Abstract
Sulforaphane is a new and effective anti-cancer component that is abundant in broccoli. In the past few years, the patterns of variability in glucosinolate content and its regulation in A. thaliana have been described in detail. However, the diversity of glucosinolate and sulforaphane contents in different organs during vegetative and reproductive stages has not been clearly explained. In this paper, we firstly investigated the transcriptome profiles of the developing buds and leaves at bolting stage of broccoli (B52) to further assess the gene expression patterns involved in sulforaphane synthesis. The CYP79F1 gene, as well as nine other genes related to glucorahpanin biosynthesis, MAM1, MAM3, St5b-2, FMO GS-OX1, MY, AOP2, AOP3, ESP and ESM1 were selected by digital gene expression analysis and were validated by quantitative real-time PCR (qRT-PCR). Meanwhile, the compositions of glucosinolates and sulforaphane were detected for correlation analysis with related genes. Finally the RNA sequencing libraries generated 147 957 344 clean reads, and 8 539 unigene assemblies were produced. In digital result, only CYP79F1, in the glucoraphanin pathway, was up-regulated in young buds but absent from the other organs, which was consistent with the highest level of sulforaphane content being in this organ compared to mature buds, buds one day before flowering, flowers and leaves. The sequencing results also presented that auxin and cytokinin might affect glucoraphanin accumulation. The study revealed that up-regulated expression of CYP79F1 plays a fundamental and direct role in sulforaphane production in inflorescences. Two genes of MAM1 and St5b-2 could up-regulated glucoraphanin generation. Synergistic expression of MAM1, MAM3, St5b-2, FMO GS-OX1, MY, ESP and ESM1 was found in sulforaphane metabolism. This study will be beneficial for understanding the diversity of sulforaphane in broccoli organs.
Highlights
In recent years, sulforaphane has attracted much interest due to its anti-cancer activity, and a growing body of epidemiological evidence has shown that increased consumption of sulforaphane or cruciferous vegetables rich in sulforaphane can lower the risk of lung [1], colon [2], pancreatic [3], breast [4], bladder [5] and prostate [6] cancers as well as some geriatric diseases such as Alzheimer’s disease [7] and cardiovascular disease [8, 9]
6656 unigenes were categorized into 4 Clusters of Orthologous Groups of Proteins (COG) classifications (S2 Fig), which was shown and validated by Venn diagram comparisons (Fig 2A) and cluster analysis of differentially expressed genes between leaves and developmental buds (Fig 2B)
Our study provided for evidence in synthesis of glucosinolate in reproductive organs.St5b-2 is numbered K11821 in the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology pathway, and it is responsible for tryptophan metabolism, glucosinolate biosynthesis, biosynthesis of secondary metabolites, and 2-Oxocarboxylic acid metabolism
Summary
Sulforaphane has attracted much interest due to its anti-cancer activity, and a growing body of epidemiological evidence has shown that increased consumption of sulforaphane or cruciferous vegetables rich in sulforaphane can lower the risk of lung [1], colon [2], pancreatic [3], breast [4], bladder [5] and prostate [6] cancers as well as some geriatric diseases such as Alzheimer’s disease [7] and cardiovascular disease [8, 9]. Sulforaphane is an isothiocyanate, and it can be synthesized from glucoraphanin through hydrolysis by myrosinase when broccoli is chewed, mechanically damaged, digested by humans, or bitten by insects [14, 15]. Glucosinolates are mainly synthesized from amino acids Met, Phe and Trp, which give rise to three groups of glucosinolates: aliphatic glucosinolates, benzenic glucosinolates and indolic glucosinolates [15, 22,23,24]. Regulation genesof glucosinolate and the pathway have been successfully identified in Arabidopsis [23, 25,26,27].Glucoraphanin belongs to aliphatic glucosinolate derived from Met. In the process of chain elongation, it starts with deamination by a BCAT4 giving rise to a 2-oxo-4-methylthiobutanoic acid. The 2-oxo-4-methylthiobutanoic acid enters a cycle of three successive transformations: condensation with acetyl-CoA by MAM1 and MAM3, isomerization by IPMI-SSU2, 3, and oxidative decarboxylation by IPM-DH, generating 2-Oxo-6-methylthiohexanoic acid [16, 28]
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