Visualization of wounding-induced root-to-shoot communication in Arabidopsis

Abstract

It is known that wounding systemically activates the expression of various defense-related genes in plants. However, most studies of wound-induced systemic response are concerned with a leaf-to-leaf response. We have recently reported that the long distance signaling was also observed in the shoots of Arabidopsis seedling with wounded roots. We identified early and late root-to-shoot responsive (RtS) genes that were upregulated in the shoots of root-wounded seedlings at 30 min and 6 h post-injury, respectively. It is likely that the primary signals were rapidly transfered from injured roots to shoots, and then these signals were converted into chemical signals. In fact, increase of JA and OPDA content activated the expression of early and late RtS genes in shoots, respectively. In addition, we visualized wound-induced root-to-shoot response by using RtS pro-moter-luciferase (Luc) transgenic plants. Analysis of the AtERF13 promoter::Luc transgenic plants clearly shows that the wound-induced root-to-shoot signaling was rapidly activated via the vascular systems.

In higher plants, “root-to-hoot” signaling plays an important role in their adaptation in response to various environmental stresses such as dehydration, flooding, larvae attack and pathogen infection.^1–4 However, little attention has been paid to root-to-shoot systemic gene expression in response to stresses.⁵ To our knowledge, there have been no report of mechanical wounding-induced root-to-shoot systemic gene expression. In previous study, we found that the systemic induction of tobacco ethylene-responsive transcription factor (ERF) genes in shoots of seedlings with wounded roots (unpublished results). Therefore, we tried to establish a model system to monitor the gene expression during wound-induced root-to-shoot communication in Arabidopsis seedlings.⁶ Then, we have performed the transcriptome analysis of wound-induced root-toshoot response in Arabidopsis.⁶ When the roots of Arabidopsis seedlings were wounded, the expression in the shoots of many genes was upregulated more than three-fold at 30 min and 6 h post-injury.⁶

JA and OPDA Regulate the Wound-Induced Root-to-Shoot Response of RtS gene

Early RtS genes encode the transcription factors, jasmonic acid (JA) biosynthetic enzymes, mediators of calcium signaling and so on.⁶ It was reported that many of these genes were also rapidly induced by JA application.⁷ Root-to-shoot response of all early RtS genes examined was significantly decreased in the JA-deficient mutant. In addition, ethylene (ET)-related genes such as 1-aminocyclopropane-1-carboxylic acid (ACC) synthase 6 (ACS6) were also induced in the shoots with wounded roots.^6,8 In fact, root-to-shoot response of some early RtS genes examined in the ET-insensitive ein3 mutant was apparently low compared with wild type plants.⁶ On the other hand, late RtS genes contain two genes encoding vegetative storage proteins (VSP1 and VSP2), which are OPDA-inducible genes.⁷ Correspondingly, JA and OPDA content were apparently increased in shoots of seedlings 30 min and 360 min, respectively (Fig. 1). Therefore, it is likely that the JA and OPDA signaling pathway is involved in the root-to-shoot systemic response of RtS gene expression. In addition, the ET signaling is at least partially involved in the regulation of early RtS gene expression.

Figure 1.

JA and OPDA were involved in the regulation of RtS gene expression during wound-induced root-to-shoot communication. When root of seedlings were wounded with a razor blade, primary signals were generated in the wounded roots, and then rapidly transferred to the shoots. In shoots of seedlings with wounded roots, primary signals were converted to chemical signals, JA and OPDA. Increase of JA and OPDA content activate the expression of RtS genes in the shoots after 30 min and 360 min of root-wounding, respectively.

Expression of most RtS genes examined was not induced in the injured roots.⁶ Correspondingly, JA and OPDA content in roots were neither increased after 30 min or 6 h of root wounding.⁶ This data implied that neither JA nor OPDA directly moved from injured roots to shoots. It is likely that physical signals such as hydraulic signals were rapidly transfer from injured roots to shoots via vascular systems, and then these signals were converted into chemical signals such JA in shoots.^9,10 Next, we tried to monitor the spatial expression pattern of RtS genes during wound-induced root-to-shoot response. For this purpose, we introduced some RtS promoter::firefly Luc fusion genes into Arabidopsis plants.

Wound-Induced Root-to-Shoot Communication Occurred via Vascular Systems

As stated above, early RtS genes contained three ERF genes (AtERF1, AtERF2 and AtERF#109) in the B3 group of AP2/ERF superfamily.¹¹ Root-to-shoot response of the AtERF13 gene, which also belonged to the B3 group, was statistically insignificant by microarray analysis, but its increase in expression was confirmed by qPCR analysis.⁶ As shown in Figure 2A, the observed 5.1-fold increases of AtERF13 promoter activities in shoots of seedlings with wounded roots is quite consistent with the increase in expression of endogenous AtERF13 mRNA by qRT-PCR (6.1-fold increase).⁶ Results suggest that the rootto-shoot response of the AtERF13 gene is mainly regulated at the transcriptional level. Therefore, AtERF13 promoter::Luc transgenic plants were subjected to further analysis.

Figure 2.

Visualization of wounding-induced root-to-shoot communication of AtERF13 expression. (A) Quantitative analysis of luciferase activity of the AtERF13 promoter::Luc plants in shoots of seedlings with wounded roots and wounded leaves (local response). Proteins were extracted from each sample, and the luciferase activity was quantified. Fold changes are relative to those of untreated control samples. Each bar shows mean of three independent samples. Error bars represent the standard deviation. Similar results were obtained from two independent AtERF13 promoter::Luc transgenic lines (#8 and #10). (B) Visualization of wounding-induced root-to-shoot communication of AtERF13 expression. Two-week-old AtERF13 promoter::Luc #8 seedlings were sprayed with luciferin (Sigma-Aldrich). After 30 min of spraying, roots of seedlings were wounded. Luminescence from seedlings was continuously captured by a charge couple device (CCD) camera. In the right part, luminescence is shown in the seedling at 0, 30 and 120 min after wounding of the roots. In the left part, luminescence is shown in a mock-treated seedling.

As shown in Figure 2B, when roots of the AtERF13 promoter::Luc transgenic plants were wounded, the promoter activity was found to be increased in vascular systems of whole shoots within 30 min. In contrast, such a systemic activation was not observed in mock-treated plants. This result suggests that primary signals generated in the roots are rapidly transmitted to shoot via vascular systems, and converted into chemical signals such as JA, ultimately leading to the transcriptional activation of AtERF13 gene. As stated above, the promoter activity was not increased in injured roots. Thus, roots are capable of sensing environmental conditions in the subterranean area and generating signals that propagate signals to shoots through vascular systems. Root-to-shoot signaling communication plays an important role in environmental stress response especially in defense response.^3,12

Addendum to: Hasegawa S, Sogabe Y, Asano T, Nakagawa T, Nakamura H, Kodama H, et al. Gene expression analysis of wounding-induced rootto-shoot communication in Arabidopsis thaliana. Plant Cell Environ. 2011;34:705–16. doi: 10.1111/j.1365-3040.