Plant Defense Stimulator Mediated Defense Activation Is Affected by Nitrate Fertilization and Developmental Stage in Arabidopsis thaliana

Abstract

Plant defense stimulators, used in crop protection, are an attractive option to reduce the use of conventional crop protection products and optimize biocontrol strategies. These products are able to activate plant defenses and thus limit infection by pathogens. However, the effectiveness of these plant defense stimulators remains erratic and is potentially dependent on many agronomic and environmental parameters still unknown or poorly controlled. The developmental stage of the plant as well as its fertilization, and essentially nitrogen nutrition, play major roles in defense establishment in the presence of pathogens or plant defense stimulators. The major nitrogen source used by plants is nitrate. In this study, we investigated the impact of Arabidopsis thaliana plant developmental stage and nitrate nutrition on its capacity to mount immune reactions in response to two plant defense stimulators triggering two major defense pathways, the salicylic acid and the jasmonic acid pathways. We show that optimal nitrate nutrition is needed for effective defense activation and protection against the pathogenic bacteria Dickeya dadantii and Pseudomonas syringae pv. tomato. Using an npr1 defense signaling mutant, we showed that nitrate dependent protection against D. dadantii requires a functional NPR1 gene. Our results indicate that the efficacy of plant defense stimulators is strongly affected by nitrate nutrition and the developmental stage. The nitrate dependent efficacy of plant defense stimulators is not only due to a metabolic effect but also invloves NPR1 mediated defense signaling. Plant defense stimulators may have opposite effects on plant resistance to a pathogen. Together, our results indicate that agronomic use of plant defense stimulators must be optimized according to nitrate fertilization and developmental stage.

Keywords: Bion; Dickeya dadantii; Methyl-jasmonate; Pseudomonas syringae pv. tomato; defense (induced); developmental stage; elicitor; nitrate.

FIGURE 1

Impact of different nitrate fertilization levels on Arabidopsis thaliana physiological traits. Plants were cultivated until the indicated developmental stages (four stages) under the indicated nutritional conditions (2, 10, 26 mM). (A) Total nitrate content was quantified in healthy plants. (B) Total amino acid content was quantified. (C) Number of leaves per plant. (D) Projected rosette surface. (E) Picture of representative plants cultivated under indicated levels of nitrate at indicated developmental stages. N = 20. Error bars represent standard deviation. Letters indicate similarities or differences based on a t-test performed to compare samples of the same stage (p < 0.05). Experiments were performed three times with similar results. Representative data are shown.

FIGURE 2

Impact of developmental stage on defense gene expression 48 h after plant defense stimulator treatment. Plants were cultivated until the indicated developmental stages (four stages) under the indicated nutritional conditions (2, 10, 26 mM). They were treated with Bion^®, MeJA or water as a control then harvested 48 h after treatment, 6 to 10 plants were harvested for each treatment in each experiment. Defense gene expression was monitored by q-RT-PCR. Data represent normalized transcript levels using Clathrin as a reference gene. (A–D) Indicate defense gene expression following water treatment, (E–H) Indicate defense gene expression following Bion^® treatment; (I–L) Indicate defense gene expression following MeJA treatment. Experiments were performed 3 to 4 times with similar results. Representative data are shown. Error bars represent standard error. Different letters represent statistically significant differences between plant defense stimulator treatments under the same nutritional condition (p < 0.05 as calculated by t-test).

FIGURE 3

Impact of nitrate nutrition on defense gene expression 48 h after plant defense stimulator treatment. Plants were cultivated until the indicated developmental stages (four stages) under the indicated nutritional conditions (2, 10, 26 mM). They were treated with Bion^®, MeJA or water as a control then harvested 48 h after treatment, 6 to 10 plants were harvested for each treatment in each experiment. Defense gene expression was monitored by q-RT-PCR. Data represent normalized transcript levels using Clathrin as a reference gene. (A–D) Indicate defense gene expression at stage 1; (E–H) Indicate defense gene expression at stage 2; (I–L) Indicate defense gene expression at stage 3; (M–P) Indicate defense gene expression at stage 4. Experiments were performed 3 to 4 times with similar results. Representative data are shown. Error bars represent standard error. Different letters represent statistically significant differences between plant defense stimulator treatments under the same nutritional condition (p < 0.05 as calculated by t-test).

FIGURE 4

Impact of nitrate fertilization on protection against the pathogenic bacterium Pseudomonas syringae pv. tomato after plant defense stimulator application. Plants were cultivated until developmental stage 2 (rosette) under the indicated nitrate nutritional conditions (2, 10, 26 mM). They were treated with Bion^®, MeJA or water as a control, then inoculated with P. syringae pv. tomato 48 h after plant defense stimulator treatment. Bacterial populations were monitored. Error bar represent standard deviation. N = 20 leaves. Different letters (A) or stars (B) represent statistically significant differences between control and plant defense stimulator treatments under the same nutritional conditions, NS represent “No Significant” differences (p < 0.05 as calculated by t-test). Experiments were performed three times with similar results. Representative data are shown.

FIGURE 5

Impact of nitrate fertilization on protection against the pathogenic bacterium D. dadantii after plant defense stimulator application on an npr1 mutant. Plants were cultivated until developmental stage 2 (rosette) under the indicated nitrate nutritional conditions (2, 10, or 26 mM). They were treated with Bion^®, MeJA or water as a control, then inoculated with D. dadantii 48 h after plant defense stimulator treatment. Symptoms were scored 48 h after inoculation. Percentage of macerated leaf surface are represented. Error bars represent standard error. N = 20 leaves. Stars represent statistically significant differences between npr1-1 mutant plants and wild type Col-0 WT (A) and between control and MeJA and Bion^®, treatment (B) for each level of fertilization. NS represent “No Significant” differences (p < 0.05 as calculated by t-test). Experiments were performed three times with similar results. Representative data are shown.

FIGURE 6

Impact of nitrate nutrition on defense gene expression 48 h after elicitor treatment. Plants were cultivated until the developmental stage 2 under the indicated nutritional conditions (2, 10, 26 mM nitrate). They were treated with Bion^®, MeJA or water as a control then harvested 48 h after treatment, 6 to 10 plants were harvested for each treatment in each experiment. Transcript levels of indictade genes, PR1 (A), PR5 (B), LOX2 (C), and PDF1.2 (D) was monitored by qRT-PCR. Data represent normalized transcript levels using Clathrin as a reference gene. Bars indicate comparisons between control and plant defense stimulator treatments under the same nutritional condition. When only a bar is visible, the difference is significant (p < 0.05 as calculated by t-test). NS: statistically Non-Significant differences between control and plant defense stimulator treatments under the same nutritional condition.