Abstract
Key messageOverexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite glucoraphanin, and the toxic and pungent sinigrin.Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties.
Highlights
Brassica oleracea (CC genome) is a member of the Brassicaceae family and one of the three diploid Brassica species in the “triangle of U” (Nagaharu 1935) that includes the diploids B. rapa (AA) and B. nigra (BB) and the allotetraploids B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC)
In order to evaluate whether the content of AGSLs in the different genotypes analysed was correlated to gene expression, the four Winspit plants plus the PSB and SAV cultivars were used to perform high-performance liquid chromatography (HPLC) analyses, microarray and qRTPCR transcription profiling assays
Variation was observed among Winspit individuals, in general the wild genotypes displayed a higher AGSL concentration compared to the two crops, for the alkenyl-GSLs gluconapin (GNP) and sinigrin (SIN) (Fig. 1a, Table S3)
Summary
Brassica oleracea (CC genome) is a member of the Brassicaceae family and one of the three diploid Brassica species in the “triangle of U” (Nagaharu 1935) that includes the diploids B. rapa (AA) and B. nigra (BB) and the allotetraploids B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC). Extended author information available on the last page of the article has undergone a specific whole-genome triplication (Lysak et al 2005). Brassica oleracea comprises many important vegetable crops including cauliflower, broccoli, cabbages, Brussels sprouts, kohlrabi and kales, which are high in carotenoids (Kopsell and Kopsell 2006) and contain diverse glucosinolates (GSLs), secondary metabolites, naturally produced by plants of the Brassicaceae family (Traka and Mithen 2009). A group of GSL products derived from enzymatic hydrolysis, isothiocyanates (ITCs), play important roles in human health and agriculture. Isothiocyanates are considered the main components responsible for the cancer-preventing properties of Brassica plants upon consumption by humans (Traka and Mithen 2009). Sulforaphane, the aliphatic ITC generated from hydrolysis of 4-methylsulphinylbutyl
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