein-coding genes in Arabidopsis was increased to 95% and 97% for Arabidopsis and Aethionema GS pathway inventory, respectively. Compared with the 22% average for all protein-coding genes in Arabidopsis, 52% and 56% of Aethionema and Arabidopsis GS loci align to ohnolog copies dating back to the last common WGD event. Although 15% of all Arabidopsis genes are organized in tandem arrays, 45% and 48% of GS loci in Arabidopsis and Aethionema descend from TD, respectively. We describe a sequential combination of TD and WGD events driving gene family extension, thereby expanding the evolutionary playground for functional diversification and thus potential novelty and success.

Yellow mustard (Sinapis alba) has a sporophytic self-incompatibility reproduction system. Genetically stable self-incompatible (SI) and self-compatible (SC) inbred lines have recently been developed in this crop. Understanding the S haplotype of different inbred lines and the inheritance of the self-(in)compatibility (SI/SC) trait is very important for breeding purposes. In this study, we used the S-locus gene-specific primers in Brassica rapa and Brassica oleracea to clone yellow mustard S-locus genes of SI lines Y514 and Y1130 and SC lines Y1499 and Y1501. The PCR amplification results and DNA sequences of the S-locus genes revealed that Y514 carried the class I S haplotype, while Y1130, Y1499, and Y1501 had the class II S haplotype. The results of our genetic studies indicated that self-incompatibility was dominant over self-compatibility and controlled by a one-gene locus in the two crosses of Y514 × Y1499 and Y1130 × Y1501. Of the five S-locus gene polymorphic primer pairs, Sal-SLGI and Sal-SRKI each generated one dominant marker for the SI phenotype of Y514; Sal-SLGII and Sal-SRKII produced dominant marker(s) for the SC phenotype of Y1501 and Y1499; Sal-SP11II generated one dominant marker for Y1130. These markers co-segregated with the SI/SC phenotype in the F2 populations of the two crosses. In addition, co-dominant markers were developed by mixing the two polymorphic primer pairs specific for each parent in the multiplex PCR, which allowed zygosity to be determined in the F2 populations. The SI/SC allele-specific markers have proven to be very useful for the selection of the desirable SC genotypes in our yellow mustard breeding program.

Environmental concerns related to synthetic pesticides and the emphasis on the adoption of an integrated pest management concept as a cardinal principle have strengthened the focus of global research and development on botanical pesticides. A scientific understanding of the mode of action of biomolecules over a range of pests is key to the successful development of biopesticides. The present investigation focuses on the in silico protein-ligand interactions of allyl isothiocyanate (AITC), a major constituent of black mustard (Brassica nigra) essential oil (MEO) against two pests, namely, Meloidogyne incognita (Mi) and Fusarium oxysporum f. sp. lycopersici (Fol), that cause severe yield losses in agricultural crops, especially in vegetables. The in vitro bioassay results of MEO against Mi exhibited an exposure time dependent on the lethal concentration causing 50% mortality (LC50) values of 47.7, 30.3, and 20.4 μg ml-1 at 24, 48, and 72 h of exposure, respectively. The study revealed short-term nematostatic activity at lower concentrations, with nematicidal activity at higher concentrations upon prolonged exposure. Black mustard essential oil displayed excellent in vitro Fol mycelial growth inhibition, with an effective concentration to cause 50% inhibition (EC50) value of 6.42 μg ml-1. In order to decipher the mechanism of action of MEO, its major component, AITC (87.6%), which was identified by gas chromatography-mass spectrometry (GC-MS), was subjected to in silico docking and simulation studies against seven and eight putative target proteins of Mi and Fol, respectively. Allyl isothiocyanate exhibited the highest binding affinity with the binding sites of acetyl cholinesterase (AChE), followed by odorant response gene-1 (ODR1) and neuropeptide G-protein coupled receptor (nGPCR) in Mi, suggesting the possible suppression of neurotransmission and chemosensing functions. Among the target proteins of Fol, AITC was the most effective protein in blocking chitin synthase (CS), followed by 2,3-dihydroxy benzoic acid decarboxylase (6m53) and trypsinase (1try), thus inferring these as the principal molecular targets of fungal growth. Taken together, the study establishes the potential of MEO as a novel biopesticide lead, which will be utilized further to manage the Mi-Fol disease complex.

Fusarium graminearum is a phytopathogenic fungus that causes Fusarium head blight in small-grain cereals, such as wheat, with significant yield reductions. Moreover, it contaminates the cereal grains with health-threatening mycotoxins, such as deoxynivalenol (DON), jeopardizing food and feed safety. Plant-based biopesticides, i.e. botanicals, have recently gained increased interest in crop protection as alternatives to synthetic chemical products. The main objective of this study was to test the control efficacy of botanicals based on white or Indian/Oriental mustard seed flours (Tillecur - Ti, Pure Yellow Mustard - PYM, Pure Oriental Mustard - POM, Oriental Mustard Bran - OMB) on F. graminearum infection and mycotoxin accumulation in wheat grain. Botanicals at 2% concentration showed a higher efficacy in inhibiting mycelium growth in vitro compared with a prothioconazole fungicide (F). In the growth chamber experiment under controlled conditions, the spraying agents reduced DON content in grain in the following order: F = Ti = PYM > POM > OMB. The antifungal activity of the botanicals may be attributed to their bioactive matrices containing isothiocyanates (ITCs) and phenolic acids. Allyl ITC was detected in POM and OMB at 8.38 and 4.48 mg g-1, while p-hydroxybenzyl ITC was found in Ti and PYM at 2.56 and 2.44 mg g-1, respectively. Considerable amounts of various phenolic acids were detected in all botanicals. Under field conditions, only the use of F significantly decreased F. graminearum infection and DON content in grain. An additional important finding of this study is that disease control was more difficult when infection was done with ascospores than conidia, which might have several potential implications considering that ascospores are more important in Fusarium head blight epidemics. Our results suggest that mustard-based botanicals are promising biopesticides for the control of Fusarium head blight in small-grain cereals, but for field applications, an appropriate formulation is necessary to stabilize and prolong the antifungal activity, especially against ascospores.

Micro RNAs (miRNAs) are a pivotal part of non-protein-coding endogenous small RNA molecules that regulate the genes involved in plant growth and development, and respond to biotic and abiotic environmental stresses posttranscriptionally.

Heavy metal exposure affect plant productivity by interfering, directly and indirectly, with photosynthetic reactions. The toxic effect of heavy metals on photosynthetic reactions has been reported in wide-ranging studies, however there is paucity of data in the literature concerning thallium (Tl) toxicity. Thallium is ubiquitous natural trace element and is considered the most toxic of heavy metals; however, some plant species, such as white mustard (Sinapis alba L.) are able to accumulate thallium at very high concentrations. In this study we identified the main sites of the photosynthetic process inhibited either directly or indirectly by thallium, and elucidated possible detoxification mechanisms in S. alba.

The specific mechanisms that result in the success of any species invasion case are difficult to document. Reproductive strategies are often cited as a primary driver of invasive success, with human activities further facilitating invasions by, for example, acting as seed vectors for dispersal via road, train, air, and marine traffic, and by producing efficient corridors for movement including canals, drainages, and roadways. Sahara mustard (Brassica tournefortii) is a facultative autogamous annual native to Eurasia that has rapidly invaded the southwestern United States within the past century, displacing natives, and altering water-limited landscapes in the southwest. We used a genotyping-by-sequencing approach to study the population structure and spatial geography of Sahara mustard from 744 individuals from 52 sites across the range of the species' invasion. We also used herbaria records to model range expansion since its initial introduction in the 1920s. We found that Sahara mustard occurs as three populations in the United States unstructured by geography, identified three introduction sites, and combined herbaria records with genomic analyses to map the spread of the species. Low genetic diversity and linkage disequilibrium are consistent with self-fertilization, which likely promoted rapid invasive spread. Overall, we found that Sahara mustard experienced atypical expansion patterns, with a relatively constant rate of expansion and without the lag phase that is typical of many invasive species.

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

Sclerotinia stem rot is one of the utmost important disease of mustard, causing considerable losses in seed yield and oil quality. The study of the genetic and proteomic basis of resistance to this disease is imperative for its effective utilization in developing resistant cultivars. Therefore, the genetic pattern of Sclerotinia stem rot resistance in Indian mustard was studied using six generations (P1, P2, F1, F2, BC1P1, and BC1P2) developed from the crossing of one resistant (RH 1222-28) and two susceptible (EC 766300 and EC 766123) genotypes. Genetic analysis revealed that resistance was governed by duplicate epistasis. Comparative proteome analysis of resistant and susceptible genotypes indicated that peptidyl-prolyl cis-trans isomerase (A0A078IDN6 PPIase) showed high expression in resistant genotype at the early infection stage while its expression was delayed in susceptible genotypes. This study provides important insight to mustard breeders for designing effective breeding programs to develop resistant cultivars against this devastating disease.