Mechanostimulation of Medicago truncatula leads to enhanced levels of jasmonic acid

Abstract

Wounding of plants leads to endogenous rise of jasmonic acid (JA) accompanied with the expression of a distinct set of genes. Among them are those coding for the allene oxide cyclase (AOC) that catalyses a regulatory step in JA biosynthesis, and for 1-deoxy-D-xylulose 5-phosphate synthase 2 (DXS2), an enzyme involved in isoprenoid biosynthesis. To address the question how roots and shoots of Medicago truncatula respond to mechanostimulation and wounding, M. truncatula plants were analysed in respect to JA levels as well as MtAOC1 and MtDXS2-1 transcript accumulation. Harvest-caused mechanostimulation resulted in a strong, but transient increase in JA level in roots and shoots followed by a transient increase in MtAOC1 transcript accumulation. Additional wounding of either shoots or roots led to further increased JA and MtAOC1 transcript levels in shoots, but not in roots. In situ hybridization revealed a cell-specific transcript accumulation of MtAOC1 after mechanostimulation in companion cells of the vascular tissue of the stem. AOC protein, however, was found to occur constitutively in vascular bundles. Further, transcript accumulation of MtDXS2-1 was similar to that of MtAOC1 in shoots, but its transcript levels were not enhanced in roots. Repeated touching of shoots increased MtAOC1 transcript levels and led to significantly shorter shoots and increased biomass. In conclusion, M. truncatula plants respond very sensitively to mechanostimulation with enhanced JA levels and altered transcript accumulation, which might contribute to the altered phenotype after repeated touching of plants.

Keywords: 1-deoxy-D-xylulose 5-phosphate synthase 2; Allene oxide cyclase; Medicago truncatula; cell specific expression; jasmonic acid; mechanostimulation; wounding.

Fig. 1.

Harvest- and wound-induced accumulation of JA in roots and shoots of 5-week-old M. truncatula plants. Plants were carefully removed from expanded clay and stayed untreated (white columns) or were wounded on roots (cross-hatched columns). From a third batch of plants shoots and roots were separated from each other and both were wounded (dark grey columns). At each time point, up to five plants were pooled, and roots and shoots were separately extracted for quantification of JA. The means ±SD are given for five biological replicates and are tested for each treatment with one-way ANOVA followed by Tukey HSD test (P <0.05). Means sharing the same letters are not significantly different.

Fig. 2.

Harvest- and wound-induced transcript accumulation of MtAOC1 in roots and shoots of 5-week-old M. truncatula plants. Relative MtAOC1 transcript levels were determined by real-time RT-PCR analysis using TaqMan probes. Plants were carefully removed from expanded clay and stayed untreated (white columns) or were wounded on roots (cross-hatched columns). From a third batch of plants, shoots and roots were separated from each other and both were wounded (dark gray columns). At each time point, up to five plants were pooled, and total RNA was extracted from roots and shoots separately. Real-time RT-PCR was carried out in triplicate for each sample. The mean MtAOC1 transcript level of plants directly after harvest (time point 0) was set to 1. Data are presented as mean values ±SD of at least three biological replicates. Different letters designate statistically different values separately for each treatment (ANOVA with Tukey HSD test, P < 0.05).

Fig. 3.

Localization of MtAOC1 transcripts in stems of M. truncatula 2 h after wounding of roots. In situ hybridization was performed on cross-sections 16 μm thick. (A) Hybridization with DIG-labelled antisense RNA for MtAOC1 shows staining in the phloem of the vascular bundles. (B) The close-up of one vascular bundle visualizes the occurrence of the signals in companion cells (arrows). (C) Control performed using DIG-labelled sense RNA shows no staining in the complete section. Scale bars represent 100 μm in all micrographs.

Fig. 4.

Immunolocalization of AOC protein in cross-sections of stem of untreated M. truncatula plants. AOC was immunolabelled by an antibody against tomato AOC followed by a secondary antibody coupled to AlexaFluor488. The occurrence of AOC is visible by the green fluorescence signal. To visualize DNA-containing organelles, sections were counterstained with DAPI (C, D). (A) Bright-field image of a cross-section. (B) The same section as in (A) illuminated for green fluorescence. The green label indicative of AOC is visible in the pith, phloem tissue, and cortex. (C, D) Confocal laser scanning micrographs of the immunolabelled sections showing the occurrence of AOC in plastids of parenchymatic cells of pith, phloem, and cortex. The blue staining visualizes the nuclei due to the fluorescence of DAPI. Scale bars represent 500 μm in A and B, 100 μm in C, and 50 μm in D.

Fig. 5.

Harvest- and wound-induced transcript accumulation of MtDXS2-1 in roots and shoots of 5-week-old M. truncatula plants. Relative MtDXS2-1 transcript levels were determined using real-time RT-PCR analysis. Plants were carefully removed from expanded clay and stayed untreated (white columns) or were wounded on roots (cross-hatched columns). From a third batch of plants shoots and roots were separated from each other and both were wounded (dark grey columns). At each time point, up to five plants were pooled, and total RNA was extracted from roots and shoots separately. Real-time RT-PCR was carried out in triplicate for each sample. The mean MtDXS2-1 transcript level of plants directly after harvest (time point 0) was set to 1. Data are presented as mean values ±SD from at least four biological replicates. Different letters designate statistically different values for each treatment (ANOVA with Tukey HSD test, P < 0.01).

Fig. 6.

Effect of repeated touching of shoots on plant growth of M. truncatula. Plants were grown without touching (A) or with repeated touch stimulation performed by touching shoots for 10 s three times per week (B). (This figure is available in colour at JXB online).