Differential impact of lipoxygenase 2 and jasmonates on natural and stress-induced senescence in Arabidopsis

Abstract

Jasmonic acid and related oxylipins are controversially discussed to be involved in regulating the initiation and progression of leaf senescence. To this end, we analyzed profiles of free and esterified oxylipins during natural senescence and upon induction of senescence-like phenotypes by dark treatment and flotation on sorbitol in Arabidopsis (Arabidopsis thaliana). Jasmonic acid and free 12-oxo-phytodienoic acid increased during all three processes, with the strongest increase of jasmonic acid after dark treatment. Arabidopside content only increased considerably in response to sorbitol treatment. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols decreased during these treatments and aging. Lipoxygenase 2-RNA interference (RNAi) plants were generated, which constitutively produce jasmonic acid and 12-oxo-phytodienoic acid but do not exhibit accumulation during natural senescence or upon stress treatment. Chlorophyll loss during aging and upon dark incubation was not altered, suggesting that these oxylipins are not involved in these processes. In contrast, lipoxygenase 2-RNAi lines and the allene oxid synthase-deficient mutant dde2 were less sensitive to sorbitol than the wild type, indicating that oxylipins contribute to the response to sorbitol stress.

Figure 1.

Comparison of oxylipin profiles and chlorophyll content of Arabidopsis leaves during natural senescence and stress-induced senescence-like phenotypes. A to D, Natural senescence. Leaves were harvested at 6, 8, and 10 weeks of plant age. E to H, Dark incubation. Leaves were detached and harvested before treatment and at 3 and 7 d of incubation in the dark. I to L, Sorbitol flotation. Leaves were detached and harvested before treatment and at 24 and 48 h of flotation on 500 mm sorbitol. M to P, Water flotation. Leaves were detached and harvested before treatment and at 24 and 48 h of flotation on water. Shown are the levels of chlorophyll (A, E, I, and M), JA and OPDA (B, F, J, and N), arabidopsides (C, G, K, and O), and the two most prominent MGDGs and DGDGs (D, H, L, and P). Data represent means of at least three biological replicates ± sd. FW, Fresh weight.

Figure 2.

Comparison of oxylipin levels and chlorophyll content of leaves of the wild type (WT) and LOX2-RNAi lines during natural senescence. Leaves were harvested at 6, 8, and 10 weeks of plant age, and levels of JA (A), OPDA (B), and chlorophyll (C) were determined. Data represent means of at least three biological replicates ± sd. FW, Fresh weight.

Figure 3.

Comparison of oxylipin levels and chlorophyll content of leaves of the wild type (WT) and LOX2-RNAi lines during dark-induced senescence-like processes. Leaves were detached and harvested before treatment and at 3 and 7 d of incubation in the dark. Levels of JA (A), OPDA (B), and chlorophyll (C) were determined. Data represent means of at least three biological replicates ± sd. FW, Fresh weight.

Figure 4.

Comparison of oxylipin levels, chlorophyll content, and gene expression of leaves of the wild type (WT) and LOX2-RNAi lines during sorbitol-induced senescence-like processes. Leaves were harvested before treatment and at 24 and 48 h of flotation on 500 mm sorbitol, and levels of JA (A), OPDA (B), chlorophyll (D), and MGDG/DGDG (E) were determined. From the same plant material, the relative expression of SAG13 and SEN1 was analyzed by quantitative RT-PCR (F). Representative photograph of leaves at the 48-h time point is shown in C. Data represent means of at least three biological replicates ± sd for the 0- and 24-h time points and six biological replicates ± sd for the 48-h time point. Asterisks represent significant differences between the wild type and LOX2-RNAi lines within a time point (one-way ANOVA; P < 0.002). Black bars represent the wild type, and gray bars represent the LOX2-RNAi lines. FW, Fresh weight.

Figure 5.

Effect of sorbitol on germination of the wild type (WT) and dde2. Seeds of the wild type and dde2 were sown on Murashige and Skoog medium with (A) and without (B) sorbitol, and germinated seedlings were counted at the time points indicated. Shown are means of three independent petri dishes ± sd with 20 seedlings each. The experiment was repeated with similar results.