An activatable fluorescence probe for rapid detection and in situ imaging of ß-galactosidase activity in cabbage roots under heavy metal stress.

ß-Galactosidase (ß-gal), an enzyme related to cell wall degradation, plays an important role in regulating cell wall metabolism and reconstruction. However, activatable fluorescence probes for the detection and imaging of ß-gal fluctuations in plants have been less exploited. Herein, we report an activatable fluorescent probe based on intramolecular charge transfer (ICT), benzothiazole coumarin-bearing ß-galactoside (BC-ßgal), to achieve distinct in situ imaging of ß-gal in plant cells. It exhibits high sensitivity and selectivity to ß-gal with a fast response (8 min). BC-ßgal can be used to efficiently detect the alternations of intracellular ß-gal levels in cabbage root cells with considerable imaging integrity and imaging contrast. Significantly, BC-ßgal can assess ß-gal activity in cabbage roots under heavy metal stress (Cd2+, Cu2+, and Pb2+), revealing that ß-gal activity is negatively correlated with the severity of heavy metal stress. Our work thus facilitates the study of ß-gal biological mechanisms.

Computer-driven Evolution of Myrosinase from the Cabbage Aphid for Efficient Production of (R)-Sulforaphane.

Myrosinase (Myr) catalyzes the hydrolysis of glucosinolates, yielding biologically active metabolites. In this study, glucoraphanin (GRA) extracted from broccoli seeds was effectively hydrolyzed using a Myr-obtained cabbage aphid (Brevicoryne brassicae) (BbMyr) to produce (R)-sulforaphane (SFN). The gene encoding BbMyr was successfully heterologously expressed in Escherichia coli, resulting in the production of 1.6 g/L (R)-SFN, with a remarkable yield of 20.8 mg/gbroccoli seeds, achieved using recombination E. coli whole-cell catalysis under optimal conditions (pH 4.5, 45 °C). Subsequently, BbMyr underwent combinatorial simulation-driven mutagenesis, yielding a mutant, DE9 (N321D/Y426S), showing a remarkable 2.91-fold increase in the catalytic efficiency (kcat/KM) compared with the original enzyme. Molecular dynamics simulations demonstrated that the N321D mutation in loopA of mutant DE9 enhanced loopA stability by inducing favorable alterations in hydrogen bonds, while the Y426S mutation in loopB decreased spatial resistance. This research lays a foundation for the environmentally sustainable enzymatic (R)-SFN synthesis.

Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

KEY MESSAGE: BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

Structure and mechanism of the plant RNA polymerase V.

In addition to the conserved RNA polymerases I to III (Pols I to III) in eukaryotes, two atypical polymerases, Pols IV and V, specifically produce noncoding RNA in the RNA-directed DNA methylation pathway in plants. Here, we report on the structures of cauliflower Pol V in the free and elongation conformations. A conserved tyrosine residue of NRPE2 stacks with a double-stranded DNA branch of the transcription bubble to potentially attenuate elongation by inducing transcription stalling. The nontemplate DNA strand is captured by NRPE2 to enhance backtracking, thereby increasing 3'-5' cleavage, which likely underpins Pol V's high fidelity. The structures also illuminate the mechanism of Pol V transcription stalling and enhanced backtracking, which may be important for Pol V's retention on chromatin to serve its function in tethering downstream factors for RNA-directed DNA methylation.

Expression of BoNOL and BoHCAR genes during postharvest senescence of broccoli heads.

BACKGROUND: Chlorophyll is the most abundant pigment on Earth, essential for the capture of light energy during photosynthesis. During senescence, chlorophyll degradation is highly regulated in order to diminish toxicity of the free chlorophyll molecule due to its photoactivity. The first step in the chlorophyll degradation pathway is the conversion of chlorophyll b to chlorophyll a by means of two consecutive reactions catalyzed by enzymes coded by NYC1 (NON-YELLOW COLORING 1), NOL (NYC1-LIKE) and HCAR. RESULTS: In this work, we studied the expression of NOL and HCAR genes during postharvest senescence of broccoli. We found that the expression of BoNOL increase during the first days of storage and then decrease. In the case of BoHCAR, its expression is maintained during the first days and then it also diminishes. Additionally, the effect of different postharvest treatments on the expression of these genes was also analyzed. It was observed that the expression of BoNOL is lower in the treatments performed with 1-methylcyclopropene (1-MCP), 6-benzylaminopurine (6-BAP) and modified atmospheres, while BoHCAR expression showed an increase in these same treatments, and a decrease in the treatment with ethylene. There were no variations in the expression of both genes in heat treatment, UV-C treatment and visible light treatment. CONCLUSIONS: These results suggest that both BoHCAR and BoNOL show a lower regulation of their expression than other genes involved in chlorophyll degradation during senescence. © 2020 Society of Chemical Industry.

The dihydroflavonol 4-reductase BoDFR1 drives anthocyanin accumulation in pink-leaved ornamental kale.

KEY MESSAGE: Overexpression and virus-induced gene silencing verified BoDFR1 conferred the anthocyanin accumulation in pink-leaved ornamental kale. Leaf color is an essential trait in the important horticultural biennial plant ornamental kale (Brassica oleracea var. acephala). The identity of the gene conferring this striking trait and its mode of inheritance are topics of debate. Based on an analysis of F1, F2, BC1P1, and BC1P2 ornamental kale populations derived from a cross between a pink-leaved P28 and white-leaved D10 line, we determined that the pink leaf trait is controlled by a semi-dominant gene. We cloned two genes potentially involved in anthocyanin biosynthesis in ornamental kale: Bo9g058630 and Bo6g100940. Based on their variation in sequence, we speculated that Bo9g058630, encoding the kale dihydroflavonol-4 reductase (BoDFR1) enzyme, plays a critical role in the development of the pink leaf trait. Indeed, an InDel marker specific for BoDFR1 completely co-segregated with the pink leaf trait in our F2 population. We then generated the 35Spro: DFR-GUS overexpression vector, which we transformed into D10. Overexpression of BoDFR1 indeed restored some anthocyanin accumulation in this white-leaved parental line. In addition, we targeted BoDFR1 in P28 using virus-induced gene silencing. Again, silencing of BoDFR1 resulted in a substantial decrease in anthocyanin accumulation. This work lays the foundation for further exploration of the mechanism underlying anthocyanin accumulation in pink-leaved ornamental kale.

Induction and Priming of Plant Defense by Root-Associated Insect-Pathogenic Fungi.

Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus, Metarhizium brunneum, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (Brassica oleracea var. botrytis) seeds were sown together with M. brunneum-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist Plutella xylostella. Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments. Metarhizium brunneum clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in M. brunneum-treated plants than in controls; before herbivory, M. brunneum-treated and control plants did not differ. Leaf consumption was slightly lower in the M. brunneum-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the Metarhizium treatment. Thus, M. brunneum associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that M. brunneum, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.

Patterns of Expansion and Expression Divergence of the Polygalacturonase Gene Family in Brassica oleracea.

Plant polygalacturonases (PGs) are closely related to cell-separation events during plant growth and development by degrading pectin. Identifying and investigating their diversification of evolution and expression could shed light on research on their function. We conducted sequence, molecular evolution, and gene expression analyses of PG genes in Brassica oleracea. Ninety-nine B. oleracea PGs (BoPGs) were identified and divided into seven clades through phylogenetic analysis. The exon/intron structures and motifs were conserved within, but divergent between, clades. The second conserved domain (GDDC) may be more closely related to the identification of PGs. There were at least 79 common ancestor PGs between Arabidopsis thaliana and B. oleracea. The event of whole genome triplication and tandem duplication played important roles in the rapid expansion of the BoPG gene family, and gene loss may be an important mechanism in the generation of the diversity of BoPGs. By evaluating the expression in five tissues, we found that most of the expressed BoPGs in clades A, B, and E showed ubiquitous expression characteristics, and the expressed BoPGs in clades C, D, and F were mainly responsible for reproduction development. Most of the paralogous gene pairs (76.2%) exhibited divergent expression patterns, indicating that they may have experienced neofunctionalization or subfunctionalization. The cis-elements analysis showed that up to 96 BoPGs contained the hormone response elements in their promoters. In conclusion, our comparative analysis may provide a valuable data foundation for the further functional analysis of BoPGs during the development of B. oleracea.

Controllable Soil Degradation Rate of 5-Substituted Sulfonylurea Herbicides as Novel AHAS Inhibitors.

Chlorsulfuron has been applied in wheat fields as a recognized herbicide worldwide, yet it was officially banned in China since 2014 for its soil persistence problem. On the basis of our previous research that 5-dimethylamino distinctively accelerated degradation rate in soils, a modified amino moiety (Ia-c) and monosubstituted amino group (Id-e) were introduced onto the fifth position of the benzene ring in sulfonylurea structures, as well as heterocyclic amino substituents (If-g) to seek a suitable soil degradation rate during such an in situ crop rotation system. Referring to the biological data and ScAHAS inhibition and ScAHAS docking results, they turned out to be AHAS inhibitors with high potent herbicidal activities. The various influence on soil degradation rate along with crop safety indicated that different substituents on the fifth position have exerted an apparent impact. Their united study of structure-activity-safety-degradation relationship has great potential to provide valuable information for further development of eco-friendly agrochemicals.

Overexpressing broccoli tryptophan biosynthetic genes BoTSB1 and BoTSB2 promotes biosynthesis of IAA and indole glucosinolates.

Tryptophan is one of the amino acids that cannot be produced in humans and has to be acquired primarily from plants. In Arabidopsis thaliana (Arabidopsis), the tryptophan synthase beta subunit (TSB) genes have been found to catalyze the biosynthesis of tryptophan. Here, we report the isolation and characterization of two TSB genes from Brassica oleracea (broccoli), designated BoTSB1 and BoTSB2. Overexpressing BoTSB1 or BoTSB2 in Arabidopsis resulted in higher tryptophan content and the accumulation of indole-3-acetic acid (IAA) and indole glucosinolates in rosette leaves. Therefore, the transgenic plants showed a series of high auxin phenotypes, including long hypocotyls, large plants and a high number of lateral roots. The spatial expression of BoTSB1 and BoTSB2 was detected by quantitative real-time PCR in broccoli and by expressing the ß-glucuronidase reporter gene (GUS) controlled by the promoters of the two genes in Arabidopsis. BoTSB1 was abundantly expressed in vascular tissue of shoots and inflorescences. Compared to BoTSB1, BoTSB2 was expressed at a very low level in shoots but at a higher level in roots. We further investigated the expression response of the two genes to several hormone and stress treatments. Both genes were induced by methyl jasmonate (MeJA), salicylic acid (SA), gibberellic acid (GA), Flg22 (a conserved 22-amino acid peptide derived from bacterial flagellin), wounding, low temperature and NaCl and were repressed by IAA. Our study enhances the understanding of tryptophan biosynthesis and its regulation in broccoli and Arabidopsis. In addition, we provide evidence that TSB genes can potentially be a good tool to breed plants with high biomass and high nutrition.

Genome-Wide Distribution, Expression and Function Analysis of the U-Box Gene Family in Brassica oleracea L.

The plant U-box (PUB) protein family plays an important role in plant growth and development. The U-box gene family has been well studied in Arabidopsis thaliana, Brassica rapa, rice, etc., but there have been no systematic studies in Brassica oleracea. In this study, we performed genome-wide identification and evolutionary analysis of the U-box protein family of B. oleracea. Firstly, based on the Brassica database (BRAD) and the Bolbase database, 99 Brassicaoleracea PUB genes were identified and divided into seven groups (I-VII). The BoPUB genes are unevenly distributed on the nine chromosomes of B. oleracea, and there are tandem repeat genes, leading to family expansion from the A. thaliana genome to the B. oleracea genome. The protein interaction network, GO annotation, and KEGG pathway enrichment analysis indicated that the biological processes and specific functions of the BoPUB genes may mainly involve abiotic stress. RNA-seq transcriptome data of different pollination times revealed spatiotemporal expression specificity of the BoPUB genes. The differential expression profile was consistent with the results of RT-qPCR analysis. Additionally, a large number of pollen-specific cis-acting elements were found in promoters of differentially expressed genes (DEG), which verified that these significantly differentially expressed genes after self-pollination (SP) were likely to participate in the self-incompatibility (SI) process, including gene encoding ARC1, a well-known downstream protein of SI in B. oleracea. Our study provides valuable information indicating that the BoPUB genes participates not only in the abiotic stress response, but are also involved in pollination.

Glycosylinositol phosphoceramide-specific phospholipase D activity catalyzes transphosphatidylation.

Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation. The available alcohol for this reaction is a primary alcohol with a chain length below C4. Neither secondary alcohol, tertiary alcohol, choline, serine nor glycerol serves as an acceptor for transphosphatidylation of GIPC-PLD. We also found that cabbage GIPC-PLD prefers GIPC containing two sugars. Neither inositol phosphoceramide, mannosylinositol phosphoceramide nor GIPC with three sugar chains served as substrate. GIPC-PLD will become a useful catalyst for modification of polar head group of sphingophospholipid.

Hydrogen gas alleviates toxic effects of cadmium in Brassica campestris seedlings through up-regulation of the antioxidant capacities: Possible involvement of nitric oxide.

Hydrogen gas (H2) has been shown as an important factor in plant tolerance to abiotic stresses, but the underlying mechanisms remain unclear. In the present study, the effects of H2 and its interaction with nitric oxide (NO) on alleviating cadmium (Cd) stress in Brassica campestris seedlings were investigated. NO donor (SNP) or hydrogen-rich water (HRW) treatment showed a significant improvement in growth of Cd-stressed seedlings. Cd treatment upregulated both endogenous NO and H2 (36% and 66%, respectively), and the increase of H2 was prior to NO increase. When treated with NO scavenger (PTIO) or NO biosynthesis enzyme inhibitors (L-NAME and Gln), HRW-induced alleviation under Cd stress was prevented. Under Cd stress, HRW pretreatment significantly enhanced the NO accumulation, and together up-regulated the activity of NR (nitrate reductase) and expression of NR. HRW induced lower reactive oxygen species (ROS), higher AsA content, enhanced activity of POD (peroxidase) and SOD (superoxide dismutase) in seedling roots were inhibited by PTIO, L-NAME and Gln. Through proteomic analysis, the level of 29 proteins were changed in response to H2 and NO-induced amelioration of Cd stress. Nearly half of them were involved in oxidation-reduction processes (about 20%) or antioxidant enzymes (approximately 20%). These results strongly indicate that in Cd-stressed seedlings, pretreatment with HRW induces the accumulation of H2 (biosynthesized or permeated), which further stimulates the biosynthesis of NO through the NR pathway. Finally, H2 and NO together enhance the antioxidant capabilities of seedlings in response to Cd toxicity.

Mutation of ACX1, a Jasmonic Acid Biosynthetic Enzyme, Leads to Petal Degeneration in Chinese Cabbage (Brassica campestris ssp. pekinensis).

Petal color, size, and morphology play important roles in protecting other floral organs, attracting pollinators, and facilitating sexual reproduction in plants. In a previous study, we obtained a petal degeneration mutant (pdm) from the 'FT' doubled haploid line of Chinese cabbage and found that the candidate gene for pdm, Bra040093, encodes the enzyme acyl-CoA oxidase1. In this study, we sought to examine the gene networks regulating petal development in pdm plants. We show that the mRNA and protein expression of Bra040093, which is involved in the jasmonic acid (JA) biosynthetic pathway, were significantly lower in the petals of pdm plants than in those of 'FT' plants. Similarly, the JA and methyl jasmonate (MeJA) contents of petals were significantly lower in pdm plants than in 'FT' plants and we found that exogenous application of these hormones to the inflorescences of pdm plants restored the 'FT' phenotype. Comparative analyses of the transcriptomes of 'FT', pdm and pdm + JA (pJA) plants revealed 10,160 differentially expressed genes (DEGs) with consistent expression tendencies in 'FT' vs. pdm and pJA vs. pdm comparisons. Among these DEGs, we identified 69 DEGs related to floral organ development, 11 of which are involved in petal development regulated by JA. On the basis of qRT-PCR verification, we propose regulatory pathways whereby JA may mediate petal development in the pdm mutant. We demonstrate that mutation of Bra040093 in pdm plants leads to reduced JA levels and that this in turn promotes changes in the expression of genes that are expressed in response to JA, ultimately resulting in petal degeneration. These findings thus indicate that JA is associated with petal development in Chinese cabbage. These results enhance our knowledge on the molecular mechanisms underlying petal development and lay the foundations for further elucidation of the mechanisms associated with floral organ development in Chinese cabbage.

Selenium spiked in soil promoted zinc accumulation of Chinese cabbage and improved its antioxidant system and lipid peroxidation.

Selenium (Se) and zinc (Zn) are necessary mineral nutrients for human body but millions of people have an inadequate intake of them, and eat food enriched with Se and Zn may minimize these problems. Chinese cabbage is an important food in people's daily life. The aim of this study was to evaluate the effects of single Se, Zn and their combination treatment in soil on their accumulation, antioxidant system and lipid peroxidation in roots and leaves of Chinese cabbage using soil pot culture experiment. When 0.5 mg kg-1 Se +30 mg kg-1 Zn and 1.0 mg kg-1 Se +30 mg kg-1 Zn were spiked in soils, Zn concentrations in roots and leaves of Chinese cabbage were significantly increased (p < 0.05) by 20.2%, 37.8% and 17.9%, 34.1% respectively compared to the treatment of 30 mg kg-1 Zn added, and the latter was significantly higher (p < 0.05) than that of former, indicating Se significantly promoted Zn accumulation. Almost all physiological indexes including POD, SOD, CAT, APX, GR, Chlorophyll a, Chlorophyll b, Carotenoids, MDA and Free proline in the treatments of Se or Zn spiked were significantly improved (p < 0.05) or basically unaffected compared to the control without Se or Zn added. The biomass change trends were similar with these indexes either. These results showed that the addition in soil of Se and Zn significantly increased their accumulation in Chinese cabbage without affected its formal growth. Particularly, the addition of Se promoted Zn accumulation. The conclusions were more important reference for the production practice of cash crop enriched of Se and Zn either.

Selenium induces changes of rhizosphere bacterial characteristics and enzyme activities affecting chromium/selenium uptake by pak choi (Brassica campestris L. ssp. Chinensis Makino) in chromium contaminated soil.

Understanding the chemical response and characteristics of bacterial communities in soil is critical to evaluate the effects of selenium (Se) supplement on plant growth and chromium (Cr)/Se uptake in Cr contaminated soil. The rhizosphere soil characteristics of pak choi (Brassica campestris L. ssp. Chinensis Makino) were investigated in soil contaminated with different levels and forms of Cr when supplemented with Se. Although inhibition of plant growth caused by Cr stress was not completely alleviated by Se, Cr content in plant tissues decreased in Cr(VI)120Se5 treatment (Cr(VI): 120 mg kg-1 soil; Se: 5 mg kg-1 soil) and its bioavailability in soil decreased in Cr(III)200Se5 (Cr(III): 200 mg kg-1 soil; Se: 5 mg kg-1 soil) treatment. Moreover, antagonism of Cr and Se on soil enzyme activities and bacterial communities were revealed. Notably, results of Cr(VI) reduction and Se metabolism functional profiles confirmed that bacterial communities play a critical role in regulating Cr/Se bioavailability. Additionally, the increases of Se bioavailability in Cr contaminated soil were ascribed to oxidation of Cr(VI) and reduction of Se reductases proportions, as well as the enhancing of pH in soil. These findings reveal that Se has the potential capacity to sustain the stability of microdomain in Cr contaminated soil.

Pol III-Dependent Cabbage BoNR8 Long ncRNA Affects Seed Germination and Growth in Arabidopsis.

Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts longer than 200 nt that are distributed widely in organisms and play many physiological roles. The BoNR8 lncRNA is a 272 nt long transcript yielded by RNA polymerase III in cabbage that was identified as the closest homolog of the AtR8 lncRNA in Arabidopsis. The BoNR8 lncRNA was expressed extensively in the epidermal tissue in the root elongation zone of germinated seeds, and its accumulation was induced by abiotic stresses, auxins and ABA. To investigate the correlation between the BoNR8 lncRNA and germination, BoNR8-overexpressing Arabidopsis plants (BoNR8-AtOX) were prepared. Three independent BoNR8-AtOX lines showed less primary root elongation, incomplete silique development and decreased germination rates. The germination efficiencies were affected strongly by ABA and slightly by salt stress, and ABA-related gene expression was changed in the BoNR8-AtOX lines.

Genome-Wide Identification and Characterization of Pectin Methylesterase Inhibitor Genes in Brassica oleracea.

The activities of pectin methylesterases (PMEs) are regulated by pectin methylesterase inhibitors (PMEIs), which consequently control the pectin methylesterification status. However, the role of PMEI genes in Brassica oleracea, an economically important vegetable crop, is poorly understood. In this study, 95 B. oleracea PMEI (BoPMEI) genes were identified. A total of 77 syntenic ortholog pairs and 10 tandemly duplicated clusters were detected, suggesting that the expansion of BoPMEI genes was mainly attributed to whole-genome triplication (WGT) and tandem duplication (TD). During diploidization after WGT, BoPMEI genes were preferentially retained in accordance with the gene balance hypothesis. Most homologous gene pairs experienced purifying selection with ω (Ka/Ks) ratios lower than 1 in evolution. Five stamen-specific BoPMEI genes were identified by expression pattern analysis. By combining the analyses of expression and evolution, we speculated that nonfunctionalization, subfunctionalization, neofunctionalization, and functional conservation can occur in the long evolutionary process. This work provides insights into the characterization of PMEI genes in B. oleracea and contributes to the further functional studies of BoPMEI genes.

Comparison of foliar silicon and selenium on cadmium absorption, compartmentation, translocation and the antioxidant system in Chinese flowering cabbage.

Silicon (Si) and selenium (Se) are beneficial for many higher plants when grown on stress conditions. However, the mechanisms underlying the differential effects between foliar Si and Se in alleviation of plant toxicity exposed to cadmium (Cd) stress are remained unclear. In this study, we investigated the discrepant mechanisms of foliar Si and Se on Cd absorption and compartmentation by roots, its translocation in xylem, and the antioxidant system within Chinese flowering cabbage (Brassica campestris L. ssp. chinensis var. utilis) under low and high Cd stress. Results showed that plant growth was significantly enhanced by foliar additions of Si or/and Se according to an increased plant tissue biomass at high Cd exposure. In addition, the foliar coupled addition of Si and Se showed little effects on the concentrations of Si or Se in plant tissues in comparison with the single addition of foliar Si or Se respectively. The foliar Si alone or combined with Se markedly reduced the Cd concentrations in plant shoots under two Cd treatments. This might be explained by the lower Cd concentrations in symplast and apoplast and the higher Cd concentrations in cell walls of plant roots, and the lower Cd concentrations in xylem sap. However, no great changes in these values were observed under the treatments of foliar Se alone. Moreover, the foliar additions of Si or/and Se all increased the antioxidant enzyme activities of SOD, CAT and APX in plant tissues, especially at high Cd dosage. No significant differences in the increasing degrees of these three antioxidant enzymes were found between the foliar Si and Se treatments. However, only the foliar Se alone or combined with Si markedly promoted the antioxidant enzyme activities of GR and DHAR in plant tissues. Our findings demonstrate that the alleviation of Cd toxicity by foliar Si maybe mainly responsible for inhibition of Cd absorption and its translocation to plant shoots, reinforcing its compartmentation into root cell walls, whilst enhancing the antioxidant enzyme system may be employed by foliar Se.

A Simple Method for On-Gel Detection of Myrosinase Activity.

Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4- is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031⁻0.25 U (pure Sinapis alba myrosinase, R² = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.