The isoleucic acid triad: distinct impacts on plant defense, root growth, and formation of reactive oxygen species

Abstract

Isoleucic acid (ILA), a branched-chain amino acid-related 2-hydroxycarboxylic acid, occurs ubiquitously in plants. It enhances pathogen resistance and inhibits root growth of Arabidopsis. The salicylic acid (SA) glucosyltransferase UGT76B1 is able to conjugate ILA. Here, we investigate the role of ILA in planta in Arabidopsis and reveal a triad of distinct responses to this small molecule. ILA synergistically co-operates with SA to activate SA-responsive gene expression and resistance in a UGT76B1-dependent manner in agreement with the observed competitive ILA-dependent repression of SA glucosylation by UGT76B1. However, ILA also shows an SA-independent stress response. Nitroblue tetrazolium staining and pharmacological experiments indicate that ILA induces superoxide formation of the wild type and of an SA-deficient (NahG sid2) line. In contrast, the inhibitory effect of ILA on root growth is independent of both SA and superoxide induction. These effects of ILA are specific and distinct from its isomeric compound leucic acid and from the amino acid isoleucine. Leucic acid and isoleucine do not induce expression of defense marker genes or superoxide production, whereas both compounds inhibit root growth. All three responses to ILA are also observed in Brassica napus.

Keywords: Brassica napus; Arabidopsis; glucosyltransferase; isoleucic acid; plant defense; reactive oxygen species; root growth; salicylic acid; superoxide anion.

Fig. 1.

Repression of bacterial pathogens upon SA and ILA application. Four-week-old soil-grown plants were watered with 10 µM SA, 250 µM ILA, the combination of 10 µM SA and 250 µM ILA, or without any addition (control). After 3 d the plants were inoculated with Pseudomonas syringae pv. tomato DC3000 (5×10⁵ cfu ml⁻¹), and the resulting P. syringae titers were determined 3 d after infection. Bars represent the means ±SE of four biological replicates. Significant differences (P_adj values) are indicated by letters according to one-way ANOVA. The experiment was independently repeated three times with similar results.

Fig. 2.

Interaction of SA and ILA in planta. PR1 expression in wild type (A) and PR1 expression in ugt76b1 (B) was investigated in leaves of 12-day-old seedlings that had been incubated 48 h with increasing concentrations of SA (0, 5, 25, 100 µM) in the absence (light-grey bars) and presence (dark grey bars) of 250 µM ILA. Expression values are normalized to S16 and UBQ5 (NRQ, normalized relative quantity); means ±SE; n=3–4.

Fig. 3.

ILA enhances superoxide radicals in leaves. NBT staining in leaves of 2-week-old seedlings was assessed as a semi-quantitative measurement of O₂⁻ formation (compare Fig. 4A, B). (A) NBT staining 48 h after treatment with 250 µM ILA, 500 µM ILA, 100 µM SA, 500 µM SA, or the combination of 100 µM SA + 250 µM ILA. Means ±SE; n=9. Significant differences (P_adj<0.05) are indicated by letters according to one-way ANOVA. (B) NBT staining in 2-week-old NahG sid2 (n=21) and wild type (WT; n=15) seedlings 48 h after treatment with 500 µM ILA. Means ±SE; differences between treated or untreated plants were analysed by Welch’s two sample t-test. (C) NBT staining detected in leaves of ugt76b1, wild type (WT) and UGT76B1 overexpressor (OE). Means ±SE; n=20–23. Significant differences (P_adj<0.05) are indicated by letters according to one-way ANOVA. The experiments were independently repeated three times with similar results. (D–F) Representative images of NBT staining of leaves of the indicated genotypes and treatments, which were analysed in (A), (B), and (C), respectively.

Fig. 4.

NADPH oxidases contribute only partially to ILA-induced superoxide formation in leaves. (A, B) NBT staining is sensitive to superoxide scavenger 4-OH-TEMPO (TEMPO) and reduced by addition of DPI. Twelve-day-old seedlings were treated with 500 µM ILA, with ILA and 4-OH-TEMPO, or with ILA and DPI for 3.5 h. NBT staining of the leaves was determined as a semi-quantitative measurement. Means ±SE; n=9. Significant differences (P_adj<0.05) are indicated by letters according to one-way ANOVA. (C) Fourteen-day-old wild type seedlings treated for 48 h either with control medium (light grey bars) or with medium containing 500 µM ILA (dark grey bars). ROS-related genes (CRK7, RBOHD, and RBOHF) were induced by exogenous ILA application in wild type. Gene expression was assessed by RT-qPCR and normalized to S16 and UBQ5. Means ±SE; n=4; differences between treated or untreated plants were analysed by Welch’s two sample t-test. *P<0.05, ***P<0.001. (D, E) O₂⁻ radical detected by NBT staining in 14-day-old wild type and rbohd rbohf seedlings treated for 48 h either with control medium (light grey bars) or with medium containing 500 µM ILA (dark grey bars). Means ±SE; n=9. Differences between treated and untreated plants were analysed by Welch’s two sample t-test. *P<0.05, ***P<0.001.

Fig. 5.

Root growth inhibition by ILA. (A) Root meristem length of 8-day-old seedlings grown on plates with 500 µM ILA or control medium. Means ±SE; n=17 (control), 15 (ILA). (B) Longitudinal extension of epidermal cells in the root differentiation zone of the seedlings grown as in (A). Means ±SE; n=257 (control), 197 (ILA). (C) Root length of ugt76b1, wild type, and UGT76B1 overexpressor (OE) plants grown on control medium and on medium containing 250 µM ILA, 10 µM SA, or the combination of ILA and SA for 10 d. Means ±SE; n=21–30. Significant differences (P_adj<0.05) are indicated by letters according to one-way ANOVA assessed for the genotypes. The experiments were independently repeated three times with similar results.

Fig. 6.

SA- and superoxide-independent root growth inhibition by ILA. (A) Root growth inhibition on media without (control) or with 500 µM ILA for ugt76b1, ugt76b1 NahG sid2, wild type, NahG sid2, and UGT76B1 overexpressor (from left to right, light grey to black bars) after 10 d. (B) NBT and DAB staining of primary root tips of wild type plants after growth on control or 500 µM ILA plates after 10 d. (C) Root length of 9-day-old ugt76b1, wild type, rbohd rbohf, and UGT76B1 overexpressor plants (from left to right, light grey to dark grey bars) on media without (control) or with 500 µM ILA. Significant differences among the genotypes of each group in (A) and (C) (P_adj<0.05) are indicated by letters according to one-way ANOVA. Means ±SE; n=13–18. (D) ILA-induced root growth inhibition in the presence of the O₂⁻ scavenger 4-OH-TEMPO. Means ±SE; n=10–32. Different lowercase letters indicate a significant difference according to a two-way ANOVA with treatment and time as discrete factors (P_adj<0.05). The experiment was independently repeated two times.

Fig. 7.

LA and Ile do not affect SA signaling and ROS induction, but also show a root inhibition effect. (A) PR1 expression in response to 500 µM Ile, 500 µM LA, and 100 µM SA as well as to the combined treatments with 250 µM Ile + 100 µM SA and 250 µM LA + 100 µM SA. PR1 expression was determined by RT-qPCR and normalized to S16 and UBQ5. Means ±SE; n=3–4. (B) Superoxide radical induction assessed by NBT staining 48 h after application of 500 µM Ile, LA, or ILA to 2-week-old seedlings (n=12). The experiment was independently repeated two times with similar results. (C) Root growth inhibition on ILA-, Ile-, and LA-containing media; a lower level of 250 µM each was used for this comparison of individually applied compounds, since 250 µM LA exerted already a very strong effect (Supplementary Fig. S7). Root length was recorded after 9 d. Data for ugt76b1 (light grey), wild type (grey), and UGT76B1 overexpressor (dark grey) were compared within the treatments; n=19–23. The experiment was independently repeated two times with similar results. Significant differences (P_adj<0.05) are indicated by letters according to one-way ANOVA. (D) Representative NBT-stained leaves after treatment with Ile, LA, and ILA.

Fig. 8.

PR1 expression, ROS induction, and root growth inhibition upon ILA treatment in Brassica napus. Seedlings were grown for 9 d on either control medium (light grey bars) or medium containing 500 µM ILA (dark grey bars). (A) BnPR1 expression level in B. napus leaves was assessed by RT-qPCR and normalized to BnUP1 and BnUBQ9. Means ±SE, n=3. (B) NBT staining of B. napus roots. Means ±SE, n=15. (C) Root length of B. napus plants. Means ±SE, n=10–11. Welch’s two sample t-test was performed to test differences between untreated and treated plants; *P<0.05, ***P<0.001.

Fig. 9.

Three separable effects of ILA on Arabidopsis plants. ILA activates plant defense and PR1 marker gene expression in an SA-dependent and UGT76B1-dependent manner. This is attributed to the inhibitory effect of ILA on the UGT76B1-catalysed SA glucosylation. In contrast to the effects of ILA on plant defense, SA and UGT76B1 are not required for the ILA-induced inhibition of root growth and formation of superoxide, since both effects are also found in NahG sid2 and ugt76b1 loss-of-function mutants. NADPH oxidases, specifically RBOHD and RBOHF, contribute to O₂⁻ production; however, this still occurs independently of RBOHs. The ILA-induced inhibition of root growth is neither linked to RBOHD/RBOHF nor blocked by pharmacologically suppressing superoxide formation.