The introgression of BjMYB113 from Brassica juncea leads to purple leaf trait in Brassica napus.

The purple leaves of Brassica napus are abundant in anthocyanins, which are renowned for their role in conferring distinct colors, stress tolerance, and health benefits, however the genetic basis of this trait in B. napus remains largely unelucidated. Herein, the purple leaf B. napus (PL) exhibited purple pigments in the upper epidermis and a substantial increase in anthocyanin accumulation, particularly of cyanidin, compared to green leaf B. napus (GL). The genetic control of the purple leaf trait was attributed to a semi-dominant gene, pl, which was mapped to the end of chromosome A03. However, sequencing of the fragments amplified by the markers linked to pl indicated that they were all mapped to chromosome B05 from B. juncea. Within this B05 chromosomal segment, the BjMYB113 gene-specific marker showed perfect co-segregation with the purple leaf trait in the F2 population, suggesting that the BjMYB113 introgression from B. juncea was the candidate gene for the purple leaf trait in B. napus. To further verify the function of candidate gene, CRISPR/Cas9 was performed to knock out the BjMYB113 gene in PL. The three myb113 mutants exhibited evident green leaf phenotype, absence of purple pigments in the adaxial epidermis, and a significantly reduced accumulation of anthocyanin compared to PL. Additionally, the genes involved in positive regulatory (TT8), late anthocyanin biosynthesis (DFR, ANS, UFGT), as well as transport genes (TT19) were significantly suppressed in the myb113 mutants, further confirming that BjMYB113 was response for the anthocyanin accumulation in purple leaf B. napus. This study contributes to an advanced understanding of the regulation mechanism of anthocyanin accumulation in B. napus.

Turning glucosinolate into allelopathic fate: investigating allyl isothiocyanate variability and nitrile formation in eco-friendly Brassica juncea from South Korea.

Leaf mustard (Brassica juncea L.) is explored for its biofumigant properties, derived from its secondary metabolites, particularly allyl isothiocyanate (AITC), produced during the enzymatic breakdown of glucosinolates like sinigrin. The research examines eight leaf mustard cultivars developed in Yeosu city, South Korea, focusing on their genetic characteristics, AITC concentration and nitriles formation rates from glucosinolates. Results indicate that the allelopathic effects, largely dependent on AITC concentration and enzymatic activity, vary across cultivar. Sinigrin and AITC constitute 79% and 36%, respectively, of glucosinolate and its hydrolysis products. The cultivar 'Nuttongii' demonstrates significant potential for inhibiting weeds, exhibiting the highest AITC concentration at 27.47 ± 6.46 µmole g-1 These outcomes highlight the importance of selecting mustard cultivars for biofumigation based on their glucosinolate profiles and hydrolysis product yields. The study also identifies a significant genetic influence on AITC and nitrile formation, suggesting that epithiospecifier protein modulation could enhance both allelopathic and other beneficial effects. Collectively, the research underscores the promise of mustard as a sustainable, environmentally friendly alternative to traditional herbicides.

Exploring the role of 28-homobrassinolide in regulation of temperature induced clastogenic aberrations and sugar metabolism of Brassica juncea L.

The present research primarily focuses on Brassica juncea's physiological and cytological responses to low and high temperature stress at 4 °C and 44 °C respectively, along with elucidating the protective role of 28-Homobrassinolide (28-homoBL). Cytological investigations performed in floral buds of Brassica juncea L. under temperature (24, 4, 44 °C) stress conditions depict the presence of some abnormalities associated with cytomixis such as chromosome stickiness or agglutination, pycnotic nature of chromatin, irregularities in spindle formation, disoriented chromatins, and non-synchronous chromatin material condensation in Brassicaceae family that subsisted at diploid level (2n = 36). Spindle abnormalities produce various size pollen grains such as sporads micronuclei at some stages of microsporogenesis, polyads, triads, dyads that irrupted the productiveness of pollen grains. Furthermore, sugars play an imperative role in protecting plants under stress besides being energy sources. Therefore, the present study revealed accumulation of total soluble sugars (TSS), with 28-homoBL treatment which pinpoints protective role of 28-homoBL under temperature stress. Sugar profiling was done by using high-performance liquid chromatography (HPLC) which helped in analyzing different sugars both quantitatively and qualitatively under 28-homoBL and temperature stress conditions. The results indicate that the 28-homoBL treatment substantially enhances plant tolerance to heat stress, as evident by higher mitotic indices, fewer chromosomal abnormalities, and significantly more sugar accumulation. The findings of the study acknowledge the potential of 28-homoBL in inducing temperature stress tolerance in B. juncea along with improving the metabolic stability thereby implying application of 28-homoBL in crop strengthening under variable temperature conditions.

Integrated transcriptomic and physio-molecular studies unveil the melatonin and PGPR induced protection to photosynthetic attributes in Brassica juncea L. under cadmium toxicity.

Cd is highly mobile, non-essential trace element, that has become serious environmental issue due to its elevated concentration in soil. The present study was taken up to work out salutary effect of melatonin (Mlt) and PGPR ((Pseudomonas putida (Pp), Pseudomonas fluorescens (Pf) in 10 days old Cd stressed (0.3 mM) Brassica juncea L. seedlings. The present work investigated growth characteristics, photosynthetic pigments, secondary metabolites in melatonin-PGPR inoculated B. juncea seedlings. It was backed by molecular studies entailing RT-PCR and transcriptomic analyses. Our results revealed, substantial increase in photosynthetic pigments and secondary metabolites, after treatment with melatonin, P.putida, P. fluorescens in Cd stressed B. juncea seedlings, further validated with transcriptome analysis. Comparative transcriptome analyses identified 455, 5953, 3368, 2238 upregulated and 4921, 430, 137, 27 down regulated DEGs, Cn-vs-Cd, Cd-vs-Mlt, Cd-vs-Mlt-Pp-Pf, Cd-vs-Mlt-Pp-Pf-Cd comparative groups respectively. In depth exploration of genome analyses (Gene ontology, Kyoto encyclopaedia of genes), revealed that Cd modifies the expression patterns of most DEGs mainly associated to photosystem and chlorophyll synthesis. Also, gene expression studies for key photosynthetic genes (psb A, psb B, CHS, PAL, and PSY) suggested enhanced expression in melatonin-rhizobacteria treated Cd stressed B. juncea seedlings. Overall, results provide new insights into probable mechanism of Mlt-PGPR induced protection to photosynthesis in Cd stressed B. juncea plants.

Lead toxicity regulation via protein degradation and tetrapyrrole biosynthesis pathways in Brassica species: A comparative quantitative analysis of proteomic study.

Understanding the heavy metals (HMs) tolerance mechanism is crucial for improving plant growth in metal-contaminated soil. In order to evaluate the lead (Pb) tolerance mechanism in Brassica species, a comparative proteomic study was used. Thirteen-day-old seedlings of B. juncea and B. napus were treated with different Pb(NO3)2 concentrations at 0, 3, 30, and 300 mg/L. Under 300 mg/L Pb(NO3)2 concentration, B. napus growth was significantly decreased, while B. juncea maintained normal growth similar to the control. The Pb accumulation was also higher in B. napus root and shoot compared to B. juncea. Gel-free proteomic analysis of roots revealed a total of 68 and 37 differentially abundant proteins (DAPs) in B. juncea and B. napus-specifically, after 300 mg/L Pb exposure. The majority of these proteins are associated with protein degradation, cellular respiration, and enzyme classification. The upregulated RPT2 and tetrapyrrole biosynthesis pathway-associated proteins maintain the cellular homeostasis and photosynthetic rate in B. juncea. Among the 55 common DAPs, S-adenosyl methionine and TCA cycle proteins were upregulated in B. juncea and down-regulated in B. napus after Pb exposure. Furthermore, higher oxidative stress also reduced the antioxidant enzyme activity in B. napus. The current finding suggests that B. juncea is more Pb tolerant than B. napus, possibly due to the upregulation of proteins involved in protein recycling, degradation, and tetrapyrrole biosynthesis pathway.

Physicochemical properties and fractal characterizations of the functionalized porous clinoptilolites for controlling alginate delivery in growing cauliflower and leaf mustard.

Using natural clinoptilolite (NCP) as a carrier and alginate (Alg)-calcium as an active species, the porous silicon calcium alginate nanocomposite (Alg-Ca-NCP) was successfully fabricated via adsorption-covalence-hydrogen bond. Its structural features and physicochemical properties were detailed investigated by various characterizations. The results indicated that Alg-Ca-NCP presented the disordered lamellar structures with approximately uniform particles in size of 300-500 nm. Specially, their surface fractal evolutions between the irregular roughness and dense structures were demonstrated via the SAXS patterns. The results elucidated that the abundant micropores of NCP were beneficial for unrestricted diffusing of Alg-Ca, which was conducive to facilitate a higher loading and sustainable releasing. The Ca content of leaf mustard treated with Alg-Ca-NCP-0.5 was 484.5 mg/100g on the 21st day, higher than that by water (CK) and CaCl2 solution treatments, respectively. Meanwhile, the prepared Alg-Ca-NCPs presented the obvious anti-aging effects on peroxidase drought stress of mustard leaves. These demonstrations provided a simple and effective method to synthesize Alg-Ca-NCPs as delivery nanocomposites, which is useful to improve the weak absorption and low utilization of calcium alginate by plants.

Applicability of Brassica juncea as a bioindicator for As contamination in soil near the abandoned mine area.

Soil monitoring in abandoned mine areas is important from the perspective of ecological and human health risk. Arsenic (As) is a predominant metalloid contaminant in abandoned mine area and its behavior has been influenced by various soil characteristics. Bioindicator can be a useful tool in terms of testing the extent to which they are uptaken by plants bioavailability. Eighteen soils near the mine tailings dam were collected to investigate the effect of As contamination on As absorption by Brassica juncea. The pH range of the experimental soils was between 4.90 and 8.55, and the total As concentrations were between 34 mg kg-1 and 3017 mg kg-1. The bioavailability of As was evaluated by Olsen method, and B. juncea was cultivated in eighteen soils for 3 weeks. Principal component analysis, correlation, and multiple regression analysis were performed to estimate a significant factor affecting As uptake by B. juncea. All statistical results indicated that As bioavailability in soil is the main factor affecting As uptake in root and shoot of B. juncea. Although translocation process, the amount of As in shoot was exponentially explained by As bioavailability in soil. This result suggests that the contamination and bioavailability of As can be confirmed only by analyzing the shoot of B. juncea, which is be easily found in environmental ecosystem, and implies the applicability of B. juncea as a bioindicator for the monitoring of As contamination and its behavior in soil ecosystem.

Zinc oxide nanoparticles and Klebsiella sp. SBP-8 alleviates chromium toxicity in Brassica juncea by regulation of antioxidant capacity, osmolyte production, nutritional content and reduction in chromium adsorption.

Heavy metals are one of the most damaging environmental toxins that hamper growth of plants. These noxious chemicals include lead (Pb), arsenic (As), nickel (Ni), cadmium (Cd) and chromium (Cr). Chromium is one of the toxic metal which induces various oxidative processes in plants. The emerging role of nanoparticles as pesticides, fertilizers and growth regulators have attracted the attention of various scientists. Current study was conducted to explore the potential of zinc oxide nanoparticles (ZnONPs) alone and in combination with plant growth promoting rhizobacteria (PGPR) Klebsiella sp. SBP-8 in Cr stress alleviation in Brassica juncea (L.). Chromium stress reduced shoot fresh weight (40%), root fresh weight (28%), shoot dry weight (28%) and root dry weight (34%) in B. juncea seedlings. Chromium stressed B. juncea plants showed enhanced levels of malondialdehyde (MDA), electrolyte leakage (EL), hydrogen peroxide (H2O2) and superoxide ion (O2• -). However, co-supplementation of ZnONPs and Klebsiella sp. SBP-8 escalated the activity of antioxidant enzymes i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) in B. juncea grown in normal and Cr-toxic soil. It is further proposed that combined treatment of ZnONPs and Klebsiella sp. SBP-8 may be useful for alleviation of other abiotic stresses in plants.

[Brassica juncea BjuWRKY71-1 accelerates flowering by regulating the expression of SOC1].

Brassica juncea (mustard) is a vegetable crop of Brassica, which is widely planted in China. The yield and quality of stem mustard are greatly influenced by the transition from vegetative growth to reproductive growth, i.e., flowering. The WRKY transcription factor family is ubiquitous in higher plants, and its members are involved in the regulation of many growth and development processes, including biological/abiotic stress responses and flowering regulation. WRKY71 is an important member of the WRKY family. However, its function and mechanism in mustard have not been reported. In this study, the BjuWRKY71-1 gene was cloned from B. juncea. Bioinformatics analysis and phylogenetic tree analysis showed that the protein encoded by BjuWRKY71-1 has a conserved WRKY domain, belonging to class Ⅱ WRKY protein, which is closely related to BraWRKY71-1 in Brassica rapa. The expression abundance of BjuWRKY71-1 in leaves and flowers was significantly higher than that in roots and stems, and the expression level increased gradually along with plant development. The result of subcellular localization showed that BjuWRKY71-1 protein was located in nucleus. The flowering time of overexpressing BjuWRKY71-1 Arabidopsis plants was significantly earlier than that of the wild type. Yeast two-hybrid assay and dual-luciferase reporter assay showed that BjuWRKY71-1 interacted with the promoter of the flowering integrator BjuSOC1 and promoted the expression of its downstream genes. In conclusion, BjuWRKY71-1 protein can directly target BjuSOC1 to promote plant flowering. This discovery may facilitate further clarifying the molecular mechanism of BjuWRKY71-1 in flowering time control, and creating new germplasm with bolting and flowering tolerance in mustard.

The correlation between flavor formation and microbial community dynamics during the fermentation of zha cai.

BACKGROUND: Zha cai, a pickled vegetable with unique flavors, is produced by fermenting fresh mustard tubers. In this study, the main physicochemical indices and volatile flavor compounds were determined in three fermentation periods. The bacterial and fungal communities in the three fermentation periods of zha cai were also monitored using high-throughput sequencing. Key microbial communities were identified based on significant correlations with flavor substances. RESULTS: Firmicutes and Proteobacteria were the main bacterial phyla found within the three fermentation periods. Lactic acid bacteria, namely Lactobacillus, was the predominant bacteria found at the genus level. Ascomycetes and Stenotrophomonas were the major fungal phyla found in the three fermentation periods. Yeast, namely Debaryomyces, was the predominant fungus found at the genus level. A total of 42 bacterial genera were negatively correlated with volatile flavor substances of zha cai, and 37 bacterial genera were positively correlated. Meanwhile, a total of 47 genera of fungi were negatively correlated with the volatile flavor substances of zha cai, while 50 genera were positively correlated. Several microbial genera were significantly correlated with volatile flavor compounds, including Lactobacillus, Halomonas, Rhodococcus, and Debaryomyces. CONCLUSION: This study identified the microbial classes that positively regulate the flavor of zha cai which could provide valuable help for flavor modulation in zha cai production. © 2024 Society of Chemical Industry.

The Mechanism of the Anti-Obesity Effects of a Standardized Brassica juncea Extract in 3T3-L1 Preadipocytes and High-Fat Diet-Induced Obese C57BL/6J Mice.

Obesity is a global health concern. Recent research has suggested that the development of anti-obesity ingredients and functional foods should focus on natural products without side effects. We examined the effectiveness and underlying mechanisms of Brassica juncea extract (BJE) in combating obesity via experiments conducted in both in vitro and in vivo obesity models. In in vitro experiments conducted in a controlled environment, the application of BJE demonstrated the ability to suppress the accumulation of lipids induced by MDI in 3T3-L1 adipocytes. Additionally, it downregulated adipogenic-related proteins peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-α (C/EBP-α), adipocyte protein 2 (aP2), and lipid synthesis-related protein acetyl-CoA carboxylase (ACC). It also upregulated the heat generation protein peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and fatty acid oxidation protein carnitine palmitoyltransferase-1 (CPT-1). The oral administration of BJE decreased body weight, alleviated liver damage, and inhibited the accumulation of lipids in mice with diet-induced obesity resulting from a high-fat diet. The inhibition of lipid accumulation by BJE in vivo was associated with a decreased expression of adipogenic and lipid synthesis proteins and an increased expression of heat generation and fatty acid oxidation proteins. BJE administration improved obesity by decreasing adipogenesis and activating heat generation and fatty acid oxidation in 3T3-L1 cells and in HFD-induced obese C57BL/6J mice. These results suggest that BJE shows potential as a natural method for preventing metabolic diseases associated with obesity.

Differential S-nitrosylation and characterization of purified S-nitrosoglutathione reductase (GSNOR) from Brassica juncea shows multiple forms of the enzyme.

S-nitrosoglutathione reductase (GSNOR). a master regulator of NO homeostasis, is a single-copy gene in most plants. In Lotus japonicus, two GSNOR isoforms were identified exhibiting similar kinetic properties but differential tissue-specific expressions. Previously, a genome-wide identification in Brassica juncea revealed four copies of GSNOR, each encoding proteins that vary in subunit molecular weights and pI. Here, we report multiple forms of GSNOR using 2D immunoblot which showed 4 immunopositive spots of 41.5 kDa (pl 5.79 and 6.78) and 43 kDa (pl 6.16 and 6.23). To confirm, purification of GSNOR using anion-exchange chromatography yielded 2 distinct pools (GSNOR-A & GSNOR-B) with GSNOR activities. Subsequently, affinity-based purification resulted in 1 polypeptide from GSNOR-A and 2 polypeptides from GSNOR-B. Size exclusion-HPLC confirmed 3 GSNORs with molecular weight of 87.48 ± 2.74 KDa (GSNOR-A); 87.36 ± 3.25 and 82.74 ± 2.75 kDa (GSNOR-B). Kinetic analysis showed Km of 118 ± 11 µM and Vmax of 287 ± 22 nkat/mg for GSNOR-A, whereas Km of 96.4 ± 8 µM and Vmax of 349 ± 15 nkat/mg for GSNOR-B. S-nitrosylation and inhibition by NO showed redox regulation of all BjGSNORs. Both purified GSNORs exhibited variable denitrosylation efficiency as depicted by Biotin Switch assay. To the best of our knowledge, this is the first report confirming multiple isoforms of GSNOR in B. juncea.

An enhanced productivity of pink oyster mushroom with improved nutritional profile, characterization and attempt for commercial exploitation.

BACKGROUND: An attempt has been made to explore the nutritional profile of pink oyster mushrooms that have been grown in various agricultural residues, including sugarcane bagasse, rice straw, coconut coir and sawdust, along with other nutrient supplements such as defatted mustard and chickpea powder, for appropriate growth and fruiting body formation in a short span of time. The spawn production was experimented with five different grain varieties. The study became interesting when the observations differed slightly from the traditional practices, with the addition of defatted mustard supplements resulting in a positive correlation with respect to reducing the fruiting time, as well as improving yield and the nutritional profile of Pleurotus djamor. RESULTS: An elevated yield of 651.93 g kg-1 was recorded in the medium where the RS and DM were used in the ratio of 1:0.01 (rice straw +1% w/w defatted mustard) bag, whereas, in terms of protein content, a maximum yield of 32.57 ± 0.79 mg g-1 was observed when SB:DM was in the same ratio (sugarcane bagasse +1% w/w defatted mustard) bag. CONCLUSION: To confer the best outcomes from the screened substrates, a series of experiments were performed by varying the concentration of RS and SB, with 1% w/w DM. It is worth noting that the highest protein content of 32.76 ± 0.38 mg g-1 was obtained along with the total yield of 702.56 ± 2.9 g kg-1 of mushroom when the ratio of RS:SB was 0.7:0.3. © 2024 Society of Chemical Industry.

Rhizobacteria Bacillus spp. mitigate osmotic stress and improve seed germination in mustard by regulating osmolyte and plant hormone signaling.

Drought, a widespread abiotic stressor, exerts a profound impact on agriculture, impeding germination and plant growth, and reducing crop yields. In the present investigation, the osmotolerant rhizobacteria Bacillus casamancensis strain MKS-6 and Bacillus sp. strain MRD-17 were assessed for their effects on molecular processes involved in mustard germination under osmotic stress conditions. Enhancement in germination was evidenced by improved germination percentages, plumule and radicle lengths, and seedling vigor upon rhizobacterial inoculation under no stress and osmotic stress conditions. Under osmotic stress, rhizobacteria stimulated the production of gibberellins and reserve hydrolytic enzymes (lipases, isocitrate lyase, and malate synthase), bolstering germination. Furthermore, these rhizobacteria influenced the plant hormones such as gibberellins and abscisic acid (ABA), as well as signalling pathways, thereby promoting germination under osmotic stress. Reduced proline and glycine betaine accumulation, and down-regulation of transcription factors BjDREB1_2 and BjDREB2 (linked to ABA-independent signalling) in rhizobacteria-inoculated seedlings indicated that bacterial treatment mitigated water deficit stress during germination, independently of these pathways. Hence, the advantageous attributes exhibited by these rhizobacterial strains can be effectively harnessed to alleviate drought-induced stress in mustard crops, potentially through the development of targeted bio-formulations.

Phytoremediation of cadmium-trichlorfon co-contaminated water by Indian mustard (Brassica juncea): growth and physiological responses.

In this study, the morphological and physiological responses of Brassica juncea to the stresses of Cadmium (Cd) and trichlorfon (TCF), and the phytoremediation potential of B. juncea to Cd and TCF were investigated under hydroponics. Results showed that Cd exhibited strong inhibition on biomass and root morphology of B. juncea as Cd concentration increased. The chlorophyll a fluorescence intensity and chlorophyll content of B. juncea decreased with the increased Cd concentration, whereas the malondialdehyde and soluble protein contents and superoxide dismutase activity increased. TCF with different concentrations showed no significant influence on these morphological and physiological features of B. juncea. The biomass and physiological status of B. juncea were predominantly regulated by Cd level under the co-exposure of Cd and TCF. B. juncea could accumulate Cd in different plant parts, as well as showed efficient TCF degradation performance. A mutual inhibitory removal of Cd and TCF was observed under their co-system. The present study clearly signified the physiological responses and phytoremediation potential of B. juncea toward Cd and TCF, and these results suggest that B. juncea can be used as an effective phytoremediation agent for the Cd-TCF co-contamination in water.

Combined pollution of heavy metals and pesticides in agricultural water systems is a common phenomenon. In previous phytoremediation studies, limited information is available on the co-contamination of heavy metals and pesticides. In this study, we aimed to investigate the concentration-dependent morphological and physiological characteristics of B. juncea under single and co-stress of Cd and trichlorfon (TCF), and the phytoremediation ability of B. juncea to remove Cd and TCF through hydroponic experiment. B. juncea exhibited efficient removal performance of Cd and TCF alone and simultaneous exposure of both pollutants, indicating that B. juncea is an effective phytoremediation agent for the Cd-TCF co-contaminated water.

Identification of nitric oxide regulated low abundant myrosinases from seeds and seedlings of Brassica juncea.

Myrosinases constitute an important component of the glucosinolate-myrosinase system responsible for interaction of plants with microorganisms, insects, pest, and herbivores. It is a distinctive feature of Brassicales. Multiple isozymes of myrosinases are present in the vacuoles. Active myrosinases are also present in the apoplast and the nucleus however, the similarity or difference in the biochemical properties with the vacuolar myrosinases are not known. Here, we have attempted to isolate, characterize, and identify myrosinases from seeds, seedlings, apoplast, and nucleus to understand these forms. 2D-CN/SDS-PAGE coupled with western blotting and MS have shown low abundant myrosinases (65/70/72/75 kDa) in seeds and seedlings and apoplast & nucleus of seedlings to exist as dimers, oligomers, and as protein complex. Nuclear membrane associated form of myrosinase was also identified. The present study for the first time has shown enzymatically active myrosinase-alpha-mannosidase complex in seedlings. Both 65 and 70 kDa myrosinase in seedlings were S-nitrosated. Nitric oxide donor treatment (GSNO) led to 25% reduction in myrosinase activity which was reversed by DTT suggesting redox regulation of myrosinase. These S-nitrosated myrosinases might be a component of NO signalling in B. juncea.

Target gene mutations endowed cross-resistance to acetolactate synthase-inhibiting herbicides in wild Brassica juncea.

Wild Brassica juncea is a troublesome weed that infests wheat fields in China. Two suspected wild B. juncea populations (19-5 and 19-6) resistant to acetolactate synthase (ALS) inhibitors were collected from wheat fields in China. To clarify their resistance profiles and resistance mechanism, the resistance levels of populations 19-5 and 19-6 to ALS-inhibiting herbicides and their underlying target-site resistance mechanism were investigated. The results showed that the 19-5 population exhibited resistance to tribenuron-methyl, pyrithiobac­sodium and florasulam, while the 19-6 population was resistant to tribenuron-methyl, pyrithiobac­sodium, imazethapyr and florasulam. Using the homologous cloning method, two ALS genes were identified in wild B. juncea, with one gene (ALS1) encoding 652 amino acids and the other (ALS2) encoding 655 amino acids. Pro-197-Arg mutation on ALS2 and Trp-574-Leu mutation on ALS1, together with the combination of these two mutations in a single plant, were observed in both 19-5 and 19-6 populations. ALS2 enzymes carrying the Pro-197-Arg mutation were cross-resistant to tribenuron-methyl, pyrithiobac­sodium, imazerthapyr and florasulam, with resistance index (RI) values of 6.23, 32.81, 7.97 and 1162.50, respectively. Similarly, ALS1 enzymes with Trp-574-leu substitutions also displayed high resistance to these four herbicides (RI values ranging from 132.61 to 3375.00). In addition, the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations increased the resistance level of the ALS enzyme to ALS inhibitors, with its RI values 3.83-214.19, 6.88-37.34, 1.91-31.82 and 2.03-5.90-fold higher than a single mutation for tribenuron-methyl, pyrithiobac­sodium, imazerthapyr and florasulam, respectively. Collectively, Pro-197-Arg mutation on ALS2, Trp-574-Leu mutation on ALS1 and the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations in wild B. juncea could endow broad-spectrum resistance to ALS inhibitors, which might provide guides for establishing effective strategies to prevent or delay such resistance evolution in this weed.

Transcriptome Dynamics of Brassica juncea Leaves in Response to Omnivorous Beet Armyworm (Spodoptera exigua, Hübner).

Cruciferous plants manufacture glucosinolates (GSLs) as special and important defense compounds against insects. However, how insect feeding induces glucosinolates in Brassica to mediate insect resistance, and how plants regulate the strength of anti-insect defense response during insect feeding, remains unclear. Here, mustard (Brassica juncea), a widely cultivated Brassica plant, and beet armyworm (Spodoptera exigua), an economically important polyphagous pest of many crops, were used to analyze the changes in GSLs and transcriptome of Brassica during insect feeding, thereby revealing the plant-insect interaction in Brassica plants. The results showed that the content of GSLs began to significantly increase after 48 h of herbivory by S. exigua, with sinigrin as the main component. Transcriptome analysis showed that a total of 8940 DEGs were identified in mustard challenged with beet armyworm larvae. The functional enrichment results revealed that the pathways related to the biosynthesis of glucosinolate and jasmonic acid were significantly enriched by upregulated DEGs, suggesting that mustard might provide a defense against herbivory by inducing JA biosynthesis and then promoting GSL accumulation. Surprisingly, genes regulating JA catabolism and inactivation were also activated, and both JA signaling repressors (JAZs and JAMs) and activators (MYCs and NACs) were upregulated during herbivory. Taken together, our results indicate that the accumulation of GSLs regulated by JA signaling, and the regulation of active and inactive JA compound conversion, as well as the activation of JA signaling repressors and activators, collectively control the anti-insect defense response and avoid over-stunted growth in mustard during insect feeding.

Discovery of Transfer Factors in Plant-Derived Proteins and an In Vitro Assessment of Their Immunological Activities.

Repeated exposure to pathogens leads to evolutionary selection of adaptive traits. Many species transfer immunological memory to their offspring to counteract future immune challenges. Transfer factors such as those found in the colostrum are among the many mechanisms where transfer of immunologic memory from one generation to the next can be achieved for an enhanced immune response. Here, a library of 100 plants with high protein contents was screened to find plant-based proteins that behave like a transfer factor moiety to boost human immunity. Aqueous extracts from candidate plants were tested in a human peripheral blood mononuclear cell (PBMC) cytotoxicity assay using human cancerous lymphoblast cells-with K562 cells as a target and natural killer cells as an effector. Plant extracts that caused PBMCs to exhibit enhanced killing beyond the capability of the colostrum-based transfer factor were considered hits. Primary screening yielded an 11% hit rate. The protein contents of these hits were tested via a Bradford assay and Coomassie-stained SDS-PAGE, where three extracts were confirmed to have high protein contents. Plants with high protein contents underwent C18 column fractionation using methanol gradients followed by membrane ultrafiltration to isolate protein fractions with molecular weights of <3 kDa, 3-30 kDa, and >30 kDa. It was found that the 3-30 kDa and >30 kDa fractions had high activity in the PBMC cytotoxicity assay. The 3-30 kDa ultrafiltrates from the top two hits, seeds from Raphanus sativus and Brassica juncea, were then selected for protein identification by mass spectrometry. The majority of the proteins in the fractions were found to be seed storage proteins, with a low abundance of proteins involved in plant defense and stress response. These findings suggest that Raphanus sativus or Brassica juncea extracts could be considered for further characterization and immune functional exploration with a possibility of supplemental use to bolster recipients' immune response.

Synergistic action of Pseudomonas fluorescens with melatonin attenuates salt toxicity in mustard by regulating antioxidant system and flavonoid profile.

Salt stress is an alarming abiotic stress that reduces mustard growth and yield. To attenuate salt toxicity effects, plant growth-promoting rhizobacteria (PGPR) offers a sustainable approach. Among the various PGPR, Pseudomonas fluorescens (P. fluorescens NAIMCC-B-00340) was chosen for its salt tolerance (at 100 mM NaCl) and for exhibiting various growth-promoting activities. Notably, P. fluorescens can produce auxin, which plays a role in melatonin (MT) synthesis. Melatonin is a pleiotropic molecule that acts as an antioxidant to scavenge reactive oxygen species (ROS), resulting in stress reduction. Owing to the individual role of PGPR and MT in salt tolerance, and their casual nexus, their domino effect was investigated in Indian mustard under salt stress. The synergistic action of P. fluorescens and MT under salt stress conditions was found to enhance the activity of antioxidative enzymes and proline content as well as  promote the production of secondary metabolites. This led to reduced oxidative stress following effective ROS scavenging, maintained photosynthesis, and improved growth. In mustard plants treated with MT and P. fluorescens under salt stress, eight flavonoids showed significant increase. Kaempferol and cyanidin showed the highest concentrations and are reported to act as antioxidants with protective functions under stress. Thus, we can anticipate that strategies involved in their enhancement could provide a better adaptive solution to salt toxicity in mustard plants. In conclusion, the combination of P. fluorescens and MT affected antioxidant metabolism and flavonoid profile that could be used to mitigate salt-induced stress and bolster plant resilience.