Glucosinolate Profiling and Expression Analysis of Glucosinolate Biosynthesis Genes Differentiate White Mold Resistant and Susceptible Cabbage Lines

Abstract

Sclerotinia stem rot (white mold), caused by the fungus Sclerotinia sclerotiorum, is a serious disease of Brassica crops worldwide. Despite considerable progress in investigating plant defense mechanisms against this pathogen, which have revealed the involvement of glucosinolates, the host⁻pathogen interaction between cabbage (Brassica oleracea) and S. sclerotiorum has not been fully explored. Here, we investigated glucosinolate profiles and the expression of glucosinolate biosynthesis genes in white-mold-resistant (R) and -susceptible (S) lines of cabbage after infection with S. sclerotiorum. The simultaneous rise in the levels of the aliphatic glucosinate glucoiberverin (GIV) and the indolic glucosinate glucobrassicin (GBS) was linked to white mold resistance in cabbage. Principal component analysis showed close association between fungal treatment and cabbage GIV and GBS contents. The correlation analysis showed significant positive associations between GIV content and expression of the glucosinolate biosynthesis genes ST5b-Bol026202 and ST5c-Bol030757, and between GBS content and the expression of the glucosinolate biosynthesis genes ST5a-Bol026200 and ST5a-Bol039395. Our results revealed that S. sclerotiorum infection of cabbage induces the expression of glucosinolate biosynthesis genes, altering the content of individual glucosinolates. This relationship between the expression of glucosinolate biosynthesis genes and accumulation of the corresponding glucosinolates and resistance to white mold extends the molecular understanding of glucosinolate-negotiated defense against S. sclerotiorum in cabbage.

Keywords: R line; S line; Sclerotinia sclerotiorum; cabbage; glucosinolates; white mold.

Figure 1

Leaf, stem and head bioassay of the susceptible line SCNU-C-033 (1, leaf; 2, stem; 3, head seen from top; 4, head seen from bottom) and resistant line SCNU-C-049 (5, leaf; 6, stem; 7, head seen from top; 8, head seen from bottom) of cabbage inoculated with S. sclerotiorum. Photographs were taken on the following days post inoculation (DPI): leaf, 5 DPI; stem, 7 DPI; head from top, 10 DPI; head from bottom, 15 DPI.

Figure 2

Contents of 11 individual glucosinolates in leaf samples from R (SCNU-C-049) and S (SCNU-C-033) lines of cabbage under different treatment conditions (C, control; M1, mock day 1; T1, treated day 1; M3, mock day 3; T3, treated day 3). The means of three biological replicates are presented. Vertical bars indicate standard deviation. Different letters indicate statistically significant differences between R and S lines and treatment interactions. Upward-pointing green arrows indicate increased glucosinolate content of R line in response to S. sclerotiorum infection. R, resistant; S, susceptible. HPLC–mass spectrometry (HPLC-MS) analysis (using an Agilent 1200 series instrument, Agilent Technologies) was conducted following Abuyusuf et al. [38].

Figure 3

Upregulation of MYB28-Bol017019, MYB34-Bol017062, ST5a-Bol026200, ST5a-Bol039395, ST5b-Bol026202 and ST5c-Bol030757 genes in white-mold-inoculated R (SCNU-C-049) line at 1 and 3 DPI compared to mock-treated plants. No similar upregulation was seen in a susceptible (S; SCNU-C-033) line. C, control; M1, mock day 1; T1, treated day 1; M3, mock day 3; T3, treated day 3. The means of three biological replicates are presented. Vertical bars indicate standard deviation. Different letters indicate statistically significant differences between R and S lines and treatment interactions. R, resistant; S, susceptible.

Figure 4

Upregulated transcription-factor- and glucosinolate-biosynthesis-related genes in white-mold-inoculated S (SCNU-C-033) cabbage at 1 DPI compared to mock-treated plants. No similar upregulation was seen in a resistant (R; SCNU-C-049) line. C, control; M1, M3, mock day 1; T1, treated day 1; M3, mock day 3; T3, treated day 3. The means of three biological replicates are presented. Vertical bars indicate standard deviation. Different letters indicate statistically significant differences between R and S lines and treatment interactions. R, resistant; S, susceptible.

Figure 5

Heat maps showing correlation between the levels of aliphatic (A) and indole (B) glucosinolate components and expression of biosynthesis genes under four specific treatments (C, control; M1, mock day 1; T1, treated day 1; M3, mock day 3; T3, treated day 3) in white-mold-inoculated R (SCNU-C-049) and S (SCNU-C-033) lines. Blue and bold letters represent statistically significant correlations (p < 0.05). For each gene and glucosinolate combination, the values indicate the Pearson correlation coefficient. Red cells represent positive correlation and green cells represent negative correlation. Yellow cells represent no significant correlation. Glucosinolate (GSL) components: GIB, glucoiberin; PRO, progoitrin; GRA, glucoraphanin; SIN, sinigrin; GNA, gluconapin; GIV, glucoiberverin; GER, glucoerucin; GBS, glucobrassicin; NGBS, neoglucobrassicin; MGBS, methoxyglucobrassicin; HGBS, hydroxyglucobrassicin. R, resistant; S, susceptible.

Figure 6

Biplot of white-mold-inoculated R (SCNU-C-049) and S (SCNU-C-033) cabbage lines and individual glucosinolate components, as determined by principal component analysis (PCA). Dark red squares denote mean PC scores of the R line, and blue circles those of the S line. Fungal treatments: C, control; M1, mock day 1; T1, treated day 1; M3, mock day 3; T3, treated day 3. Glucosinolate (GSL) components: GIB, glucoiberin; PRO, progoitrin; GRA, glucoraphanin; SIN, sinigrin; GNA, gluconapin, GIV, glucoiberverin; GER, glucoerucin; GBS, glucobrassicin; NGBS, neoglucobrassicin; MGBS, methoxyglucobrassicin; HGBS, hydroxyglucobrassicin. R, resistant; S, susceptible.

Figure 7

White mold disease progress in resistant (R; SCNU-C-049) and sensitive (S; SCNU-C-033) lines of cabbage. The third-youngest leaf was infected at the of ninth leaf stage of each plant. Infected leaves were examined at 0 hours post inoculation (HPI), and then from 1 to 5 days post inoculation (DPI). C, control plant leaf (no inoculation). ‘Mock’ treatment(s) - were those done with mycelium-free agar plugs.