Arabidopsis myrosinases link the glucosinolate-myrosinase system and the cuticle

Abstract

Both physical barriers and reactive phytochemicals represent two important components of a plant's defence system against environmental stress. However, these two defence systems have generally been studied independently. Here, we have taken an exclusive opportunity to investigate the connection between a chemical-based plant defence system, represented by the glucosinolate-myrosinase system, and a physical barrier, represented by the cuticle, using Arabidopsis myrosinase (thioglucosidase; TGG) mutants. The tgg1, single and tgg1 tgg2 double mutants showed morphological changes compared to wild-type plants visible as changes in pavement cells, stomatal cells and the ultrastructure of the cuticle. Extensive metabolite analyses of leaves from tgg mutants and wild-type Arabidopsis plants showed altered levels of cuticular fatty acids, fatty acid phytyl esters, glucosinolates, and indole compounds in tgg single and double mutants as compared to wild-type plants. These results point to a close and novel association between chemical defence systems and physical defence barriers.

Figure 1. Scanning electron micrographs of abaxial leaf surface of WT, and tgg1, tgg2 single and tgg1 tgg2 double mutants of Arabidopsis.

(a,b) WT: the non-stomatal pavement cells showing the characteristic jigsaw puzzle shape. (c,d) tgg1 single mutant: the pavement cells appeared relatively bigger, but showed jigsaw puzzle shape, and the stomatal guard cells appeared bigger (marked by white rectangles). (e,f) tgg2 single mutant: the pavement cells appeared bigger and flattened, showing irregular jigsaw puzzle shape, and the stomatal guard cells also appeared bigger (marked by white rectangles). (g,h) tgg1 tgg2 double mutant: the pavement cells appeared overlapping each other, collapsed at some places, and showed irregular jigsaw puzzle shape, and the stomatal guard cells appeared smaller, closed and sunken (marked by white rectangles). (a–h) (Scale bars, 50 μm).

Figure 2. Scanning electron micrographs of stomatal guard cells of WT and tgg1 tgg2 double mutant.

(a–d) WT. (e–h) tgg1 single mutant: the stomatal guard cells appear bigger. (i-l), tgg2 single mutant: the stomatal guard cells appear bigger. (m–p) tgg1 tgg2 double mutant: stomatal guard cells appear smaller, tightly closed and sunken showing variations as compared to the normal and open stomatal guard cells in the WT, and tgg1 and tgg2 single mutants. (a,b,e,h–j,l) (Scale bars, 5 μm). (c,d,f,g,k,m–p) (Scale bars, 2 μm).

Figure 3. Measures of stomatal size in WT, tgg1, tgg2 single mutants, and tgg1 tgg2 double mutant.

Average guard cell length and average guard cell width (stomatal aperture) were used as measures of stomatal size. Different letters above the bars indicate significant differences between WT, tgg1, tgg2 single mutants and tgg1 tgg2 double mutant (Kruskal-Wallis test, P < 0.05), followed by pairwise Wilcoxon Mann-Whitney tests and Bonferroni correction, P < 0.00417). Error bars represent the means ± SE (n = 100).

Figure 4. Ultrastructure differences in cuticle of rosette leaves of WT, and tgg1, tgg2 single and tgg1 tgg2 double mutants of Arabidopsis as examined through transmission electron microscopy (TEM).

(a,b) WT: The cuticle is visible as a continuous, condensed and dark electron dense apposition on the cell wall. (c,d) tgg1 single mutant, (e,f) tgg2 single mutant, (g,h) tgg1 tgg2 double mutant. The cuticle in tgg1, tgg2 single and tgg1 tgg2 double mutants is visible as a disrupted and irregular electron-dense apposition on the cell wall. Bars = 2 μm (X 18,500 magnification in a,c,e and g), =500 nm (X 68,000 magnification in b,d,f and h). Cu, cuticle; and CW, cell wall.

Figure 5. 2D principal component analysis (PCA) of 111 compounds (obtained from FAME (F), leaf cutin (C) and LCMS profiling (L) of WT and> tgg1, tgg2 single and tgg1 tgg2 double mutants of Arabidopsis (n = 5), based on log₂ ratio amended concentration levels (median) for single metabolites (Supplementary Table S4).

A total of 53.2% of variation is explained by PC1 and PC2. (a) PCA plot showing differences among wild-type and tgg single and double mutants for metabolic profiles. (b) Loading plot of metabolites explaining the observed variation in PCA plot, indicated by coloured lines for the different compound groups following the colour scheme used in Fig. 6. glucosinolates; sinapoyl esters; fatty alcohol; fatty acid ester; FAs; monoglycerides; phenolics; flavonol glycosides; hydroxycinnamic acids; indole compounds; diterpene alcohol; fatty acid phytyl esters; polyols; aldehydes; carbohydrate.

Figure 6. Hierarchical cluster analysis (HCA) (Pearson correlation) of 111 compounds (obtained from FAME (F), leaf cutin (C) and LCMS profiling (L)) of WT and tgg1, tgg2 single and tgg1 tgg2 double mutants of Arabidopsis.

Heat map visualization is based on log₂(n) ratio amended concentration levels (median) for single metabolites. Bluish colours indicate lower concentration levels, and reddish colours enhanced metabolite levels (see colour scale). Compound group colour code: glucosinolates; sinapoyl esters; fatty alcohol; fatty acid ester; FAs; monoglycerides; phenolics; flavonol glycosides; hydroxycinnamic acids; indole compounds; diterpene alcohol; fatty acid phytyl esters; polyols; aldehydes; carbohydrate.

Figure 7. Metabolic levels (log₂ ratio) of glucosinolates obtained from LC-TOF–MS analyses of WT and tgg single and double mutants.

Figure 8. Double bond index (DBI) of fatty acids obtained from FAME and leaf cutin analysis; and metabolic levels (log₂ ratio) of selected compounds obtained from FAME, leaf cutin and LC-TOF–MS analyses of WT and tgg single and double mutants.

(a) DBI of fatty acids. (b) Metabolic levels of some of the differentially accumulated fatty acids between the WT and tgg mutants (P < 0.05). (c) Metabolic levels of differentially accumulated fatty alcohol, 1-dodecanol between the WT and tgg mutants (P < 0.05). (d) Metabolic levels of some of the differentially accumulated indole compounds between the WT and tgg mutants (P < 0.05), (e) Metabolic levels of differentially accumulated compound sinapoylmalate between the WT and tgg mutants (P < 0.05).