Four genes encoding MYB28, a major transcriptional regulator of the aliphatic glucosinolate pathway, are differentially expressed in the allopolyploid Brassica juncea

Abstract

Glucosinolates are Capparales-specific secondary metabolites that have immense potential in human health and agriculture. Unlike Arabidopsis thaliana, our knowledge about glucosinolate regulators in the Brassica crops is sparse. In the current study, four MYB28 homologues were identified (BjuMYB28-1,-2,-3,-4) from the polyploid Brassica juncea, and the effects of allopolyploidization on the divergence of gene sequence, structure, function, and expression were assessed. The deduced protein sequences of the four BjuMYB28 genes showed 76.1-83.1% identity with the Arabidopsis MYB28. Phylogenetic analysis revealed that the four BjuMYB28 proteins have evolved via the hybridization and duplication processes forming the B. juncea genome (AABB) from B. rapa (AA) and B. nigra (BB), while retaining high levels of sequence conservation. Mutant complementation and over-expression studies in A. thaliana showed that all four BjuMYB28 genes encode functional MYB28 proteins and resulted in similar aliphatic glucosinolate composition and content. Detailed expression analysis using qRT-PCR assays and promoter-GUS lines revealed that the BjuMYB28 genes have both tissue- and cell-specific expression partitioning in B. juncea. The two B-genome origin BjuMYB28 genes had more abundant transcripts during the early stages of plant development than the A-genome origin genes. However, with the onset of the reproductive phase, expression levels of all four BjuMYB28 increased significantly, which may be necessary for producing and maintaining high amounts of aliphatic glucosinolates during the later stages of plant development. Taken together, our results suggest that the four MYB28 genes are differentially expressed and regulated in B. juncea to play discrete though overlapping roles in controlling aliphatic glucosinolate biosynthesis.

Keywords: Brassica juncea; MYB28; expression partitioning; glucosinolates; transcription factor..

Fig. 1.

Amino acid sequence alignment of BjuMYB28 proteins. The sequence alignment of the four BjuMYB28 proteins with the known aliphatic glucosinolate-regulating MYB proteins of A. thaliana namely, AtMYB28, AtMYB29, and AtMYB76 was performed using Clustal W. Consensus sequences for R2 and R3 domains (Dubos et al., 2010) are marked as solid lines. The putative nuclear localization signal (LKKRL) is also marked (NLS).

Fig. 2.

Evolutionary relationships of BjuMYB28 proteins. Phylogenetic analysis of BjuMYB28 proteins with the MYB proteins involved in aliphatic glucosinolate biosynthesis from A. thaliana (At, open diamond), A. lyrata (Ala, open triangle), C. rubella (Cru, open inverted triangle), T. halophila (Tha, open square), B. rapa (Bra, closed triangle), B. nigra (Bni, closed inverted triangle), B. oleracea (Bol, closed circle), and B. juncea (Bju, closed diamond) genomes was performed using the MEGA5 program (Tamura et al., 2011). The evolutionary history was inferred using the maximum likelihood method (Saitou and Nei, 1987). The percentage of replicate trees in which the associated proteins clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Fig. 3.

Subcellular localization of BjuMYB28-YFP fusion constructs in onion epidermal cells. The YFP filter, bright field, and merged images of (A) the YFP positive control vector, (B) BjuMYB28-2:YFP, and (C) AtMYB28:YFP fusion proteins are shown.

Fig. 4.

Functional complementation analysis of BjuMYB28 genes in the Arabidopsis myb28 mutant (BRC_H161b). Glucosinolate accumulation in the rosette leaves of Arabidopsis myb28 mutant over-expressing BjuMYB28 genes. The glucosinolate content and profile (in nmol mg^–1 dry weight) was determined in 25-d-old rosette leaves. The individual graphs show the accumulation of (A) total aliphatic glucosinolates (GSLs); (B) the predominant GSL, 4MSOB; (C) short and long chain GSLs (3MSOP, 5MSOP, 8MSOO); and (D) total indolic glucosinolate. Two independent mutant-complemented lines for each BjuMYB28 gene were analysed and the average foliar glucosinolates are represented along with their standard errors. Asterisks indicate significant differences in glucosinolate content compared with the Arabidopsis mutant background (P <0.05, in Fishers LSD test determined by ANOVA). Abbreviations: 4-methylsulphinylbutyl-glucosinolate (4MSOB), 3-methylsulphinylpropyl-glucosinolate (3MSOP), 5-methylsulphinylpentyl-glucosinolate (5MSOP), 8-methylsulphinyloctyl-glucosinolate (8MSOO).

Fig. 5.

Glucosinolate accumulation in rosette leaves of BjuMYB28 over-expressing Arabidopsis lines (Col-0). The glucosinolate content and profile (in nmol mg^–1 dry weight) was determined in 25-d-old rosette leaves. The individual graphs show the accumulation of (A) total aliphatic glucosinolates (GSLs), (B) the predominant GSL, 4MSOB, (C) short chain aliphatic glucosinolates including 3MSOP, 4MTB, and 5MSOP, and (D) long-chain aliphatic glucosinolates including 6MSOH, 7MSOH, and 8MSOO. Abbreviations used are given in the Materials and methods. At least two independent over-expression lines for each BjuMYB28 were analysed and the average foliar glucosinolate are represented together with their standard error. Asterisks indicate significant differences in glucosinolate content compared with the Arabidopsis Col-0 wild-type background (P <0.05, in Fishers LSD test determined by ANOVA). Abbreviations: 4-methylsulphinylbutyl-glucosinolate (4MSOB), 3-methylsulphinylpropyl-glucosinolate (3MSOP), 4-methylthiobutyl-glucosinolate (4MTB), 5-methylsulphinylpentyl-glucosinolate (5MSOP), 6-methylsulphinylhexyl-glucosinolate (6MSOH), 7-methylsulphinylheptyl-glucosinolate (7MSOH), 8-methylsulphinyloctyl-glucosinolate (8MSOO).

Fig. 6.

Expression profile of BjuMYB28 genes in organs of B. juncea and its progenitor genomes. Expression profile of MYB28 genes across various developmental stages/tissue types in (A) B. rapa (A genome), (B) B. nigra (B genome) and B. juncea (AB genome). The stages are defined as: root (15 d), cotyledons, seedling (7 d), leaf (15 d), stem (30 d), flowers (open), silique (10 d post-anthesis). Real-time quantitative PCR (qRT-PCR) was conducted and expression values across different tissue types were normalized against Actin gene expression (set at 100). Each bar represents the mean (± standard error) of three independent biological replicates. Different letters on the top indicate significant differences at P <0.05 in Tukey’s post hoc test.

Fig. 7.

Expression profile of BjuMYB28 genes in different tissue types of B. juncea. Expression of the BjuMYB28 genes was performed across B. juncea (A) developing leaf stages and (B) developing stages of siliques. The stages are defined as: primary leaf (15 d), young leaf (30 d), mature leaf (60 d), flag (inflorescence) leaf, silique 5, 10, 15, and 30 dpa (days post-anthesis). qRT-PCR was conducted and expression values across different tissue types were normalized against B. juncea Actin gene expression (set at 100). Each bar represents the mean (±standard error) of three independent biological replicates. Different letters on the top indicate significant differences at P <0.05 in Tukey’s post hoc test.

Fig. 8.

Histochemical GUS staining of Promoter_BjuMYB28 -GUS transgenic Arabidopsis lines during different developmental stages and wounding. (A) Two-week old seedlings, (B) 4-week-old rosette leaves, (C) flowers, (D) immature silique, (E) mature silique, and (E) cut ends of leaf of Pro:GUS plants for all four BjuMYB28 homologues. Two independent single copy transgenic lines of each BjuMYB28 homologues were tested for the GUS histochemical assay in the T₃ generation.

Fig. 9.

Graphical comparison of expression profiles of BjuMYB28 genes across plant developmental stages in B. juncea. (A) The mean normalized expression value of AtMYB28 (identifier 247549_at) were obtained by normalizing absolute expression values to median across different tissue types available in the AtGenExpress Visualization tool (www.arabidopsis.org/), and plotted. (B) Graphical representation of the expression profiles of the four MYB28 homologues in B. juncea during the corresponding developmental stages. The colour of the box (data summarized from Figs 6 and 7) represents the comparative expression score of BjuMYB28 genes. The two ‘A’ and ‘B’ subgenome specific homologues are also marked. (C) The comparative scoring index was constructed from the fold expression values of BjuMYB28 genes obtained using real-time expression data as indicated.