The Defense Metabolite, Allyl Glucosinolate, Modulates Arabidopsis thaliana Biomass Dependent upon the Endogenous Glucosinolate Pathway

Abstract

Glucosinolates (GSLs) play an important role in plants as direct mediators of biotic and abiotic stress responses. Recent work is beginning to show that the GSLs can also inducing complex defense and growth networks. However, the physiological significance of these GSL-induced responses and the molecular mechanisms by which GSLs are sensed and/or modulate these responses are not understood. To identify these potential mechanisms within the plant and how they may relate to the endogenous GSLs, we tested the regulatory effect of exogenous allyl GSL application on growth and defense metabolism across sample of Arabidopsis thaliana accessions. We found that application of exogenous allyl GSL had the ability to initiate changes in plant biomass and accumulation of defense metabolites that genetically varied across accessions. This growth effect was related to the allyl GSL side-chain structure. Utilizing this natural variation and mutants in genes within the GSL pathway we could show that the link between allyl GSL and altered growth responses are dependent upon the function of known genes controlling the aliphatic GSL pathway.

Keywords: Arabidopsis; GSL-induced responses; allyl GSL; defense metabolism; plant growth.

Figure 1

Differential plant biomass responses of Arabidopsis accessions to exogenous allyl GSL is sugar dependent. (A) Quantification of biomass of 15-day-old seedlings from seven A. thaliana natural accessions fed with 50 μM of allyl GSL using MS media differing in sucrose concentrations. The accessions Bay-0, Col-0, Ler-0, and Tsu-1 do not synthesize endogenous allyl GSL, while the accessions Cvi, Kas, and Sha produce endogenous allyl GSL. Asterisks indicates significant effect of the exogenous allyl GSL treatment on that accession under that condition, ^*P ≤ 0.05 and ^**P ≤ 0.01 from the ANOVA analysis (Table S3). The error bars represent standard deviation from three independent experiments with each bar showing the average biomass of at least 60 individual plants across these experiments. (B) Representative photographs of seven Arabidoposis accessions feed with or without 50 μM of allyl GSL at 1% of sucrose concentration.

Figure 2

Differential plant biomass responses of Ler-0 accession fed with 50 μM of allyl and 4MSB GSLs using MS media at 1% of sucrose concentration. The bar chart represents the mean fw (mg tissue/plant) and the error bars represent the standard deviation within each treatment (MS + allyl GSL; MS + 4MSB GSL). Letters indicate significant differences (P ≤ 0.05) between treatments using ANOVA and post-hoc t-test.

Figure 3

Allyl GSL accumulation. (A) The average of exogenous allyl GSL accumulation within leaves of 15-day-old seedlings was directly measured using HPLC. Shown are the accessions that are genetically incapable of synthesizing endogenous allyl GSL. Thus, detected allyl must have been taken up from the surrounding media, transported via the vasculature to the leaf and provides a direct measurement of the accumulation. Averages with the same letter are not significantly different at P ≤ 0.05 using a Tukey's post hoc t-test within the ANOVA. The error bars represent standard deviation from three independent experiments where 12 plants were separately measured per experiment. (B) Average of the total endogenous aliphatic GSL and accumulated allyl content within the accessions that do not synthesize endogenous allyl GSL grown in MS with allyl at 1% of sucrose on the growing media. The error bars represent standard deviation across accessions and experiments as described in part (A).

Figure 4

Differential GSL responses of Arabidopsis accessions to exogenous allyl GSL. Quantification of GSLs in 15-day-old seedlings from seven A. thaliana natural accessions fed with 50 μM of allyl GSL using MS media with 1% sucrose. Asterisks indicate a significant effect of the exogenous allyl GSL treatment on the accumulation of the shown GSL within the specific accession using a post-hoc Tukey test within the ANOVA analysis (^*P ≤ 0.05 and ^**P ≤ 0.01). The error bars represent standard deviation from three independent experiments with each bar showing the average biomass of at least 12 individual plants across these experiments. The GSL are as follows (A) Short-chain GSL content. (B) Long-chain GSL content. (C) Indolic GSL content. (D) Accumulation of the dominant short-chain GSL within each accession as shown by 3-hydroxypropyl (3OHP), 4-methylsulfinylbutyl (4MSB) and but-3-enyl GSL. (E) 8-methylsulfinyloctyl GSL (8 MSB) content. (F) qRT-PCR expression levels of five selected GSL biosynthetic genes in seedlings of Col-0 grown in the presence and absence of exogenous 50 μM of allyl GSL at 1% sucrose. The error bars represent standard deviation from two independent experiments with three biological replicates per experiment. None of the differences were statistically significant (ANOVA, P ≤ 0.05).

Figure 5

Natural variation in Arabidopsis biomass and GSL acumulation in response to exogenous allyl GSL. Histograms showing the frequency distribution of the relative responses to exogenous allyl GSL (50 μM of allyl GSL using MS media with 1% sucrose) across the 96 Arabidopsis natural accessions for the following phenotypes (A) plant biomass (B) total aliphatic GSL (C) total indolic GSL. The relative response of each phenotype to the allyl treatment within each accession was determined as (Phenotype in plants treated with allyl—phenotype in plants from the control treatement)/((0.5 × [Phenotype in plants treated with allyl + phenotype in plants from the control treatement]).

Figure 6

GSL chemotype interacts with exogenous Allyl treatment to influence plant biomass of Arabidopsis accessions. Quantification of fw (mg tissue/plant) from 15-day-old seedlings from 96 Arabidopsis natural accessions fed with 50 μM of allyl GSL using MS media with 1% sucrose. The population was grouped by their GSL chemotype profile generated by the combination of variation at GS-AOP (alkenyl, hydroxyalkyl, and methylsulfinyl) GS-Elong (3C vs. 4C). The sub-populations generated are labeled by their predominant glucosinolate; AOP1 C3 accumulates 3-methylsulfinylpropyl, AOP1 C4 accumulates 4-methylsulfinylbutyl, AOP2 C3 accumulates allyl. AOP2 C4 accumulates But-3-enyl and AOP3 C3 accumulates 3-hydroxypropyl (Table S1). None of the studied accessions displayed the AOP3 C4 chemotype accumulating 4-hydroxybutyl. The bar chart represents the mean fw and the error bars represent the standard deviation among the accessions within each chemotype. ^*shows GSL haplotypes that had a significant difference in plant biomass response to exogenous allyl GSL using post hoc Tukey's t-test with P ≤ 0.05 from the ANOVA analysis.

Figure 7

Model results comparing relative plant biomass response to relative GSL content response to exogenous allyl GSL treatment. Scatter plots with 95% confidence intervals on the partial correlations between relative plant biomass response (y axis) and the relative GSL content differences (x axis) for traits showing significance in the stepwise linear regression analysis (Table 3). See Table S2 for abbreviations.

Figure 8

Plant biomass responses and GSL content variation among GSL mutant genotypes treated with exogenously applied allyl GSL. (A) Quantification of 15-day-old fw (mg/plant) seedlings from seven GSL mutant genotypes and wild-type (Col-0) fed with 50 μM of allyl glucosinolate using MS media with 1% sucrose. (B) Ratio of 4-methylsulfinylbutyl (4MSB)/4-methylthiobutyl (4MTB) calculated as 4MSB/(4MSB + 4MTB). (C) Allyl GSL accumulation average of the evaluated genotypes. The bar chart represents the mean and standard deviation. Each genotype within each treatment has a minimum of 60 independent plants measured. These plants were grown in a randomized block design with six individuals per genotype per treatment and 10 separate independent experiments. Means with the same letter show if the genotype's response to the treatment was statistically similar to Col-0 (a) or different from Col-0 (b) at P ≤ 0.05 from the two-way ANOVA analysis (Table S8).