Chromosome-scale reference genome of broccoli (Brassica oleracea var. italica Plenck) provides insights into glucosinolate biosynthesis.

Broccoli (Brassica oleracea var. italica Plenck) is an important vegetable crop, as it is rich in health-beneficial glucosinolates (GSLs). However, the genetic basis of the GSL diversity in Brassicaceae remains unclear. Here we report a chromosome-level genome assembly of broccoli generated using PacBio HiFi reads and Hi-C technology. The final genome assembly is 613.79 Mb in size, with a contig N50 of 14.70 Mb. The GSL profile and content analysis of different B. oleracea varieties, combined with a phylogenetic tree analysis, sequence alignment, and the construction of a 3D model of the methylthioalkylmalate synthase 1 (MAM1) protein, revealed that the gene copy number and amino acid sequence variation both contributed to the diversity of GSL biosynthesis in B. oleracea. The overexpression of BoMAM1 (BolI0108790) in broccoli resulted in high accumulation and a high ratio of C4-GSLs, demonstrating that BoMAM1 is the key enzyme in C4-GSL biosynthesis. These results provide valuable insights for future genetic studies and nutritive component applications of Brassica crops.

Preharvest Methyl Jasmonate Treatment Increased Glucosinolate Biosynthesis, Sulforaphane Accumulation, and Antioxidant Activity of Broccoli.

Broccoli is becoming increasingly popular among consumers owing to its nutritional value and rich bioactive compounds, such glucosinolates (GSLs) and hydrolysis products, which are secondary metabolites for plant defense, cancer prevention, and higher antioxidant activity for humans. In this study, 40 µmol/L methyl jasmonate (MeJA) was sprayed onto broccoli from budding until harvest. The harvested broccoli florets, stem, and leaves were used to measure the contents of GSLs, sulforaphane, total phenolics, and flavonoids, as well as myrosinase activity, antioxidant activity, and gene expression involved in GSL biosynthesis. The overall results revealed that GSL biosynthesis and sulforaphane accumulation were most likely induced by exogenous MeJA treatment by upregulating the expression of CYP83A1, SUR1, UGT74B1, and SOT18 genes. Exogenous MeJA treatment more remarkably contributed to the increased GSL biosynthesis in broccoli cultivars with low-level GSL content (Yanxiu) than that with high-level GSLs (Xianglv No.3). Moreover, MeJA treatment had a more remarkable increasing effect in broccoli florets than stem and leaves. Interestingly, total flavonoid content substantially increased in broccoli florets after MeJA treatment, but total phenolics did not. Similarly, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, trolox-equivalent antioxidant capacity (ABTS), and ferric-reducing antioxidant power (FRAP) were higher in broccoli floret after MeJA treatment. In conclusion, MeJA mediated bioactive compound metabolism, had positive effects on GSL biosynthesis, sulforaphane, and flavonoids accumulation, and showed positive correlation on inducing higher antioxidant activities in broccoli floret. Hence, preharvest supplementation with 40 µM MeJA could be a good way to improve the nutritional value of broccoli florets.

Effects of LED illumination spectra on glucosinolate and sulforaphane accumulation in broccoli seedlings.

Glucosinolates (GSLs) are well known for plant defense and human nutrition. In this study, broccoli seedlings were illuminated under different LED light, including white, red, blue, and 75% red + 25% blue (200 mmol·m-2·s-1) for 4 weeks to investigate the effects of LED light on GSLs and sulforaphane biosynthesis. Results showed that red light promoted GSL biosynthesis and sulforaphane accumulation because red light could induce SOT18 expression to advance aliphatic GSLs biosynthesis, whereas the high tryptophan content and the upregulation of CYP79B2, CYP79B3, and CYP83B1 were attributed to indole GSL biosynthesis. Low-level methionine content and downregulated SOT18 were the main factors inhibiting GSLs and sulforaphane accumulation under blue LED illumination. BoHY5 gene expression was induced significantly and the yeast one-hybrid assay demonstrated BoHY5 could bind to SOT18 promoter. Consequently, BoHY5 inhibited SOT18 expression, and played a negative role in the GSL biosynthetic network.

Effect of selenium-sulfur interaction on the anabolism of sulforaphane in broccoli.

The effects of S (as sulphate) and Se (as selenite) treatment (S mM/Se µM: 1/0, 1/50, 1/100, 1/150, 4/0, 4/50, 4/100, and 4/150) on the production of sulforaphane (an anticancer compound), the accumulation of its precursor substance, and the expression of genes related to glucoraphanin biosynthesis in broccoli were examined. Sulforaphane yield and myrosinase activity increased significantly with the combined application of 4 mM S and 100 µM Se on broccoli. Furthermore, the concentrations of glucoraphanin (a sulforaphane precursor) and methionine (a glucoraphanin substrate) slightly changed after Se application. And the strong anticancer activity of compound Se-SMC was further improved. Analysis of related gene expression showed that MY, which encodes myrosinase, was strongly induced by Se treatment. Thus, the myrosinase activity induced by Se treatment is the dominant factor affecting sulforaphane yield from glucoraphanin hydrolyzation. Selenium-sulfur biofortification provides a technical support for the cultivation of broccoli with high sulforaphane and high anti-cancer selenium compounds.

Comparative transcriptome analyses of genes involved in sulforaphane metabolism at different treatment in Chinese kale using full-length transcriptome sequencing.

BACKGROUND: Sulforaphane is a natural isothiocyanate available from cruciferous vegetables with multiple characteristics including antioxidant, antitumor and anti-inflammatory effect. Single-molecule real-time (SMRT) sequencing has been used for long-read de novo assembly of plant genome. Here, we investigated the molecular mechanism related to glucosinolates biosynthesis in Chinese kale using combined NGS and SMRT sequencing. RESULTS: SMRT sequencing produced 185,134 unigenes, higher than 129,325 in next-generation sequencing (NGS). NaCl (75 mM), methyl jasmonate (MeJA, 40 µM), selenate (Se, sodium selenite 100 µM), and brassinolide (BR, 1.5 µM) treatment induced 6893, 13,287, 13,659 and 11,041 differentially expressed genes (DEGs) in Chinese kale seedlings comparing with control. These genes were associated with pathways of glucosinolates biosynthesis, including phenylalanine, tyrosine and tryptophan biosynthesis, cysteine and methionine metabolism, and glucosinolate biosynthesis. We found NaCl decreased sulforaphane and glucosinolates (indolic and aliphatic) contents and downregulated expression of cytochrome P45083b1 (CYP83b1), S-alkyl-thiohydroximatelyase or carbon-sulfur lyase (SUR1) and UDP-glycosyltransferase 74B1 (UGT74b1). MeJA increased sulforaphane and glucosinolates contents and upregulated the expression of CYP83b1, SUR1 and UGT74b1; Se increased sulforaphane; BR increased expression of CYP83b1, SUR1 and UGT74b1, and increased glucosinolates contents. The desulfoglucosinolate sulfotransferases ST5a_b_c were decreased by all treatments. CONCLUSIONS: We confirmed that NaCl inhibited the biosynthesis of both indolic and aliphatic glucosinolates, while MeJA and BR increased them. MeJA and BR treatments, conferred the biosynthesis of glucosinolates, and Se and MeJA contributed to sulforaphane in Chinese kale via regulating the expression of CYP83b1, SUR1 and UGT74b1.