Exogenous hormones influence Brassica napus leaf cuticular wax deposition and cuticle function

Abstract

Background: Cuticular waxes cover plant surface and play important roles in protecting plants from abiotic and biotic stresses. The variations of wax deposition and chemical compositions under changing environments have been shown to be related to plant adaptations. However, it is still not clear whether the wax depositions could be adjusted to increase plant adaptations to stressed conditions.

Methods: In this study, exogenous methyl jasmonate (MeJA), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and salicylic acid (SA) were applied to test their effects on cuticular wax deposition in two Brassica napus cultivars, Zhongshuang 9 (ZS9, low wax coverage ) and Yuyou 19 (YY19, high wax coverage). Next, we measured the water loss rate and the transcriptional expression of genes involved in wax biosynthesis as well as genes related to disease defense.

Results: Seven wax compound classes, including fatty acids, aldehydes, alkanes, secondary alcohols, ketones, and unbranched as well as branched primary alcohols, were identified in B. napus leaf wax mixtures. MeJA, SA and ACC treatments had no significant effect on total wax amounts in YY19, whereas ACC reduced total wax amounts in ZS9. Overall, hormone treatments led to an increase in the amounts of aldehydes and ketones, and a decrease of secondary alcohol in ZS9, whereas they led to a decrease of alkane amounts and an increase of secondary alcohol amounts in YY19. Concomitantly, both cultivars also exhibited different changes in cuticle permeability, with leaf water loss rate per 15 min increased from 1.57% (averaged across treatments) at 1.57% (averaged across treatments) at 15 min to 3.12% at 30 min for ZS9 (except for ACC treated plant) and decreased for YY19. MeJA-treated plants of both cultivars relatively had higher water loss rate per 15 min when compared to other treatments. Conclusion. Our findings that B. napus leaf wax composition and cuticle permeability are altered by exogenous SA, MeJA and ACC suggest that the hormone treatments affect wax composition, and that the changes in wax profiles would cause changes in cuticle permeability.

Keywords: Brassica napus; Cuticular wax; Permeability; Phytohormone; Water loss rate.

Figure 1. Cuticular wax biosynthetic pathways.

Text in rectangular box denotes wax compound classes. Small text outside rectangular box denotes genes involved in wax production.

Figure 2. Effects of SA (A), MeJA (B) and ACC (C) on cuticular wax associated gene expression in Zhongshuang 9 (ZS9) and Yuyou 19 (YY19).

Plants were treated with 0.2 mM SA, 0.1 mM MeJA or 0.2 mM ACC for 21 d and plants treated with 1% MS were as control. The data represented the average of three biological replicates plus/minus standard deviation. Bars with asterisk represent significance at P < 0.05 according to student T test when compared with the control for each gene.

Figure 3. Total wax amounts on Brassica napus leaves treated with stress hormones.

(A) Cultivar Zhongshuang 9 (ZS9) and (B) Cultivar Yuyou 19 (YY19). Plants were treated with 0.2 mM SA, 0.1 mM MeJA or 0.2 mM ACC for 21 d, then leaves were extracted with chloroform and resulting wax solutions analyzed using GC-FID. The data represent the average of four biological replicates plus/minus standard deviation, and different lowercase letters above the data bars represent significance at P < 0.05 according to least significant difference test using one-way ANOVA analysis.

Figure 4. Amounts of wax compositions on Brassica napus leaves treated with stress hormones.

(A) Cultivar Zhongshuang 9 (ZS9) and (B) Cultivar Yuyou 19 (YY19). Plants were treated with 0.2 mM SA, 0.1 mM MeJA or 0.2 mM ACC for 21 d, then leaves were extracted with chloroform and resulting wax solutions analyzed using GC-FID. The data represent the average of four biological replicates plus/minus standard deviation, and different lowercase letters above the data bars represent significance at P < 0.05 according to least significant difference test using one-way ANOVA analysis.

Figure 5. Water loss rates of isolated Brassica napus leaves treated with stress hormones.

(A) Cultivar Zhongshuang 9 (ZS9) and (B) Cultivar Yuyou 19 (YY19). Plants were treated with 0.2 mM SA, 0.1 mM MeJA or 0.2 mM ACC for 21 d, then water loss of isolated leaves was monitored every 15 min over 2.5 h. The water loss at each time point was expressed as the percentage of the water lost relative to the total water content. Data are shown as average of seven replicates plus/minus standard deviation.