Activation of indolic glucosinolate pathway by extracellular ATP in Arabidopsis

Dear Editor,

Extracellular ATP (eATP) is a purinergic signal recognized by plasma membrane-localized transmembrane receptors, such as P2X or P2Y receptors found in mammals (Verkhratsky and Burnstock, 2014), and the P2K receptors found in plants (Choi et al., 2014; Pham et al., 2020). In mammals, eATP and purinoceptors are the basis of intercellular signaling used to regulate diverse processes including neuronal signaling, apoptosis, and inflammation (Verkhratsky and Burnstock, 2014). In plants, eATP and P2K1 appear to influence root growth (Weerasinghe et al., 2009; Zhu et al., 2017), but are currently best characterized as a damage associated molecular pattern signal and recognition system contributing to plant defense (Tanaka et al., 2014). In this study, we found that the indolic glucosinolate pathway is induced by eATP in Arabidopsis (Arabidopsis thaliana) and may play a role as a biochemical defense when plants are damaged by pathogens and herbivores.

Accumulating evidence from our group and others suggests that eATP is released to trigger plant responses to various biotic stresses and touch/wounding (Dark et al., 2011; Ramachandran et al., 2019). Recently, the P2K1 overexpression line OxP2K1 was reported to enhance plant resistance against various foliar pathogens, such as Phytophthora brassicae (biotrophic oomycete), Pseudomonas syringae (hemibiotrophic bacterium), Botrytis cinerea (necrotrophic fungus), and Rhizoctonia solani (necrotrophic root fungus), whereas a knockout mutant line of P2K1, dorn1-3, showed increased susceptibility (Bouwmeester et al., 2011; Wang et al., 2014, 2016; Balagué et al., 2017; Chen et al., 2017; Tripathi et al., 2018; Kumar et al., 2020). As shown in Table 1 and Supplemental Figures S1–S4, a similar trend was observed with Sclerotinia sclerotiorum (necrotrophic fungus) and Phytophthora capsici (hemi-biotrophic oomycete), overexpression of P2K1 made the plants more resistant to the parasitic nematode Meloidogyne javanica, and, interestingly, pre-treatment of Arabidopsis with eATP made the plants more resistant to the turnip mosaic virus (TuMV). These data suggest that eATP plays an important role in plant defense against a broad range of pathogens. Furthermore, OxP2K1 showed enhanced resistance against Pieris rapae (specialist) and Spodoptera exigua (generalist) (Supplemental Figure S5), suggesting that eATP also plays an important role in the plant defense response against wounding caused by herbivorous insects. How eATP signaling contributes to defense against varied pests is not well understood.

Table 1.

P2K1-mediated eATP signaling enhances plant resistance against multiple pathogens

Categories	Pathogens	Host plants	ATP treatment	P2K1 involvement	References
Viruses	TuMV	Arabidopsis	Reduced infection	N.D.	In this study (Supplemental Figure S1)
Bacteria	P. syringae	Arabidopsis	Reduced infection	Knockout mutant is susceptible; Overexpression line is resistant	Bouwmeester et al., 2011; Balagué et al., 2017; Chen et al., 2017;
Fungi	B. cinerea	Arabidopsis	Reduced infection	Knockout mutant is susceptible; Overexpression line is resistant	Tripathi et al., 2018
	R. solani	Arabidopsis	Reduced infection	Knockout mutant is susceptible; Overexpression line is resistant	Kumar et al., 2020
	S. sclerotiorum	Arabidopsis	Reduced infection	Overexpression line is resistant	In this study (Supplemental Figure S2)
Oomycetes	P. brassicae	Arabidopsis	N.D.	Overexpression line is resistant	Bouwmeester et al., 2011
	P. infestans	Potato, N. benthamiana	N.D.	Overexpression line is resistant	Bouwmeester et al., 2014; Wang et al., 2014, 2016
	P. capsici	Arabidopsis, N. benthamiana	Reduced infection	Knockout mutant is susceptible; Overexpression line is resistant	In this study (Supplemental Figure S3); Wang et al., 2014, 2016
Nematodes	M. javanica	Arabidopsis	N.D.	Overexpression line is resistant	In this study (Supplemental Figure S4)
Insects	P. rapae	Arabidopsis	N.D.	Overexpression line is resistant	In this study (Supplemental Figure S5)
	S. exigua	Arabidopsis	N.D.	Overexpression line is resistant	In this study (Supplemental Figure S5)

N.D., not determined.

Detailed analysis in our previous eATP-induced transcriptomes (Jewell et al., 2019) highlighted several secondary metabolite pathways (Supplemental Figure S6) based on surveys in the PlantCyc database in the Plant Metabolic Network (https://pmn.plantcyc.org/). Notably, one of these pathways was the indolic glucosinolate biosynthesis pathway (Figure 1A), which produces a set of defense-related metabolites found in order Brassicales and known to deter attack by a broad range of pathogens and herbivores (Wittstock and Burow, 2010). Most of the genes involved in glucosinolate biosynthesis were significantly upregulated in ATP-induced transcriptomes (Figure 1B). These gene expression levels were further exaggerated in OxP2K1, whereas they were attenuated in dorn1-3. This correlation between P2K1 function and gene expression of glucosinolate biosynthesis genes is also corroborated by transcriptome coexpression data (ATTED-II; https://atted.jp/), in which glucosinolate biosynthesis genes were overrepresented in the top 100 list of P2K1-associated coexpression genes (Supplemental Data Set S1).

Figure 1.

Genes in the indole glucosinolate pathway are induced after eATP treatment. A, Overview of genes and metabolites in indolic glucosinolate metabolic pathway. B, Expression of glucosinolate biosynthetic genes 30 min after eATP treatment. Colors represent log2 fold change and asterisks indicate significant changes (*, **, and *** represent false-discovery rate with P < 0.05, P < 0.01, and P < 0.001, respectively).

We next performed untargeted metabolic profiling by liquid chromatography-quadrupole time-of-flight-mass spectrometry (UPLC-qTOF-MS) from ATP-treated leaf materials. We compared methanolic extracts of 4-week-old intact leaves infiltrated with or without ATP for 6 or 24 h. In this experiment, 0.5 mM of ATP was used since we previously found that ATP-induced transcriptional changes are maximal at this concentration with no impact of pH changes (Jewell et al., 2019). Overall, 7,169 and 6,805 metabolic features (mass-to-change ratio [m/z] ions) were detected in negative and positive ionization mode, respectively. Partial least-squares discriminant analysis (PLS-DA) was used as a supervised multivariate analysis to compare general features of the metabolites identified in ATP- versus mock-treated samples. For example, data at 24 h after ATP treatment are shown in Figure 2A, which indicated a substantial separation in features identified between the different samples. Among these, we noted an increase among the glucosinolate/camalexin-related compounds 4-benzoyloxybutyl glucosinolate, raphanusamic acid, and indole-3-carboxaldehyde (Figure 2B andSupplemental Figure S7). In other respects, the eATP-induced metabolomes included increased metabolites in arabidopside B (Supplemental Figure S8A) and several cutin monomers (Supplemental Figure S8B), and a decrease in several phenylpropanoids (Supplemental Figure S9).

Figure 2.

Accumulation of indolic glucosinolate metabolites upon ATP treatment. Untargeted (A and B) and targeted metabolomics (C) were performed using UPLC-qTOF-MS in 4-week-old plant leaves treated with 2-morpholinoethanesulfonic acid (MES) buffer (mock) or 0.5 mM ATP. A, PLS-DA of untargeted metabolite profiles of WT leaf tissues at 24 h after infiltration with or without 0.5 mM ATP. B, Abundance (in normalized arbitrary units) of a putative glucosinolate-related metabolite, 4-benzoyloxylbutyl glucosinolate, was identified at 6 and 24 h after infiltration of wild-type leaves with or without 0.5 mM ATP. This tentative identification was based on accurate mass and isotope model with a database search in KNApSAcK (http://kanaya.naist.jp/KNApSAcK/). Data represent mean ± sem, n = 6, **P < 0.01, Fisher’s least significant difference (LSD) for comparison with control at each time point. mAU, milliabsorbance units. C, Relative amounts of glucosinolates in the leaves of wild type, dorn1-3, and OxP2K1 at 24 and 48 h after infiltration with or without 0.5 mM ATP. Data represent mean ± sem, n = 6. Statistical results with ANOVA of the pooled data are shown in Supplemental Table S1. Data from another replicate experiment are shown in Supplementary Figure S9. 14a and 14b, C6-aliphatic glucosionolates; 16a, C7-aliphatic glucosinolate; 8MTO, 8-methylthiooctyl; and 7MTH, 7-methylthioheptyl.

To examine accumulation of 16 different glucosinolate metabolites triggered by eATP perception, we infiltrated ATP into leaves of wild type, dorn1-3, and OxP2K1, and harvested tissue at 24 and 48 h. A factorial analysis of variance (ANOVA) of the pooled data revealed significant effects of ATP treatment (P < 0.05). When we consider the metabolites together (Figure 2C;Supplemental Figure S10 and Supplemental Table S1), glucosinolates were more induced in OxP2K1 at the earlier time point of 24 h (both Trials #1 and #2) and occasionally at 48 h (Trial #1), while their accumulations were higher in wild type in comparison to dorn1-3 at 48 h (Trial #1). In summary, these data suggest that P2K1-mediated eATP signaling activates the glucosinolate biosynthesis pathway.

In this study, we found that eATP induces glucosinolate biosynthesis in Arabidopsis (Figure 2), likely via an up-regulation of its biosynthetic genes (Figure 1), potentially revealing secondary metabolites for eATP-induced defense against a wide array of pathogens and pests (Table 1). To corroborate our conclusions, testing glucosinolate mutants and/or varying timepoints of prior ATP treatment followed by pathogen attack are required in the future.

In other plants, eATP as a general defense-inducing signal is suggested by the ability to enhance tobacco resistance to tobacco mosaic virus and P. syringae pv. tabaci (Chivasa et al., 2009). Additionally, over-expression of the Arabidopsis P2K1 gene in the non-glucosinolate-producing plants, potato and Nicotiana benthamiana confers resistance to P. capsici and to Phytophthora infestans (Table 1). Thus, it would be interesting to profile the induced defensive compounds of other plants in response to eATP in the future. These studies will further advance our understanding of plant defense mechanisms induced by eATP signaling that is evolutionary conserved within the plant kingdom (Tanaka and Heil, 2021).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. eATP reduces infection of TuMV.

Supplemental Figure S2. eATP reduces S. sclerotiorum infection in Arabidopsis leaves.

Supplemental Figure S3. eATP reduces P. capsici infection in Arabidopsis leaves.

Supplemental Figure S4. P2K1 overexpression line (OxP2K1) is resistant to parasitic nematode infection.

Supplemental Figure S5. Feeding assay on the dorn1-3 mutant and the OxP2K1 line with the crucifer specialist herbivore P. rapae and the generalist herbivore S. exigua.

Supplemental Figure S6. Mapping of eATP-induced transcriptomes on Arabidopsis metabolic pathways.

Supplemental Figure S7. Putative glucosinolate-related metabolites increased in eATP-treated Arabidopsis plants.

Supplemental Figure S8. Representative metabolites increased in eATP-treated Arabidopsis plants.

Supplemental Figure S9. Representative metabolites decreased in eATP-treated Arabidopsis plants.

Supplemental Figure S10. Targeted metabolomics for glucosinolate pathway metabolites (second trial).

Supplemental Table S1. Summary of statistical analysis of Figure 2C and Supplemental Figure S10.

Supplemental Data Set S1. Top 100 coexpressed genes to LecRK-I.9 (ATTED-II).

 

J.B.J., D.T., and K.T. designed the research. J.B.J., A.B., D.T., C.G., C.O., and K.T. performed the research. A.B. and D.R.G. contributed analytic tools. J.B.J., A.B., D.T., C.G., C.O., and K.T. analyzed the data. J.B.J., A.B., D.T., C.G., C.O., H.R.P., D.R.G., and K.T. wrote the manuscript.

Funding

This work was supported by the National Science Foundation (grant no. IOS-1557813) and USDA NIFA (Hatch project #1015621).

Conflict of interest statement. None declared.