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C-repeat binding factor (CBF) dependent cold stress signaling cascade is well studied in the model plant arabidopsis but is relatively lesser studied in the crop plants. In the present study, two novel isoforms of an upstream regulator of CBF, Inducer of CBF expression (ICE), BjICE46 (1314 bp, accession number HQ446510) and BjICE53 (1494 bp, accession number HQ857208) were isolated from Brassica juncea seedlings. Genomic clones of both the isoforms (accession numbers HQ433510 and JX571043) showed three introns, out of which one intron was spanning the bHLH (basic helix-loop-helix) domain. Interestingly, the constitutive expression of BjICE53 was 21 fold higher than BjICE46. Real time quantitative expression (RT-qPCR) showed BjICE53 to be cold induced but non-responsive to phytohormones. Interestingly, BjICE46 was salinity stress induced and showed upregulation with methyl jasmonate (MeJa) and abscisic acid (ABA). This was supported by the presence of ABA, MeJa and defense related cis- acting regulatory elements in the promoter region of BjICE46. The downstream transcription factor BjCBF (645 bp) was also isolated. The promoter region of BjCBF showed three E-boxes, the binding site for ICE. BjCBF was expressed and purified from E. coli and binding of purified BjCBF with the DRE/CRT elements (present in the promoter of cold responsive genes) was EMSA confirmed. Overall, this study shows that ICE-CBF pathway is conserved in Brassica juncea along with the differential regulation of the ICE isoforms indicating cross-talk between cold and defense signaling., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The spread of food allergens is a topic of global importance due to its impact on public health. National and International regulations ask food producers and manufacturers to declare product compositions on the label, especially in case of processed raw materials. Wheat flour ( Triticum aestivum ) can be contaminated by a wide range of species belonging to the Brassicaceae in the field or during grain harvests, storage, and processing. Among them, mustards ( Brassica nigra , Brassica juncea and Sinapis alba ) are well known allergenic species. Often, food quality laboratories adopt an ELISA approach to detect the presence of mustard species. However, this approach shows cross-reactivity with other non-allergenic species such as Brassica napus (rapeseed). In the last few years, DNA barcoding was proposed as a valid identification method, and it is now commonly used in the authentication of food products. This study aims to set up an easy and rapid DNA-based tool to detect mustard allergenic species. DNA barcoding ( matK and ITS2) and chromosome markers (A6, B, C1 genome regions) were selected, and specific primers were validated on incurred reference food matrices. The developed test was proven to be able to distinguish mustard from rapeseed and wheat, overcoming cross-reactivity with Brassica napus ., Competing Interests: FEM2-Ambiente s.r.l., provided support in the form of a salary for authors J.F., V.M. and F.D.M., whereas BARILLA G. e R. FRATELLI Spa, provided support in the form of a salary for authors and R.P., F.L., M.S., S.Z. and A.L. Both the companies only provided financial support in the form of research materials.

Background: Abiotic stresses like drought, heat, cold and salinity cause major productivity loss in the rapeseed-mustard crops (Brassica). Major efforts have been made in the past to identify genes that provide resistance against such stresses. Superoxide dismutase (SOD) proteins, member of the metallo-enzyme family play vital role in protecting plants against abiotic stresses. In the present study, genome-wide analysis of abiotic stress responsive SOD gene family has been done in B. juncea and B. rapa., Results: A total of 29 and 18 SOD genes were identified in B. juncea and B. rapa respectively and chromosome location mapping indicated their wide distribution across genome. On the basis of domain composition, the SODs were phylogenetically classified into sub-groups which was also substantiated by the gene structure and sub-cellular locations of SOD proteins. Functional annotation of SODs was also done by Gene Ontology (GO) mapping and the result was corroborated by the identified cis-regulatory elements in the promoter region of SOD genes. Based on FPKM analysis of SRA data available for drought, heat and salt stress, we identified 14 and 10 abiotic stress responsive SOD genes in B. rapa and B. juncea respectively. The differential expression analysis under drought and heat stress of identified abiotic-stress responsive SOD genes was done through quantitative Real Time PCR., Conclusion: We identified abiotic-stress responsive genes that could help in improving the plant tolerance against abiotic stresses. This was the first study to describe the genome-wide analysis of SOD gene family in B. rapa and B. juncea, and the results will help in laying basic ground for future work of cloning and functional validation of SOD genes during abiotic stresses leading to Brassica crop improvement.

The antioxidant defense system of Brassica juncea under Cd stress was examined on supplementation of earthworms in the rhizosphere at different concentrations of Cd (0.50, 0.75, 1.00 and 1.25 mM i.e. 56, 84, 112 and 140 mg kg -1 respectively). Seedlings were raised in small pots containing soil spiked with Cd and earthworms under controlled conditions for 15 days. Improved Cd accumulation, as well as enhanced plant dry weight and metal tolerance were observed following the addition of earthworms. Earthworm supplementation reduced reactive oxygen species (ROS) generation by 7.3% for hydrogen peroxide (H 2 O 2 ), 7.1% for superoxide anion (O 2 - ), and 8.4% for malondialdehyde (MDA) in plants treated with 1.25 mM (140 mg kg -1 ) Cd. Confocal microscopy revealed improved cell viability and reduced H 2 O 2 content due to enhanced antioxidative activity. Activity and expression levels of genes coding for antioxidative enzymes (superoxide dismutase; SOD, catalase; CAT, guaicol peroxidase; POD, glutathione reductase; GR, and glutathione-S-transferase; GST) were higher in plants raised in soils inoculated with earthworms, with expression of SOD increasing by 58.8%, CAT by 75%, POD by 183%, GR by 106.6%, and GST by 11.8%. Moreover, plant pigment (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) concentrations increased by 8%, 9.1%, 9.1%, and 7.7% respectively, in plants grown in soils supplemented with earthworms. The results of our study suggest that the addition of earthworms to soil increases antioxidative enzyme activities, gene expression in plants, and ROS inhibition, which enhances tolerance to Cd during the phytoextraction process., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Two mutations Leu498 and Glu425 in the PDS gene were identified as the main cause conferring resistance to diflufenican and picolinafen in two oriental mustard populations P3 and P40. As mutations are suspected to affect fitness, this study was designed to test this hypothesis using the F 2 of two crosses P3.2 (P3♂ × S♀) and P40.5 (P40♂ × S♀) of oriental mustard. The F 2 plants, which segregated for target-site point mutations of PDS gene (Leu498 and Glu425) grown in monoculture and under competition with wheat in pot-trials and evaluated for growth and fecundity. All F 2 individuals were genotyped by using Cleaved Amplified Polymorphic Sequence (CAPS) technique. Regression analysis showed no fitness cost in the resistant plants because no significant difference was identified in seed and biomass production within RR, RS and SS individuals. The absence of measurable negative effects on fitness associated mutations suggests that the frequency of the PDS resistance alleles will not decline in the absence of selection pressure of PDS-inhibitors., (Copyright © 2019. Published by Elsevier Inc.)

Seed size and seed metabolites have been the targets of direct or indirect selection during domestication and subsequent crop breeding. Understanding these traits and associated genetics can prove very useful for plant translational research. Large germplasm assemblage (235) of Brassica juncea and its progenitor species (B. rapa and B. nigra) was evaluated to establish seed trait variations for seed size and seed metabolites. Seeds were smallest in B. nigra and largest in B. juncea. Australian B. juncea and Indian B. rapa var brown sarson types averaged more seed oil content. Seed size and oil content were generally higher in modern cultivars in comparison to the land races. Allelic diversity for known associated genes for seed-size and oil-content (AP2, ARF2, TTG2, GRF2, GL2, CYP78A5, CYP78A6, MINI3, IKU2, IKU1, BRI1, DGAT, GPDH, LPAAT, GPAT and DA1) was studied so as to infer the effect of domestication on seed traits. Three genes (IKU1, IKU2, AP2) in B. rapa, two (TTG2 and GL2) in B. nigra and two (IKU1 and GRF2) in natural B. juncea were identified as targets of selection on the basis of Fst outlier and/or sequence diversity tests. We report parallel divergence for seed traits between B. juncea and B. rapa. Directional selection appeared stronger for seed-size as compared to correlated seed metabolites. Positive selection on seed-size is likely to have played a significant role in structuring regional variation in the germplasm.

Abiotic stress tolerance is one of the target trait in crop breeding under climate change scenario. Selection of suitable gene pools among available germplasm is first requisite for any crop improvement programme. Drought and salinity traits, being polygenic, are most difficult to target. The present investigation aimed at exploring and assessment of the genetic variability in Indian mustard at molecular level. A total of twenty-five genotypes and five related species were used. Sixty-three molecular markers including sequence related amplified polymorphism (SRAP) markers along with twenty-three expressed sequence tag-simple sequence repeats (EST-SSRs) were used for diversity analysis. Thirty-seven SRAPs and 18 EST-SSRs showed amplification producing a total of 423 alleles of which 422 were polymorphic. These markers gave an overall polymorphism of 99.78%, with 99.67% polymorphism in SRAPs and 100% polymorphism in EST-SSRs. The study revealed the genetic relationships among different genotypes of B. juncea and related species which could be used for Indian mustard improvement for targeting drought and salinity tolerance in future. Four SRAP and two EST-SSRs identified unique bands which may be related to abiotic stress tolerance. EST sequence BRMS-040 (IM7) was similar to Brassica and radish sequences related to PR-5 (pathogenesis-related) protein.

Tumorous stem mustard is a characteristic vegetable in Southeast Asia, as are its sprouts. The purple color of the purple variety 'Zi Ying' leaves is because of anthocyanin accumulation. The ways in which this anthocyanin accumulation is affected by the environment and hormones has remained unclear. Here, the impacts of sucrose, methyl jasmonate (MeJA), light, and dark on the growth and anthocyanin production of 'Zi Ying' sprouts were explored. The results showed that anthocyanins can be enhanced by sucrose in sprouts under light condition, and MeJA can promote anthocyanins production under light and dark conditions in sprouts. The anthocyanin biosynthetic regulatory genes BjTT8, BjMYB1, BjMYB2 and BjMYB4, and the EBGs and LBGs were upregulated under light conditions, while BjTT8, BjMYB1, and BjMYB2 and anthocyanin biosynthetic genes BjF3H and BjF3'H were upregulated under DM condition. These results indicate that sucrose and methyl jasmonate can stimulate the expression of genes encoding components of the MBW complex (MYB, bHLH, and WD40) and that they transcriptional activated the expression of LBGs and EBGs to promote the accumulation of anthocyanins in 'Zi Ying' sprouts. Our findings enhance our understanding of anthocyanin accumulation regulated by sucrose and MeJA in 'Zi Ying', which will help growers to produce anthocyanin-rich foods with benefits to human health.

Phytoremediation is an efficient and promising cleanup technology to extract or inactivate heavy metals and several organic and inorganic pollutants from soil and water. In this study, different Brassica nigra L. ecotypes, including Diyarbakır, collected from mining areas were exposed to different concentrations of copper and harvested after 72 h of Cu stress for the assessment of phytoremediation capacity. The Diyarbakır ecotype was called as "metallophyte" because of surviving at 500 μM Cu. To better understand Cu stress mechanism, ArabidopsisATH1 genome array was used to compare the gene expression in root and shoot tissues of B. nigra under 25 μM Cu. The response to Cu was much stronger in roots (88 genes showing increased or decreased mRNA levels) than in leaf tissues (24 responding genes). These genes were classified into the metal transport and accumulation-related genes, signal transduction and metabolism-related genes, and transport facilitation genes. Glutathione pathway-related genes (γ-ECS, PC, etc.) mRNAs were identified as differentially expressed in root and shoot tissues. QRT-PCR validation experiments showed that γ-ECS and PC expression was upregulated in the shoot and leaf tissues of the 100 μM Cu-subjected B. nigra-tolerant ecotype. This is the first study showing global expression profiles in response to Cu stress in B. nigra by Arabidopsis genome array. This work presented herein provides a well-illustrated insight into the global gene expression to Cu stress response in plants, and identified genes from microarray data will serve as molecular tools for the phytoremediation applications in the future.

SOC1, a MADS-box type II transcription factor, integrates environmental and endogenous cues to promote flowering in angiosperms. Recent reports implicating SOC1 in roles beyond floral transition prompted functional characterization of SOC1 in polyploid rapeseed mustard genomes. Gene characterization in Brassicas necessitates analysis of composite homeolog function. While insertional mutagenesis is untenable in Brassicas owing to gene redundancy, gain-of-function approach entails serial characterization of individual homeologs. Herein, we demonstrate modulated floral promotive effects in natural variants of Brassica SOC1 and provide lateral branching as a probable outcome of polyploidy-induced gene diversification. Ectopic expression of two B genome specific SOC1 variants in Arabidopsis thaliana resulted in differential floral acceleration and manifestation of multiple vegetative rosettes. Characterization of composite homeolog function in B. juncea via introgression of Brassica SOC1 specific artificial miRNA, designed to target homeologs, also exhibited modifications in floral transition and lateral branching. Comprehensive analysis of field performance of B. juncea transgenics displayed altered fitness across 11 agronomic traits. Crucially, reduced SOC1 levels directly impacted two developmental traits, namely, flowering time and number of lateral branches which in turn influenced several dependent agronomic traits. While delayed flowering and crop maturity resulted in altered fatty acid composition with higher SFA and lower PUFA in transgenics relative to controls, reduction in overall count of lateral branches caused a concomitant decrease in silique count which ultimately impacted total seed yield in transgenics. Statistical analysis revealed number of secondary branches as the most critical trait influencing seed yield. Based on our findings, we propose enhancing levels Brassica SOC1, a key target, for achieving earliness in flowering, improved seed yield and oil quality, and studying trait trade-offs.

Natural structural variants of regulatory proteins causing quantitative phenotypic consequences have not been reported in plants. Herein, we show that 28 natural structural variants of FT homeologs, isolated from 6 species of Brassica, differ with respect to amino-acid substitutions in regions critical for interactions with FD and represent two evolutionarily distinct categories. Analysis of structural models of selected candidates from Brassica juncea (BjuFT_AAMF1) and Brassica napus (BnaFT_CCLF) predicted stronger binding between BjuFT and Arabidopsis thaliana FD. Over-expression of BjuFT and BnaFT in wild type and ft-10 mutant backgrounds of Arabidopsis validated higher potency of BjuFT in triggering floral transition. Analysis of gain-of-function and artificial miRNA mediated silenced lines of B. juncea implicated Brassica FT in multiple agronomic traits beyond flowering, consistent with a pleiotropic effect. Several dependent and independent traits such as lateral branching, silique shape, seed size, oil-profile, stomatal morphology and plant height were found altered in mutant lines. Enhanced FT levels caused early flowering, which in turn was positively correlated to a higher proportion of desirable fatty acids (PUFA). However, higher FT levels also resulted in altered silique shape and reduced seed size, suggesting trait trade-offs. Modulation of FT levels for achieving optimal balance of trait values and parsing pair-wise interactions among a reportoire of regulatory protein homeologs in polyploid genomes are indeed future areas of crop research., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Aims: Since pollen flow or seed dispersal can contribute to transgene persistence in the environment, the sympatric presence of transgenic crops with their wild relatives is an ecological concern. In this study, we tested the hypothesis that proximate growth of a herbivore-resistant Bt crop and wild relatives coupled with the presence of herbivores can increase relative frequency of crop-to-wild transgene flow persistence outside of cultivation., Methods: We conducted a field experiment using insect enclosures with and without herbivores with cultivated Bt-transgenic Brassica napus (Bt OSR) and wild brown mustard (Brassica juncea) in pure and mixed stands. Low-density diamondback moth (Plutella xylostella) caterpillar infestation treatments were applied and transgene flow and reproductive organs were measured., Key Results: Bt-transgenic B. napus produced more ovules and pollen than wild mustard, but the pollen to ovule (P/O) ratio in the two species was not significantly different. Low-level herbivory had no effects on fitness parameters of Bt OSR or wild brown mustard or on the transgene flow frequency. All progeny from wild brown mustard containing the Bt transgene came from mixed stands, with a gene flow frequency of 0.66 %. In mixed stands, wild brown mustard produced less pollen and more ovules than in pure stands of brown mustard. This indicates a decreased P/O ratio in a mixed population scenario., Conclusions: Since a lower P/O ratio indicates a shift in sex allocation towards relatively greater female investment and a higher pollen transfer efficiency, the presence of transgenic plants in wild populations may further increase the potential transgene flow by altering reproductive allocation of wild species.

Gene flow from genetically modified crops has been studied for more than 20 years, but public concern still remains. A lot of data on this matter is obtained on the territory of EU and the USA, but in the majority of countries, such experiments were never carried out. Here, we present the first study of interspecific and intraspecific hybridization of transgenic Brassica napus on the territory of Russia. The experiment was conducted using two different models of coexistence. Cross-pollination with related species was more frequent in mixed than that in separated populations. We observed maximum 4.1% of transgenic seeds in the progeny of Brassica rapa and 0.6% in the progeny of Brassica juncea. The highest intraspecific hybridization rate of 0.67% was observed in separated populations. DNA fragments, typical to both parents, were present in the genome of the hybrids. The risk of gene flow in Russia is relatively low, but it will be problematic to do environmental monitoring on such a big territory. However, instead of banning the cultivation of genetically modified crops, some new varieties with visually detectable selective traits could be designed and approved for cultivation.

SaNRAMP3 gene cloned from a Zn/Cd hyperaccumulator Sedum alfredii was ectopicly expressed in Brassica juncea, a fast-growing and high-biomass crop plant. In a tissue culture experiment, transgenic plants were grown on MS medium with 0, 25, 50, 100, 200 μM Cd. It was shown that, at the same Cd treatment, the Cd tolerance of transgenic plants had no significant difference with those of wild-type plants (WT). However, the shoot Cd content and accumulation were improved significantly while the root Cd content and accumulation were descended significantly by SaNRAMP3 gene expression, which obviously enhanced the Cd root-to-shoot translocation factor (TF). In the hydroponic experiment, plants were cultured in nutrition solution with 0, 2.5, 25 μM Cd. Data showed that the Cd tolerance of transgenic plants had no significant difference with that of WT under the same Cd exposure. Whereas, the shoot Cd content and accumulation was increased 1.43-1.81 times and the TF was enhanced 3.09-3.51 times by SaNRAMP3 gene expression. Those results indicated that ectopic expression of SaNRAMP3 in B. juncea didn't lead to Cd sensitivity, but enhanced Cd root-to-shoot transport, so that increased shoot Cd accumulation. This study provided a possibility to improve phytoextraction efficiency of heavy metal through gene engineering., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Premise of Study: Mechanisms by which invasive species succeed across multiple novel environmental contexts are poorly understood. Functional traits show promise for identifying such mechanisms, yet we lack knowledge of which functional traits are critical for success and how they vary across invaded ranges and with environmental features. We evaluated the widespread recent invasion of Sahara mustard (Brassica tournefortii) in the southwestern United States to understand the extent of functional trait variation across the invaded range and how such variation is related to spatial and climatic gradients., Methods: We used a common garden approach, growing two generations of plants in controlled conditions sourced from 10 locations across the invaded range. We measured variation within and among populations in phenological, morphological, and physiological traits, as well as performance., Key Results: We found nine key traits that varied among populations. These traits were related to phenology and early growth strategies, such as the timing of germination and flowering, as well as relative allocation of biomass to reproduction and individual seed mass. Trait variation was related most strongly to variation in winter precipitation patterns across localities, though variations in temperature and latitude also had significant contributions., Conclusions: Our results identify key functional traits of this invasive species that showed significant variation among introduced populations across a broad geographic and climatic range. Further, trait variation among populations was strongly related to key climatic variables, which suggests that population divergence in these traits may explain the successful colonization of Sahara mustard across its invaded US range., (© 2018 Botanical Society of America.)

Key Message: Transgenic Brassica juncea plants expressing Colocasia esculenta tuber agglutinin (CEA) shows the non-allergenic nature of the expressed protein leading to enhanced mortality and reduced fecundity of mustard aphid-Lipaphis erysimi. Lipaphis erysimi (common name: mustard aphid) is the most devastating sucking insect pest of Indian mustard (Brassica juncea L.). Colocasia esculenta tuber agglutinin (CEA), a GNA (Galanthus nivalis agglutinin)-related lectin has previously been reported by the present group to be effective against a wide array of hemipteran insects in artificial diet-based bioassays. In the present study, efficacy of CEA in controlling L. erysimi has been established through the development of transgenic B. juncea expressing this novel lectin. Southern hybridization of the transgenic plants confirmed stable integration of cea gene. Expression of CEA in T 0 , T 1 and T 2 transgenic plants was confirmed through western blot analysis. Level of expression of CEA in the T 2 transgenic B. juncea ranged from 0.2 to 0.47% of the total soluble protein. In the in planta insect bioassays, the CEA expressing B. juncea lines exhibited enhanced insect mortality of 70-81.67%, whereas fecundity of L. erysimi was reduced by 49.35-62.11% compared to the control plants. Biosafety assessment of the transgenic B. juncea protein containing CEA was carried out by weight of evidence approach following the recommendations by FAO/WHO (Evaluation of the allergenicity of genetically modified foods: report of a joint FAO/WHO expert consultation, 22-25 Jan, Rome, http://www.fao.org/docrep/007/y0820e/y0820e00.HTM , 2001), Codex (Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome; Codex, Codex principles and guidelines on foods derived from biotechnology, Food and Agriculture Organization of the United Nations, Rome, 2003) and ICMR (Indian Council of Medical Research, guidelines for safety assessment of food derived from genetically engineered plants, http://www.icmr.nic.in/guide/Guidelines%20for%20Genetically%20Engineered%20Plants.pdf , 2008). Bioinformatics analysis, pepsin digestibility, thermal stability assay, immuno-screening and allergenicity assessment in BALB/c mice model demonstrated that the expressed CEA protein from transgenic B. juncea does not incite any allergenic response. The present study establishes CEA as an efficient insecticidal and non-allergenic protein to be utilized for controlling mustard aphid and similar hemipteran insects through the development of genetically modified plants.

HDA9 (a RPD3-like histone deacetylase) belongs to the histone deacetylase family which is involved in flowering time control through repression of AGL19 and FT, but it is still elusive that whether and how HDA9 directly interacts with flowering signal integrators of SOC1 and AGL24 in Brassica juncea. In this study, BjuHDA9 (a homologous HDA9) was cloned from B. juncea and ubiquitously expressed in root, stem, cauline leaf, flower bud and opening flower. BjuHDA9 was highly induced by short-day photoperiod. Yeast two-hybrid and pull-down assays demonstrated that BjuHDA9 could not interact with BjuSOC1 and BjuAGL24 proteins. Whereas, BjuHDA9 directly interacted with promoters of BjuSOC1 and BjuAGL24 via yeast one-hybrid and Dual-Glo ® Luciferase assays. It suggested that the histone deacetylase BjuHDA9 was probably involved in flowering time control by binding to promoter regions of BjuSOC1 and BjuAGL24. This study will provide valuable information for elucidating the molecular mechanism of BjuHDA9 in regulating flowering time., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Nitrate is the main source of inorganic nitrogen for plants, which also act as signaling molecule. Present study was aimed to understand nitrate regulatory mechanism in Brassica juncea cultivars, with contrasting nitrogen-use-efficiency (NUE) viz. Pusa Bold (PB, high-NUE) and Pusa Jai Kisan (PJK, low-NUE), employing RNA-seq approach. A total of 4031, 3874 and 3667 genes in PB and 2982, 2481 and 2843 genes in PJK were differentially expressed in response to early, low (0.25 mM KNO 3 ), medium (2 mM KNO 3 ) and high (4 mM KNO 3 ) nitrate treatments, respectively, as compared to control (0 mM KNO 3 ). Genes of N-uptake (NRT1.1, NRT1.8, and NRT2.1), assimilation (NR1, NR2, NiR, GS1.3, and Fd-GOGAT) and remobilization (GDH2, ASN2-3 and ALaT) were highly-upregulated in PB than in PJK in response to early nitrate treatments. We have also identified transcription factors and protein kinases that were rapidly induced in response to nitrate, suggesting their involvement in nitrate-mediated signaling. Co-expression network analysis revealed four nitrate specific modules in PB, enriched with GO terms like, "Phenylpropanoid pathway", "Nitrogen compound metabolic process" and "Carbohydrate metabolism". The network analysis also identified HUB transcription factors like mTERF, FHA, Orphan, bZip and FAR1, which may be the key regulators of nitrate-mediated response in B. juncea.

Lead (Pb) is considered one the most hazardous pollutant, and its accumulation in soil and plants is of prime concern. To understand the role of plant hormones in combating heavy metal stress, the present study was planned to assess the interactive effects of 24-epibrassinolide (EBL) (10 -7  M) and salicylic acid (SA) (1 mM) in regulating growth, pigment contents, antioxidative defense response, and gene expression in Brassica juncea L. seedlings exposed to different concentrations of Pb metal (0.25, 0.50, and 0.75 mM). Reduction in root and shoot lengths, chlorophyll and carotenoid content, and non-enzymatic antioxidants like glutathione, ascorbic acid, and tocopherol in response to Pb toxicity was observed. The enzymatic antioxidants such as guaiacol peroxidase (POD), ascorbate peroxidase (APOX), glutathione peroxidase (GR), dehydroascorbate reductase (DHAR), monodehydroascorbate redductase (MDHAR), glutathione-S-transferease (GST), and glutathione peroxidase (GPOX) were lowered in response to Pb treatments. Other antioxidative enzymes including superoxide dismutase (SOD), catalase (CAT), and polyphenol oxidase (PPO) enhanced under metal stress. Co-application of EBL + SA to 0.75 mM Pb-treated seedlings resulted in improvement of root and shoot lengths, chlorophyll, and carotenoid contents. Similarly, glutathione, ascorbic acid, and tocopherol contents were also elevated. Enzymatic antioxidants were also significantly enhanced in response to pre-sowing combined treatment of both hormones. Gene expression analysis suggested elevation in expression of CAT, POD, GR, DHAR, and GST genes by application of EBL. Our results reveal that Pb metal toxicity caused adverse impact on B. juncea L. seedlings, but pre-soaking treatment with EBL and SA individually and in combination help seedlings to counter the ill effects of Pb by improving growth, contents of pigment, and modulation of antioxidative defense system. The combined application of EBL and SA was found to be more effective in ameliorating Pb stress as compared to their individual treatments.

Flower color in Brassica spp. is an important trait and considered a major visual signal for insect-pollinated plants. In previous study, we isolated and identified two genes (BjPC1 and BjPC2) that control the flower color in B. juncea, and mapped BjPC1 to a 0.13-cM region. In this study, we report the fine-mapping and candidate analysis of BjPC2. We conducted whole-genome resequencing, using bulked segregant analysis (BSA) to determine the BjPC2 candidate intervals. Crossing, allelism testing, and repeated full-sib mating were used to generate XG3, a near isogenic line (NIL) population that segregated on the BjPC2 locus. Through a genome-wide comparison of single nucleotide polymorphism (SNP) profiles between the yellow- and white-flowered bulks, a candidate interval for BjPC2 was identified on chromosome B04 (2.45 Mb). The BjPC2 linkage map was constructed with the newly developed simple sequence repeat (SSR) markers in the candidate interval to narrow the candidate BjPC2 region to 31-kb. Expression profiling and RNA-seq analysis partially confirmed that the AtPES2 homolog, BjuB027334 is the most promising candidate gene for BjPC2. Furthermore, analyses with high pressure liquid chromatography and transmission electron microscopy demonstrated that BjPC2 might be important in xanthophyll esterification, a process that limits xanthophyll degradation and increases sequestration. Overall, we mapped the BjPC2 to a 31-kb region on the B04 in B. juncea and identified BjuB027334 as a valuable candidate gene. Our results provide a basis for understanding the molecular mechanisms underlying the white-flowered trait and for molecular marker-assisted selection of flower color in B. juncea breeding.

Multilocular trait has recently attracted considerable attention for its potential to increase yield. Our previous studies indicated that two genes (Bjln1 and Bjln2) are responsible for multilocular siliques in Brassica juncea and the Bjln1 gene has been delimited to a 208-kb region. In present study, the Bjln1 gene was successfully isolated using the map-based cloning method. Complementation test indicated that the BjuA07.CLV1 (equivalent to BjLn1) could rescue the multilocular phenotype and generate bilocular siliques. Two amino acids changes at positions 28 and 63 in BjuA07.clv1 as well as a 702-bp deletion in its promoter have been proved to affect the carpel numbers. Microscopic analyses suggested that BjuA07.CLV1 is involved in the maintenance of shoot and floral meristem size. The expression level of BjuA07.clv1 was significantly reduced in the SAM. Furthermore, WUS, CLV2, CLV3, RPK2 and POL, key genes in the CLV/WUS signal pathway, showed lower expression level in the multilocular plants. These data suggest that the mutations in the CDS and promoter of BjuA07.clv1 reduced its function and expression level, which disturbed CLV/WUS signal pathway, thereby leading to the enlargement of the shoot and floral meristem and resulting in the multilocular siliques.

DNA J HOMOLOG 3 (J3) is a special transcriptional regulator in flowering time control, but the molecular mechanism of J3 in regulating flowering time has not been thoroughly revealed in B. juncea which is one important oilseed and vegetable crop. In this study, J3 gene was cloned from B. juncea (BjuJ3). Phylogenetic relationship analysis showed that the BjuJ3 had high amino acid sequence similarity (>93%) with other Brassica plants. The BjuJ3-transgenic tobacco plants exhibited early flowering, suggesting that BjuJ3 was an activator of flowering time. The qRT-PCR analysis found that BjuJ3 could be ubiquitously induced by the long-day and vernalization treatments in all the tissues of B. juncea. Yeast two-hybrid assays and GST pull-down experiments revealed that BjuJ3 could not directly interact with BjuSOC1, BjuSVP and BjuAGL24. Whereas, yeast one-hybrid and Dual-Glo ® Luciferase assays found that BjuJ3 could not interact with BjuAGL24 promoter but could specifically bind to BjuSOC1-1 which is one of truncated fragments of BjuSOC1 promoter. Our research will provide valuable information for unraveling regulatory mechanisms of flowering time in B. juncea., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cytoplasmic male sterility (CMS) is primarily caused by chimeric genes located in the mitochondrial genomes. In Brassica juncea, orf288 has been identified as a CMS-associated gene in the hau CMS line; however, neither the specific abortive stage nor the molecular function of the gene have been determined. We therefore characterized the hau CMS line, and found that defective mitochondria affect the development of archesporial cells during the L2 stage, leading to male sterility. The expression level of the orf288 transcript was higher in the male-sterility line than in the fertility-restorer line, although no significant differences were apparent at the protein level. The toxicity region of ORF288 was found to be located near the N-terminus and repressed growth of Escherichia coli. However, transgenic expression of different portions of ORF288 indicated that the region that causes male sterility resides between amino acids 73 and 288, the expression of which in E. coli did not result in growth inhibition. Transcriptome analysis revealed a wide range of genes involved in anther development and mitochondrial function that were differentially expressed in the hau CMS line. This study provides new insights into the hau CMS mechanism by which orf288 affects the fertility of Brassica juncea., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The study focuses on potential of combined pre-soaking treatment of 24-Epibrassinolide (EBL) and Salicylic acid (SA) in alleviating Pb phytotoxicity in Brassica juncea L. plants. The seeds after treatment with combination of both the hormones were sown in mixture of soil, sand and manure (3:1:1) and were exposed to Pb concentrations (0.25mM, 0.50mM and 0.75mM). After 30 days of growth, the plants were harvested and processed, for quantification of various metabolites. It was found that pre-sowing of seeds in combination of EBL and SA, mitigated the adverse effects of metal stress by modulating antioxidative defense response and enhanced osmolyte contents. Dry matter content and heavy metal tolerance index were enhanced in response to co-application of EBL and SA. The levels of superoxide anions, hydrogen peroxide and malondialdehyde were lowered by the combined treatment of hormones. Enhancement in activities of guaiacol peroxidase, catalase, glutathione reductase and glutathione-s-transferase were recorded. Contents of glutathione, tocopherol and ascorbic acid were also enhanced in response to co-application of both hormones. Expression of POD, CAT, GR and GST1 genes were up-regulated whereas SOD gene was observed to be down-regulated. Contents of proline, trehalose and glycine betaine were also reported to be elevated as a result of treatment with EBL+SA. The results suggest that co-application of EBL+SA may play an imperative role in improving the antioxidative defense expression of B. juncea plants to combat the oxidative stress generated by Pb toxicity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aim of the present study was to explore the effect of exogenous application of castasterone (CS) on physiologic and biochemical responses in Brassica juncea seedlings under copper (Cu) stress. Seeds were pre-soaked in different concentrations of CS and grown for 7 days under various levels of Cu. The exposure of B. juncea to higher levels of Cu led to decrease of morphologic parameters, with partial recovery of length and fresh weight in the CS pre-treated seedlings. Metal content was high in both roots and shoots under Cu exposure while the CS pre-treatment reduced the metal uptake. Accumulation of hydrogen peroxide (H 2 O 2 ) and superoxide anion radical (O 2 - ) were chosen as stress biomarker and higher levels of H 2 O 2 (88.89%) and O 2 - (62.11%) showed the oxidative stress in metal treated B. juncea seedlings, however, CS pre-treatment reduced ROS accumulation in Cu-exposed seedlings. The Cu exposures lead to enhance the plant's enzymatic and non-enzymatic antioxidant system. It was observed that enzymatic activities of ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), glutathione perxoidase (GPOX) and gultrathione-s-transferase increased while activity of monodehydroascorbate reductase (MDHAR) decreased under Cu stress. The pre-treatment with CS positively affected the activities of enzymes. RT-PCR analysis showed that mRNA transcript levels were correlated with total enzymatic activity of DHAR, GR, GST and GSH. Increase in the gene expression of DHAR (1.85 folds), GR (3.24 folds), GST-1 (2.00 folds) and GSH-S (3.18 folds) was noticed with CS pre-treatment. Overall, the present study shows that Cu exposure induced severe oxidative stress in B. juncea plants and exogenous application of CS improved antioxidative defense system by modulating the ascorbate-glutathione cycle and amino acid metabolism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

KEY MESSAGE: oxa CMS is a new cytoplasmic male sterility type in Brassica juncea. oxa CMS is a cytoplasmic male sterility (CMS) line that has been widely used in the production and cultivation of stem mustard in the southwestern China. In this study, different CMS-type specific mitochondrial markers were used to confirm that oxa CMS is distinct from the pol CMS, ogu CMS, nap CMS, hau CMS, tour CMS, Moricandia arvensis CMS, orf220-type CMS, etc., that have been previously reported in Brassica crops. Pollen grains of the oxa CMS line are sterile with a self-fertility rate of almost 0% and the sterility strain rate and sterility degree of oxa CMS is 100% due to a specific flower structure and flowering habit. Scanning electron microscopy revealed that most pollen grains in mature anthers of the oxa CMS line are empty, flat and deflated. Semi-thin section further showed that the abortive stage of anther development in oxa CMS is initiated at the late uninucleate stage. Abnormally vacuolated microspores caused male sterility in the oxa CMS line. This cytological study combined with marker-assisted selection showed that oxa CMS is a novel CMS type in stem mustard (Brassica juncea). Interestingly, the abortive stage of oxa CMS is later than those in other CMS types reported in Brassica crops, and there is no negative effect on the oxa CMS line growth period. This study demonstrated that this novel oxa CMS has a unique flower structure with sterile pollen grains at the late uninucleate stage. Our results may help to uncover the mechanism of oxa CMS in Brassica juncea.

Annexins belong to a plasma membrane binding (in a calcium dependent manner), multi-gene family of proteins, which play ameliorating roles in biotic and abiotic stresses. The expression of annexin AnnBj2 of Indian mustard is tissue specific with higher expression in roots and under treatments with sodium chloride and abscisic acid (ABA) at seedling stage. The effect of constitutive expression of AnnBj2 in mustard was analyzed in detail. AnnBj2 OE (over expression) plants exhibited insensitivity to ABA, glucose and sodium chloride. The insensitivity/tolerance of the transgenic plants was associated with enhanced total chlorophylls, relative water content, proline, calcium and potassium with reduced thiobarbituric acid reactive substances and sodium ion accumulation. The altered ABA insensitivity of AnnBj2 OE lines is linked to downregulation of ABI4 and ABI5 transcription factors and upregulation of ABA catabolic gene CYP707A2. Furthermore, we found that overexpression of AnnBj2 upregulated the expression of ABA-dependent RAB18 and ABA-independent DREB2B stress marker genes suggesting that the tolerance phenotype exhibited by AnnBj2 OE lines is probably controlled by both ABA-dependent and -independent mechanisms., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Flowering plants have evolved diverse mechanisms that promote outcrossing. The most widespread of these outbreeding devices are self-incompatibility systems, the highly selective prefertilization mating barriers that prevent self-fertilization by disrupting pollen-pistil interactions. Despite the advantages of outcrossing, loss of self-incompatibility has occurred repeatedly in many plant families. In the mustard family, the highly polymorphic receptors and ligands that mediate the recognition and inhibition of self-pollen in self-incompatibility have been characterized and the 3D structure of the receptor-ligand complex has been solved. Sequence analyses and empirical studies in self-incompatible and self-compatible species are elucidating the genetic basis of switches from the outcrossing to selfing modes of mating and beginning to provide clues to the diversification of the self recognition repertoire., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A promoter trap mutant line of Arabidopsis carrying a promoterless β-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5' and 3' promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Main Conclusion: Transgenic mustard plants ( Brassica juncea ) expressing non-allergenic and biologically safe RiD peptide show higher tolerance against Lipaphis erysimi. Rorippa indica defensin (RiD) has previously been reported as a novel insecticidal protein derived from a wild crucifer Rorippa indica. RiD was found to have an effective insecticidal property against mustard aphid, Lipaphis erysimi. In the present study, RiD was highly upregulated in R. indica during aphid infestation initiating a defense system mediated by jasmonic acid (JA), but not by salicylic acid (SA)/abscisic acid (ABA). RiD has also been assessed for biosafety according to the FAO/WHO guideline (allergenicity of genetically modified foods; Food And Agriculture Organisation of the United Nations, Rome, Italy, 2001) and Codex Alimentarius Guideline (Guidelines for the design and implementation of national regulatory food safety assurance programme associated with the use of veterinary drugs in food producing animals. Codex Alimentarius Commission. GL, pp 71-2009, 2009). The purified protein was used to sensitize BALB/c mice and they showed normal histopathology of lung and no elevated IgE level in their sera. As the protein was found to be biologically safe and non-allergenic, it was used to develop transgenic Brassica juncea plants with enhanced aphid tolerance, which is one of the most important oilseed crops and is mostly affected by the devastating pest-L. erysimi. The transgene integration was monitored by Southern hybridization, and the positive B. juncea lines were further analyzed by Western blot, ELISA, immunohistolocalization assays and in planta insect bioassay. Transgenic plants expressing RiD conferred a higher level of tolerance against L. erysimi. All these results demonstrated that RiD is a novel, biologically safe, effective insecticidal agent and B. juncea plants expressing RiD are important components of integrated pest management.

Objectives: To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H 2 O 2 , by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR)., Results: Under H 2 O 2 stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H 2 O 2 level decreased. In addition, under H 2 O 2 stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains., Conclusion: By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H 2 O 2 stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

Differential accumulation of plant defence metabolites has been suggested to have important ecological consequence in the context of plant-insect interactions. Feeding of generalist pests on Brassica juncea showed a distinct pattern with selective exclusion of leaf margins which are high in glucosinolates. Molecular basis of this differential accumulation of glucosinolates could be explained based on differential expression profile of BjuMYB28 homologues, the major biosynthetic regulators of aliphatic glucosinolates, as evident from quantitative real-time PCR and promoter:GUS fusion studies in allotetraploid B. juncea. Constitutive overexpression of selected BjuMYB28 homologues enhanced accumulation of aliphatic glucosinolates in B. juncea. Performance of two generalist pests, Helicoverpa armigera and Spodoptera litura larvae, on transgenic B. juncea plants were poor compared to wild-type plants in a no-choice experiment. Correlation coefficient analysis suggested that weight gain of H. armigera larvae was negatively correlated with gluconapin (GNA) and glucobrassicanapin (GBN), whereas that of S. litura larvae was negatively correlated with GNA, GBN and sinigrin (SIN). Our study explains the significance and possible molecular basis of differential distribution of glucosinolates in B. juncea leaves and shows the potential of overexpressing BjuMYB28 for enhanced resistance of Brassica crops against the tested generalist pests., (© 2017 John Wiley & Sons Ltd.)

Linoleic acid (C18:2) is an important polyunsaturated fatty acid in the seed oil of many crops. Here, we report that mutations in the promoter, intron and CDS of the FAD2 genes SalFAD2.LIA1 and SalFAD2.LIA2 generate three alleles LIA 1a , LIA 1b and lia 1 and two alleles LIA 2 and lia 2 , respectively, controlling the C18:2 variation (4.4-32.7%) in yellow mustard. The allelic effect on increasing C18:2 content is LIA 1a  > LIA 1b  > lia 1 , LIA 2  > lia 2 , and LIA 1a  > LIA 2 . The five FAD 2 alleles each contain two exons, one intron and a promoter adjacent to exon 1. LIA 1a has a 1152 bp CDS, a 1221 bp intron with promoter function and a 607 bp promoter. Compared with LIA 1a , the intron of LIA 1b has reduced promoter activity and that of LIA 2 and lia 2 has no promoter function due to extensive SNP and indel mutations. lia 1 differed from LIA 1b by having an insertion of 1223 bp retrotransposon in its intron. lia 2 with mutations in the promoter has reduced promoter activity compared with LIA 2 . This study revealed that complex quantitative variation of trait phenotype in plants could be modulated by multiple alleles of oligogenic loci resulting from mutations in the regulatory region and CDS.

Sinapine is a major anti-nutritive compound that accumulates in the seeds of Brassica species. When ingested, sinapine imparts gritty flavuor in meat and milk of animals and fishy odor to eggs of brown egg layers, thereby compromising the potential use of the valuable protein rich seed meal. Sinapine content in Brassica juncea germplasm ranges from 6.7 to 15.1 mg/g of dry seed weight (DSW) which is significantly higher than the prescribed permissible level of 3.0 mg/g of DSW. Due to limited natural genetic variability, conventional plant breeding approach for reducing the sinapine content has largely been unsuccessful. Hence, transgenic approach for gene silencing was adopted by targeting two genes-SGT and SCT, encoding enzymes UDP- glucose: sinapate glucosyltransferase and sinapoylglucose: choline sinapoyltransferase, respectively, involved in the final two steps of sinapine biosynthetic pathway. These two genes were isolated from B. juncea and eight silencing constructs were developed using three different RNA silencing approaches viz. antisense RNA, RNAi and artificial microRNA. Transgenics in B. juncea were developed following Agrobacterium-mediated transformation. From a total of 1232 independent T0 transgenic events obtained using eight silencing constructs, 25 homozygous lines showing single gene inheritance were identified in the T2 generation. Reduction of seed sinapine content in these lines ranged from 15.8% to 67.2%; the line with maximum reduction had sinapine content of 3.79 mg/g of DSW. The study also revealed that RNAi method was more efficient than the other two methods used in this study.

Sclerotinia stem rot (Sclerotinia sclerotiorum) is a major disease of Brassica oilseeds. As suitable donors to develop resistant cultivars are not available in crop Brassicas, we introgressed resistance from a wild Brassicaceae species, B. fruticulosa. We produced 206 B. juncea-B. fruticulosa introgression lines (ILs). These were assessed for pollen grain fertility, genome size variations and resistance responses to Sclerotinia following stem inoculations under disease-conducive conditions. Of these, 115 ILs showing normal fertility and genome size were selected for cytogenetic characterization using florescent genomic in situ hybridization (Fl-GISH). B. fruticulosa segment substitutions were indicated in 28 ILs. These were predominantly terminal and located on B-genome chromosomes. A final set of 93 highly fertile and euploid (2n = 36) ILs were repeat-evaluated for their resistance responses during 2014-15. They were also genotyped with 202 transferable and 60 candidate gene SSRs. Association mapping allowed detection of ten significant marker trait associations (MTAs) after Bonferroni correction. These were: CNU-m157-2, RA2G05, CNU-m353-3, CNU-m442-5, ACMP00454-2, ACMP00454-3, EIN2-3-1, M641-1, Na10D09-1 and Na10D11-1. This is the first time such a molecular mapping technique has been deployed with introgression lines carrying genomic segments from B. fruticulosa, and the first to show that they possess high levels of resistance against S. sclerotiorum.

Background: In nature, plants are frequently exposed to simultaneous biotic stresses that activate distinct and often antagonistic defense signaling pathways. How plants integrate this information and whether they prioritize one stress over the other is not well understood., Results: We investigated the transcriptome signature of the wild annual crucifer, Brassica nigra, in response to eggs and caterpillars of Pieris brassicae butterflies, Brevicoryne brassicae aphids and the bacterial phytopathogen Xanthomonas campestris pv. raphani (Xcr). Pretreatment with egg extract, aphids, or Xcr had a weak impact on the subsequent transcriptome profile of plants challenged with caterpillars, suggesting that the second stress dominates the transcriptional response. Nevertheless, P. brassicae larval performance was strongly affected by egg extract or Xcr pretreatment and depended on the site where the initial stress was applied. Although egg extract and Xcr pretreatments inhibited insect-induced defense gene expression, suggesting salicylic acid (SA)/jasmonic acid (JA) pathway cross talk, this was not strictly correlated with larval performance., Conclusion: These results emphasize the need to better integrate plant responses at different levels of biological organization and to consider localized effects in order to predict the consequence of multiple stresses on plant resistance.

Brassica juncea is a chief oil yielding crop in many parts of the world including India. With advancement of molecular techniques, RT-qPCR based study of gene-expression has become an integral part of experimentations in crop breeding. In RT-qPCR, use of appropriate reference gene(s) is pivotal. The virtue of the reference genes, being constant in expression throughout the experimental treatments, needs to be validated case by case. Appropriate reference gene(s) for normalization of gene-expression data in B. juncea during the biotic stress of aphid infestation is not known. In the present investigation, 11 reference genes identified from microarray database of Arabidopsis-aphid interaction at a cut off FDR ≤0.1, along with two known reference genes of B. juncea, were analyzed for their expression stability upon aphid infestation. These included 6 frequently used and 5 newly identified reference genes. Ranking orders of the reference genes in terms of expression stability were calculated using advanced statistical approaches such as geNorm, NormFinder, delta Ct and BestKeeper. The analysis suggested CAC, TUA and DUF179 as the most suitable reference genes. Further, normalization of the gene-expression data of STP4 and PR1 by the most and the least stable reference gene, respectively has demonstrated importance and applicability of the recommended reference genes in aphid infested samples of B. juncea., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

As a desirable agricultural trait, multilocular trait of rapeseed (Brassica rapa; Brassica napus; Brassica juncea), always represents higher yield per plant compared with bilocular plants. We previously isolated a trilocular gene locus, Bjmc1, and identified a set of molecular markers linked to the trilocular gene. With a map-based cloning, we identified that the BjMc1 was located in B genome of Brassica juncea, and it was a CLAVATA1 (CLV1) gene homologue. The insertion of a copia-LTR retrotransposable element 1 (RTE1) into the coding region of BjMc1 interrupted its transcription in rapeseed, leading to the trilocular phenotype. Phylogenetic analysis showed that Mc1 genes were conserved and widespread in land plants. Two amino acid sites had undergone positive selection in the ancestor of Mc1 genes, and then purifying selection was the dominant force after the divergence of dicots and monocots from their common ancestor in the evolutionary process, indicating that Mc1 genes are conserved in modern land plants. Our results provided new insights in molecular regulatory mechanism of multilocularity in rapeseed, and better understanding of molecular mechanism in crop yield improvement.

LEAFY plays a central role in regulation of flowering time and floral meristem identity in plants. Unfortunately, LFY function remains uncharacterized in agronomicaly important Brassicas. Herein, we illustrate fine-mapping of expression domains of LFY in 15 cultivars of 6 Brassica species and describe gain-of-function phenotypes in Arabidopsis and Brassica. We depict early flowering and altered fatty-acid composition in transgenic seed. The cDNA encoding BjuLFY (417aa) shared only 85% identity with reported homolog of B.juncea implying distinctness. Quantitative RT-PCR based coarse expression mapping of BjuLFY in tissue samples representing 3 time points at specific days after sowing (DAS), pre-flowering (30 DAS), flowering (75 DAS) and post-flowering (110 DAS), depicted an intense pulse of BjuLFY expression restricted to primary floral buds (75 DAS) which subsided in secondary floral buds (110 DAS); expression in root samples was also recorded implying neo-functionalization. Fine-mapping of expression during flowering confirmed tightly regulated LFY expression during early stages of bud development in 15 cultivars of 6 Brassica species implying functional conservation. Ectopic expression of BjuLFY in A. thaliana and B. juncea caused floral meristem defects and precocious flowering. B. juncea transgenics (T 1 ) over-expressing BjuLFY flowered 20days earlier produced normal flowers. GC-MS analysis of mature seed from Brassica transgenics showed an altered fatty-acid profile suggestive of seed maturation occurring at lower temperatures vis-à-vis control. Our findings implicate BjuLFY as a regulator of flowering in B. juncea and suggest its application in developing climate resilient crops., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Activation of stress tolerance mechanisms demands transcriptional reprogramming. Salt stress, a major threat to plant growth, enhances ROS production and affects transcription through modulation of miRNAs and hormones. The present study delineates salt stress ameliorating action of thiourea (TU, a ROS scavenger) in Brassica juncea and provides mechanistic link between redox, microRNA and hormones. The ameliorative potential of TU towards NaCl stress was related with its ability to decrease ROS accumulation in roots and increase Na + accumulation in shoots. Small RNA sequencing revealed enrichment of down-regulated miRNAs in NaCl + TU treated roots, indicating transcriptional activation. Ranking analysis identified three key genes including BRX4, CBL10 and PHO1, showing inverse relationship with corresponding miRNA expression, which were responsible for TU mediated stress mitigation. Additionally, ABA level was consistently higher till 24 h in NaCl, while NaCl + TU treated roots showed only transient increase at 4 h suggesting an effective stress management. Jasmonate and auxin levels were also increased, which prioritized defence and facilitated root growth, respectively. Thus, the study highlights redox as one of the "core" components regulating miRNA and hormone levels, and also strengthens the use of TU as a redox priming agent for imparting crop resilience to salt stress.

Background: Pesticides cause oxidative stress to plants and their residues persist in plant parts, which are a major concern for the environment as well as human health. Brassinosteroids (BRs) are known to protect plants from abiotic stress conditions including pesticide toxicity. The present study demonstrated the effects of seed-soaking with 24-epibrassinolide (EBR) on physiological responses of 10-day old Brassica juncea seedlings grown under imidacloprid (IMI) toxicity., Results: In the seedlings raised from EBR-treated seeds and grown under IMI toxicity, the contents of hydrogen peroxide (H 2 O 2 ) and superoxide anion (O . 2 - ) were decreased, accompanied by enhanced activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST), guaiacol peroxidase (POD) and the content of glutathione (GSH). As compared to controls, the gene expressions of SOD, CAT, GR, POD, NADH (NADH-ubiquinone oxidoreductase), CXE (carboxylesterase), GSH-S (glutathione synthase), GSH-T (glutathione transporter-1), P450 (cytochrome P450 monooxygenase) and GST1-3,5-6 were enhanced in the seedlings raised from EBR-treated seeds and grown in IMI supplemented substratum. However, expression of RBO (respiratory burst oxidase, the gene responsible for H 2 O 2 production) was decreased in seedlings raised from EBR treated seeds and grown under IMI toxicity. Further, the EBR seed treatment decreased IMI residues by more than 38% in B. juncea seedlings., Conclusions: The present study revealed that EBR seed soaking can efficiently reduce oxidative stress and IMI residues by modulating the gene expression of B. juncea under IMI stress. In conclusion, exogenous EBR application can protect plants from pesticide phytotoxicity.

Key Message: Seed weight QTL identified in different populations were synthesized into consensus QTL which were shown to harbor candidate genes by in silico mapping. Allelic variation inferred would be useful in breeding B. juncea lines with high seed weight. Seed weight is an important yield influencing trait in oilseed Brassicas and is a multigenic trait. Among the oilseed Brassicas, Brassica juncea harbors the maximum phenotypic variation wherein thousand seed weight varies from around 2.0 g to more than 7.0 g. In this study, we have undertaken quantitative trait locus/quantitative trait loci (QTL) analysis of seed weight in B. juncea using four bi-parental doubled-haploid populations. These four populations were derived from six lines (three Indian and three east European lines) with parental phenotypic values for thousand seed weight ranging from 2.0 to 7.6 g in different environments. Multi-environment QTL analysis of the four populations identified a total of 65 QTL ranging from 10 to 25 in each population. Meta-analysis of these component QTL of the four populations identified six 'consensus' QTL (C-QTL) in A3, A7, A10 and B3 by merging 33 of the 65 component Tsw QTL from different bi-parental populations. Allelic diversity analysis of these six C-QTL showed that Indian lines, Pusajaikisan and Varuna, hold the most positive allele in all the six C-QTL. In silico mapping of candidate genes with the consensus QTL localized 11 genes known to influence seed weight in Arabidopsis thaliana and also showed conserved crucifer blocks harboring seed weight QTL between the A subgenomes of B. juncea and B. rapa. These findings pave the way for a better understanding of the genetics of seed weight in the oilseed crop B. juncea and reveal the scope available for improvement of seed weight through marker-assisted breeding.

Whole-genome shotgun reads were analyzed to determine the repeat sequence composition in the genome of black mustard, Brassica nigra (L.) Koch. The analysis showed that satellite DNA sequences are very abundant in the black mustard genome. The distribution pattern of 7 new tandem repeats (BnSAT13, BnSAT28, BnSAT68, BnSAT76, BnSAT114, BnSAT180, and BnSAT200) on black mustard chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of BnSAT13 and BnSAT76 provided useful cytogenetic markers; their position and fluorescence intensity allowed for unambiguous identification of all 8 somatic metaphase chromosomes. A karyotype showing the location and fluorescence intensity of these tandem repeat sequences together with the position of rDNAs and centromeric retrotransposons of Brassica (CRB) was constructed. The establishment of the FISH-based karyotype in B. nigra provides valuable information that can be used in detailed analyses of B. nigra accessions and derived allopolyploid Brassica species containing the B genome., (© 2017 S. Karger AG, Basel.)

Key Message: Under severe drought conditions, Brassica juncea shows differential methylation and demethylation events, such that certain epialleles are silenced and some are activated. The plant employed avoidance strategy by delaying apoptosis through the activation of several genes. Harsh environmental conditions pose serious threat to normal growth and development of crops, sometimes leading to their death. However, plants have developed an essential mechanism of modulation of gene activities by epigenetic modifications. Brassica juncea is an important oilseed crop contributing effectively to the economy of India. In the present investigation, we studied the changes in the methylation level of various stress-responsive genes of B. juncea variety RH30 by methylation-dependent immune-precipitation-chip in response to severe drought. On the basis of changes in the number of differential methylation regions in response to drought, the promoter regions were designated as hypermethylated and hypomethylated. Gene body methylation increased in all the genes, whereas promoter methylation was dependent on the function of the gene. Overall, the genes responsible for delaying apoptosis were hypomethylated and many genes responsible for normal routine activities were hypermethylated at promoter regions, thereby suggesting that these may be suspending the activities under harsh conditions.

Plants have evolved adaptive mechanisms that allow them to tolerate a continuous range of abiotic and biotic stressors. Tropospheric ozone (O 3 ), a global anthropogenic pollutant, directly affects living organisms and ecosystems, including plant-herbivore interactions. In this study, we investigate the stress responses of Brassica nigra (wild black mustard) exposed consecutively to O 3 and the specialist herbivore Pieris brassicae Transcriptomics and metabolomics data were evaluated using multivariate, correlation, and network analyses for the O 3 and herbivory responses. O 3 stress symptoms resembled those of senescence and phosphate starvation, while a sequential shift from O 3 to herbivory induced characteristic plant defense responses, including a decrease in central metabolism, induction of the jasmonic acid/ethylene pathways, and emission of volatiles. Omics network and pathway analyses predicted a link between glycerol and central energy metabolism that influences the osmotic stress response and stomatal closure. Further physiological measurements confirmed that while O 3 stress inhibited photosynthesis and carbon assimilation, sequential herbivory counteracted the initial responses induced by O 3 , resulting in a phenotype similar to that observed after herbivory alone. This study clarifies the consequences of multiple stress interactions on a plant metabolic system and also illustrates how omics data can be integrated to generate new hypotheses in ecology and plant physiology., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Brassica juncea L. is the second most important edible oil seed crop of India. In the current study, we report the complete chloroplast genome sequence of Indian mustard, Brassica juncea L. The size of the complete chloroplast genome was found to be 153 483 bp long with an overall GC content of 36.36%. A large single copy (LSC) region of 83 286 bp and a short single copy region (SSC) region of 17 775 bp were separated by a pair of inverted repeat (IR) regions of 26 211 bp. A total of 113 unique genes were annotated, which includes 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Either one or two introns were present in nine protein-coding genes and six tRNA genes. Phylogenetic analysis with the complete chloroplast genomes of other related species revealed that B. juncea is much closer to Brassica rapa.