Transcriptome Profiling of the Potato Exposed to French Marigold Essential Oil with a Special Emphasis on Leaf Starch Metabolism and Defense against Colorado Potato Beetle

Abstract

Flower strips of French Marigold are commonly used pest repellents in potato fields. However, the effect of French Marigold volatiles on potato metabolism, physiology and induced defense is unknown. Thus, a microarray transcriptome analysis was performed to study the effects of French Marigold essential oil (EO) on laboratory-grown potato. After 8 h of exposure to EO, with gas chromatography/mass spectrometry (GC/MS)-detected terpinolene and limonene as dominant compounds, 2796 transcripts were differentially expressed with fold change >2 compared to expression in controls. A slightly higher number of transcripts had suppressed expression (1493 down- vs. 1303 up-regulated). Since transcripts, annotated to different photosynthesis-related processes, were mostly down-regulated, we selected a set of 10 genes involved in the leaf starch metabolism pathway, and validated microarray patterns using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Except for decreased synthesis and induced decomposition of starch granule in leaves, 8 h long EO exposure slightly elevated the accumulation of sucrose compared to glucose and fructose in subjected potato plants. An in vitro feeding bioassay with Colorado potato beetle showed that EO-induced alternations on transcriptional level and in the sugars' metabolism caused the enhancement of feeding behavior and overall development of the tested larvae. Results of comprehensive analysis of transcriptional responses in potato exposed to French Marigold EO provide a basis for further elucidation of molecular mechanisms underlying eco-physiological interactions in companion planting cropping systems.

Keywords: Colorado potato beetle; French Marigold; essential oil; microarray; potato; starch metabolism.

Figure 1

Annotations of the acknowledged DE transcripts to Gene Ontology (GO) categories: BP—biological process, CC—cellular component and MF—molecular function. Top 10 categories for BP and MF, and top 9 categories for CC, per the total number of DE transcripts, with significant over representation, are presented. (A) Number of down- (blue) and up -regulated (red) DE transcripts in each category. Category over representation p-values are indicated with asterisks: ***—p-value < 10^{− 10}; **—p-value < 10⁻⁵; *—p-value < 10⁻³. (B) Percentage of DE transcripts from the total number of transcripts in the annotated category. (C) Boxplots of transcript log₂FC values for down- (blue) and up -regulated (red) DE transcripts in the corresponding category.

Figure 2

Annotations of the acknowledged DE transcripts to Interpro and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associations. Top 15 categories per the total number of DE transcripts, with significant over representation are presented. (A) Number of down- (blue) and up -regulated (red) DE transcripts in each category. Category over representation p-values are indicated with asterisks: ***—p-value <10⁻¹⁰; **—p-value <10⁻⁵; *—p-value <10⁻³. (B) Percentage of DE transcripts from the total number of transcripts in the annotated category. (C) Boxplots of transcript log₂FC values for down- (blue) and up -regulated (red) DE transcripts in the corresponding category.

Figure 3

(A) Proposed starch metabolism pathway and associated gene expression in potato leaves exposed to French Marigold EO for 8 h. Expression patterns of DE transcripts (p ≤ 0.05) obtained by cDNA microarray analysis were presented as a single rectangle on color-coded heat maps (blue, down-regulated; red, up-regulated). Fruc-6P, Fructose-6-phosphate; PGI, phosphoglucoisomerase; Glc-6P, glucose 6-phosphate; Glc-1P, glucose 1-phosphate; PGM, phosphoglucomutase; AGPase, ADP-glucose pyrophosphorylase; ADP-Glc, ADP-glucose; SS, starch synthase; GBSS, granule-bound starch synthase; SBE, starch branching enzyme; GWD, glucan, water dikinase; AMY, α-amylase; LSF, Like starch-excess Four; BAM, β-amylase; DPE, disproportionating enzyme; GLT, glucose transporter; MEX1, maltose transporter; Mal, maltose; Glc, glucose. RT-qPCR obtained expression profiles of potato leaf genes involved in starch (B) biosynthesis and (C) degradation after exposure to French Marigold EO for different time periods (4, 8 and 12 h). The fold change (FC) of the genes expression were obtained after ΔΔCt normalization to the expression of reference 18S gene and to the expression in non-exposed controls (left bars; log₂FC = 0) for each time point, and presented after log₂ transformation. For each gene at each time point, Welch’s t-test was used, and the obtained p-values were jointly adjusted using the FDR method for multiple comparisons. Asterisks denote adjusted p ≤ 5 × 10⁻² (n = 3).

Figure 4

Effect of French Marigold EO exposure of potato plants on feeding Colorado potato beetles (CPB) growth and developmental dynamics. (A) Feeding of CPB larvae from 2nd instar placed on potato plants previously exposed to EO for 8 h, throughout 3rd and 4th instar larvae on defoliated potato plants after 8 days of feeding. (B) Larval growth was assessed through daily weight measures for larvae fed on EO-exposed (EO) and control (C) potato plants. (C) Duration of 2nd through 4th larval stages. All values are expressed as mean ± SE (n = 25). Means denoted with * are significantly different according to the least significant difference (LSD) test (p ≤ 5 × 10⁻²), within each time point.