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. 2016 Mar;90(4-5):503-16.
doi: 10.1007/s11103-016-0437-z. Epub 2016 Jan 28.

Functional analysis of three BrMYB28 transcription factors controlling the biosynthesis of glucosinolates in Brassica rapa

Mi-Suk Seo  1 , Mina Jin  2 , Jin-Hyuk Chun  3 , Sun-Ju Kim  4 , Beom-Seok Park  5 , Seong-Han Shon  6 , Jung Sun Kim  7
Affiliations

Functional analysis of three BrMYB28 transcription factors controlling the biosynthesis of glucosinolates in Brassica rapa

Mi-Suk Seo et al. Plant Mol Biol. 2016 Mar.

Abstract

Glucosinolates (GSLs) are secondary metabolites that have anticarcinogenic activity and play defense roles in plants of the Brassicaceae family. MYB28 is known as a transcription factor that regulates aliphatic GSL biosynthesis in Arabidopsis thaliana. Brassicaceae plants have three orthologous copies of AtMYB28 derived from recent genome triplication. These BrMYB28 genes have a high level of sequence homology, with 81-87% similarities in the coding DNA sequence compared to Arabidopsis. Overexpression of three paralogous BrMYB28 genes in transgenic Chinese cabbage increased the total GSL content in all T1 generation plants and in two inbred lines of homozygous T2 plants. The highest total GSL contents were detected in homozygous T2 lines overexpressing BrMYB28.1, which showed an approximate fivefold increase compared to that of nontransgenic plants. The homozygous T2 lines with overexpressed BrMYB28.1 also showed an increased content of aliphatic, indolic, and aromatic GSLs compared to that of nontransgenic plants. Furthermore, all of the three BrMYB28 genes were identified as negative regulators of BrAOP2 and positive regulators of BrGSL-OH in the homozygous T2 lines. These data indicate the regulatory mechanism of GSL biosynthesis in B. rapa is unlike that in A. thaliana. Our results will provide useful information for elucidating the regulatory mechanism of GSL biosynthesis in polyploid plants.

Keywords: Brassica rapa; Chinese cabbage; Glucosinolates; Transcription factor BrMYB28s; Transgenic plats.

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Figures

Fig. 1
Fig. 1
Amino acid sequence alignment of MYB28 proteins that regulate the glucosinolate biosynthesis pathway of B. rapa and A. thaliana. The R2 and R3 binding domains are boxed in white and black, respectively
Fig. 2
Fig. 2
Phylogenetic analysis of MYB TFs related to the glucosinolate biosynthesis pathway in B. rapa and Arabidopsis. This tree was constructed using MEGA, version 6, software. Bootstrap values with 1000 replicates are denoted as percentages
Fig. 3
Fig. 3
Expression analysis of BrMYB28 TFs in various organs and developmental stages. A RT-PCR analysis of BrMYB28 TFs in different types of tissues. S, seed; SL, seedling; L, mature leaf (3-week-old vegetative stage); FB, floral bud; ST, stamen; C, carpel; R, root. The PCR products are approximately 1 kb for BrMYB28 genes. BrActin1 is approximately 500 bp and serves as an internal control. B Microarray expression analysis of BrMYB28 TFs in different growth stages. BLCS0D, seeds; BLCS2D, seedling (2 days old); BLCS1W, whole plant, 1-week-old vegetative stage (7 days old); BLCS2W, whole plant, 2-week-old vegetative stage (14 days old); BLCC0D, whole plant, 3-week-old vegetative stage (21 days old); BLCC1D, whole plant, 1 day after light chilling at 4 °C (22 days old); BLCC1W, whole plant, 1 week after light chilling at 4 °C (28 days old); BLCC4W, whole plant, 4 weeks after light chilling at 4 °C (56 days old); BLCC7W, whole plant, 7 weeks after light chilling at 4 °C (70 days old); BLCA1D, whole plant, 1 day after greenhouse growth (71 days old); BLCA2D, whole plant, 2 days after greenhouse growth (72 days old); BLCA1W, whole plant, 1 week after greenhouse growth (77 days old); BLCA2W, whole plant, 2 weeks after greenhouse growth (84 days old); BLCA3W, whole plant, 3 weeks after greenhouse growth (91 days old)
Fig. 4
Fig. 4
Schematic diagram of part of the T-DNA region of the binary vector and PCR analysis of transgenic Chinese cabbage. A T-DNA region of binary vector construct used for Agrobacterium-mediated transformation. LB, left border; RB, right border; 35S Pro, CaMV 35S promoter; Pnos, Nos promoter; Tnos, Nos terminator, HPT, hygromycin resistance gene. B Agrobacterium-mediated transformation of BrMYB28 genes in Chinese cabbage. a Hypocotyl explants of B. rapa used for transformation. b Hygromycin-resistant callus induced from hypocotyl in selection medium containing 10 mg/L hygromycin. c Hygromycin resistance shoot regenerated from callus in regeneration medium containing 10 mg/L hygromycin. d The hygromycin-resistant plantlets were transferred to soil in pots and grown to maturity in a greenhouse with non-transgenic plants (leftmost panel). C, D Detection of the hpt gene (C) and the three BrMYB28 genes (D) in hygromycin-resistant plants (T1) by PCR analysis. The PCR products were identified at 757 bp for the hpt gene and 1454 bp for BrMYB28.1, 1581 bp for BrMYB28.2, and 1821 bp for BrMYB28.3. M, molecular weight marker; P, plasmid DNA; NC, CC, nontransgenic NW line (NC) and CT001 line (CC); N1-1,2, NW BrMYB28.1 gene transgenic plants; N2-1, NW BrMYB28.2 gene transgenic plant; N3-1,2, NW BrMYB28.3 gene transgenic plants; C1-1–4, CT001 BrMYB28.1 gene transgenic plants; C2-1–2; CT001 BrMYB28.2 gene transgenic plants; C3-1–3, CT001 BrMYB28.3 gene transgenic plants
Fig. 5
Fig. 5
Expression of genes involved in glucosinolate biosynthesis in the 6-week-old leaves of T1 transgenic NW (A) and CT001 (B) plants. Relative expression was determined in triplicate measurements in three independent biological replicates. The Bractin gene was used as a quantitative control. NC, CC, non-transgenic plants; N1-1,2, NW BrMYB28.1 gene transgenic plants; C1-1–4, CT001 BrMYB28.1 gene transgenic plants; N2-1, NW BrMYB28.2 gene transgenic plant; C2-1–3, CT001 BrMYB28.2 gene transgenic plants; N3-1,2, NW BrMYB28.3 gene transgenic plants; C3-1–3, CT001 BrMYB28.3 gene transgenic plants
Fig. 6
Fig. 6
HPLC analysis of GSL content in leaves of T1 transgenic NW (A) and CT001 (B) plants. Values are the means of 3 replications. Bars represent the standard error of the mean. NC, CC, non-transgenic plants; N1-1, 2, NW BrMYB28.1 gene transgenic plants; C1-1–5, CT001 BrMYB28.1 gene transgenic plants; N2-1, NW BrMYB28.2 gene transgenic plant; C2-1, 2, CT001 BrMYB28.2 gene transgenic plants; N3-1,2, NW BrMYB28.3 gene transgenic plants; C3-1–3, CT001 BrMYB28.3 gene transgenic plants
Fig. 7
Fig. 7
Expression of genes involved in glucosinolate biosynthesis in the 6-week-old leaves of T2 transgenic NW (A) and CT001 (B) plants. Relative expression was determined in triplicate measurements in three independent biological replicates. The Bractin gene was used as a quantitative control. NC, CC, non-transgenic plants; N-1-2-1–N-1-2-3, NW BrMYB28.1 gene transgenic plants; C-1-1-1–C-1-2-4, CT001 BrMYB28.1 gene transgenic plants; N-2-1-1, NW BrMYB28.2 gene transgenic plant; N-3-1-1, NW BrMYB28.3 gene transgenic plant; C-3-1-1, CT001 BrMYB28.3 gene transgenic plants
Fig. 8
Fig. 8
Expression profiling for glucosinolate biosynthesis-related genes in T2 homozygous transgenic lines. NC, CC, non-transgenic plants; NC, CC, non-transgenic plants; N-1-2-1–N-1-2-3, NW BrMYB28.1 gene transgenic plants; C-1-1-1–C-1-2-4, CT001 BrMYB28.1 gene transgenic plants; N-2-1-1, NW BrMYB28.2 gene transgenic plant; N-3-1-1, NW BrMYB28.3 gene transgenic plant; C-3-1-1, CT001 BrMYB28.3 gene transgenic plants
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