Engineered coumarin accumulation reduces mycotoxin-induced oxidative stress and disease susceptibility

Abstract

Coumarins can fight pathogens and are thus promising for crop protection. Their biosynthesis, however, has not yet been engineered in crops. We tailored the constitutive accumulation of coumarins in transgenic Nicotiana benthamiana, Glycine max and Arabidopsis thaliana plants, as well as in Nicotiana tabacum BY-2 suspension cells. We did so by overexpressing A. thaliana feruloyl-CoA 6-hydroxylase 1 (AtF6'H1), encoding the key enzyme of scopoletin biosynthesis. Besides scopoletin and its glucoside scopolin, esculin at low level was the only other coumarin detected in transgenic cells. Mechanical damage of scopolin-accumulating tissue led to a swift release of scopoletin, presumably from the scopolin pool. High scopolin levels in A. thaliana roots coincided with reduced susceptibility to the root-parasitic nematode Heterodera schachtii. In addition, transgenic soybean plants were more tolerant to the soil-borne pathogenic fungus Fusarium virguliforme. Because mycotoxin-induced accumulation of reactive oxygen species and cell death were reduced in the AtF6'H1-overexpressors, the weaker sensitivity to F. virguliforme may be caused by attenuated oxidative damage of coumarin-hyperaccumulating cells. Together, engineered coumarin accumulation is promising for enhanced disease resilience of crops.

Keywords: coumarin; disease resistance; mycotoxin; reactive oxygen species; soybean; stress tolerance.

Figure 1

Transient overexpression of AtF6‘H1 causes scopoletin accumulation in N. benthamiana. Scopoletin (a) and AtF6‘H1 mRNA transcript (b) in N. benthamiana leaves overexpressing AtF6‘H1 or C‐ or N‐terminal fusions of AtF6‘H1 with GFP, RFP or FLAG controlled by the constitutive CaMV 35S promoter. P19 silencing inhibitor was co‐expressed in all cases. Expression of P19 only served as negative control. Shown are means ± SD of three independent experiments at 3 days after transient transformation. (c) Photos of transiently transformed leaves with characteristic fluorescence of scopoletin (in UV light; upper panel), GFP (middle panel) and RFP (lower panel). (d) AtF6‘H1‐GFP co‐localizes with cytoplasmic RFP in transiently transformed epidermal cells of N. benthamiana. GFP and RFP fluorescence were analysed by confocal laser scanning microscopy. (e, f) Accumulation of GFP and RFP and fusions of both fluorescent proteins and FLAG with AtF6‘H1 was verified by SDS‐PAGE and immunodetection using antibodies specific to GFP, RFP and FLAG.

Figure 2

Stable overexpression of AtF6‘H1 in Arabidopsis and soybean results in constitutive coumarin accumulation. Representative photos of adaxial and abaxial sides of detached leaves of Arabidopsis (a) and soybean (b) wild‐type and AtF6‘H1‐overexpressing transgenic lines were inspected in ambient or UV light. Only the transgenic plants showed characteristic blue fluorescence of leaves in UV light. (c) Scopoletin and scopolin content in roots and leaves of wild‐type Arabidopsis (left panel) and soybean (right panel) and two independent events overexpressing AtF6’H1, respectively. Coumarin concentration in methanolic extracts of 3‐to‐6‐week‐old plants was quantified by HPLC. Shown is the average + SD of three independent experiments.

Figure 3

Scopoletin and scopolin mainly accumulate within the cells. (a) AtF6‘H1‐expressing tobacco BY‐2 cells, but not the untransformed wild type, and their culture medium fluoresce in UV light. Tobacco BY‐2 cells were separated from the growth medium by vacuum filtration at 7 days after subculture. Cells and medium individually inspected in ambient and UV light. (b) Scopoletin and scopolin content of AtF6‘H1‐overexpressing BY‐2 cells and their culture medium relative to the total amount of scopoletin and scopolin in the batch culture. Average values and SD of three technical replicates are shown.

Figure 4

Upon destruction of AtF6‘H1‐overexpressing leaves scopolin is hydrolysed to scopoletin. Leaves of transgenic soybean (a) and Arabidopsis (b) plants were homogenized and analysed for their content of scopolin and scopoletin at the indicated times after tissue disruption (hpt). Average values + SD of three biological replicates are shown.

Figure 5

Transgenic Arabidopsis plants with high scopolin levels in root are less susceptible to H. schachtii. Wild‐type Arabidopsis, f6‘h1 mutants, AtF6‘H1 overexpressors and azygous Arabidopsis plants were grown on agar plates. At 12 days after inoculation with nematodes infection, severity and root content of scopolin were determined. High scopolin levels in roots exceeding 200 μg/g FW coincided with reduced female numbers. Shown are (a) the root scopolin content and infection scores from individual plates with two plants per genotype, respectively. The average infection severity (b) of groups containing more (group >200) or less (group <200) than 200 μg/g FW scopolin in the root. The asterisk marks statistically significant differences between groups >200 and <200 (t‐test; P < 0.05). Three independent experiments with at least four plates (2 plants per plate) per genotype were performed. Data from (a and b) are derived from identical experiments but illustrated in different way.

Figure 6

Stable overexpression of AtF6‘H1 in soybean induces SDS tolerance. Wild‐type plants and two independent AtF6‘H1‐overexpressing soybean events were hydroponically grown for 7 days prior to root inoculation with spores of F. virguliforme. (a) Wild‐type and transgenic plants show similar root rotting at 14 days after inoculation. (b) Shoot biomass (fresh weight) of infected wild‐type plants or transgenic plants was calculated in comparison with mock‐inoculated plants 14 days after inoculation. Shown are min‐to‐max boxplots with all data points, average values (+) and SD of three independent experiments with nine plants per genotype and treatment. Asterisks indicate significant differences to the wild‐type control according to Dunnett's multiple comparisons test with P ≤ 0.05. (c) Abundance of F. virguliforme DNA in infected roots of transgenic and wild‐type plants at 14 days after inoculation. DNA was extracted from ≥22 plants per genotype for fungal DNA quantification by qRT‐PCR (real‐time quantitative reverse transcription PCR). Shown are min‐to‐max boxplots with all data points, average values (+) and SD of three independent experiments. Differences among wild‐type and transgenic plants were not significant according to Dunnett's multiple comparisons test.

Figure 7

AtF6‘H1 overexpression is associated with decreased ROS accumulation upon mycotoxin exposure. Leaf discs from trifoliate leaves of 19‐day‐old wild‐type or transgenic soybean were floated on (a) water or (b) 30 μg/mL FA. ROS accumulation was recorded over time by luminol‐amplified chemiluminescence. Average values ± SD from three independent experiments are shown. (c) Area under the curve (AUC) values (mean ± SEM) were calculated from relative luminescence intensities (RLUs) as measured by the ROS assay for (a) water and (b) FA treatment. Asterisks mark statistical difference relative to the respective treatment in wild‐type cells (Sidak's multiple comparisons test, P < 0.0001). (d) Treatment with FA and DON induces ROS production in wild‐type BY‐2 cells but not in RFP‐AtF6’H1‐overexpressing cells. Cells were treated with 30 μg/mL FA or 50 μg/mL DON or water 7 days after subculture and ROS levels quantified by measuring fluorescence intensity in a plate reader following addition of the dye 2′,7′‐dichlorofluorescin diacetate (H2DCFDA). Average values and SD from three independent experiments are shown. Asterisks mark statistical difference relative to the respective treatment in wild‐type cells (Sidak's multiple comparisons test, P < 0.0001). (e) Viability of wild‐type and transgenic BY‐2 cells was analysed by confocal laser scanning microscopy 4 h after treatment with FA, DON or water (control) and addition of FDA and propidium iodide. (f) Differences were semi‐quantitively measured by counting pixels in the respective FDA or PI channels and calculating ratios. Shown are average values and SD from three independent experiments. Asterisks mark statistical difference relative to the respective control treatment (Dunnett's multiple comparisons test, P < 0.001).