Soil water stress affects both cuticular wax content and cuticle-related gene expression in young saplings of maritime pine (Pinus pinaster Ait)

Abstract

Background: The cuticle is a hydrophobic barrier located at the aerial surface of all terrestrial plants. Recent studies performed on model plants, such as Arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. Although forest trees will face more intense drought stresses (in duration and intensity) with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported.

Results: This aspect was investigated in a conifer, maritime pine (Pinus pinaster Ait.), in a factorial design with two genetic units (two half-sib families with different growth rates) and two treatments (irrigated vs non-irrigated), in field conditions. Saplings were grown in an open-sided greenhouse and half were irrigated three times per week for two growing seasons. Needles were sampled three times per year for cuticular wax (composition and content) and transcriptome (of 11 genes involved in cuticle biosynthesis) analysis. Non-irrigated saplings (i) had a higher cuticular wax content than irrigated saplings and (ii) overexpressed most of the genes studied. Both these trends were more marked in the faster growing family.

Conclusions: The higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. This study provides also a set of promising candidate genes for future forward genetic studies in conifers.

Figure 1

Total height of the sapling over the two growing seasons. Dotted and solid lines represent the non-irrigated and irrigated treatments, respectively. Triangles and squares represent the “V-” and “V+” families, respectively. Abbreviations are as follows: T: treatment effect, G: family effect. **P value < 0.001 and ***P value < 0.0001.

Figure 2

Predawn leaf water potential over the two growing seasons studied (2008 and 2009). Dotted and solid lines represent the non-irrigated and irrigated treatments, respectively. Triangles and squares represent the “V-” and “V+” families, respectively. Standard deviations were obtained from 6 independent measurements. Abbreviations: T: treatment effect. ** P value < 0.01, ***P value < 0.001 and ****P value < 0.0001.

Figure 3

Changes in cuticular wax content in 2008 and 2009. Quantification was performed using three biological replicates. Abbreviations: V + NI: “V+” family, non-irrigated; V-NI: “V-” family, non-irrigated; V + I: “V+” family, irrigated; V-I: “V-” family, irrigated, T: treatment effect, G: family effect, GT: interaction effect, *P value < 0.1, ** P value < 0.01, ***P value < 0.001 and ****P value < 0.0001.

Figure 4

Expression profile of the most relevant candidate genes identified over the two growing seasons. Expression profiles for 2008 are on the left, whereas those for 2009 are on the right. Squares and red lines are used for the “V+” family for the non-irrigated treatment and triangles and orange lines are used for the “V-” family for the non-irrigated treatment. Squares and dark blue lines are used for the “V+” family for the irrigated treatment and triangles and light blue lines are used for the “V-” family for the irrigated treatment. Error bars represent the standard deviation (N = 3). Results of two-way ANOVA are also indicated above each sampling point. Abbreviations are as follows: T: treatment effect, G: family effect, TG: interaction effect. * P value < 0.01, **P value < 0.001 and ***P value < 0.0001.

Figure 5

Schematic representation of the metabolic pathway involved in cuticular wax biosynthesis. A significant effect over the two growing seasons was observed for genes in red. For genes in blue, a significant effect was found only in 2008, and genes in black displayed very little variation of expression.