Diverse Allyl Glucosinolate Catabolites Independently Influence Root Growth and Development

Abstract

Glucosinolates (GSLs) are sulfur-containing defense metabolites produced in the Brassicales, including the model plant Arabidopsis (Arabidopsis thaliana). Previous work suggests that specific GSLs may function as signals to provide direct feedback regulation within the plant to calibrate defense and growth. These GSLs include allyl-GSL, a defense metabolite that is one of the most widespread GSLs in Brassicaceae and has also been associated with growth inhibition. Here we show that at least three separate potential catabolic products of allyl-GSL or closely related compounds affect growth and development by altering different mechanisms influencing plant development. Two of the catabolites, raphanusamic acid and 3-butenoic acid, differentially affect processes downstream of the auxin signaling cascade. Another catabolite, acrylic acid, affects meristem development by influencing the progression of the cell cycle. These independent signaling events propagated by the different catabolites enable the plant to execute a specific response that is optimal to any given environment.

Figure 1.

Effect of allyl-GSL on root growth and auxin responses. A, Effect of allyl-GSL and auxin (IAA) on root length of Arabidopsis seedlings. Seedlings were grown vertically on MS medium supplemented with IAA (0.1–0.5 μm) or IAA and allyl-GSL (50 and 100 μm). Root length was measured at 14 d. Data are least-squared means over two independent experimental replicates with 15 to 18 seedlings per condition per experiment. Significance was tested via two-way ANOVA (for detailed statistics, see Supplemental Table S1). B, Phenotypes of 14-d-old plants grown with or without IAA and allyl-GSL. C, Effect of allyl-GSL and IAA on number of root curls within 1 cm of the root tip. Seedlings were grown vertically on MS medium for 4 d, then transferred to medium with IAA (0.1–0.5 μm) or IAA and allyl-GSL (50 μm). After another day the plates were tilted back 45° and photographed 3 d later. Results are least-squared means over two independent experimental replicates, with 15 to 18 seedlings per condition per experiment. Significance was tested via two-way ANOVA (***P < 0.0001 relative to control seedlings; quadratic curves were fitted to the model; for detailed statistics, see Supplemental Table S1). D, Phenotypes of seedlings grown with or without allyl-GSL.

Figure 2.

Effect of allyl-GSL breakdown products and auxin on root length. A, Allyl-GSL biosynthesis and breakdown pathway. GSOH, GLUCOSINOLATE HYDROXYLASE; MSB, methylsulfinylbutyl; MTB, methylthiobutyl; NSP, nitrile specifier protein. B and C, Effect of allyl-GSL catabolites and IAA on root length. Arabidopsis seedlings were grown vertically on MS medium supplemented with IAA (0.1–0.5 μm) or IAA and the different allyl-GSL catabolites: 50 μm of allyl-GSL, allyl-nitrile, or allyl-isothiocyanate (allyl ITC; B) and 50 μm allyl-GSL, butenoic acid, acrylic acid, or raphanusamic acid (C). At 14 d old, their root lengths were measured. Results are least-squared means over at least two independent experimental replicates, with 15 to 18 seedlings per condition per experiment. Significance was tested via two-way ANOVA (***P < 0.0001 relative to control seedlings. Quadratic curves were fitted to the model; for detailed statistics, see Supplemental Table S1). D, Arabidopsis seedlings were grown vertically on clean MS medium or MS media supplemented with 0.5 μm IAA and 50 μm allyl-GSL, as indicated. After 7 d, the seedlings were transferred to MS medium with treatments as indicated. At day 14 they were photographed, and the root lengths were measured. Results are least-squared means over two independent experimental replicates, with 15 to 18 seedlings per condition per experiment. Significance was tested by Student’s t-test; ***P < 0.0001; error bars represent the means ± se. E, Seedlings were grown on MS medium with or without 50 μm allyl-GSL. Seven-day-old seedlings were stained with trypan blue and photographed. Two experiments were conducted, with 5 to 10 replicates in every experiment for each treatment.

Figure 3.

Effect of allyl-GSL catabolites on GSL accumulation. Arabidopsis seedlings were grown on MS medium supplemented with 50 μm of allyl-GSL breakdown products. After 14 d, GSL content was measured using HPLC. Results are least-squared means over at least two independent experimental replicates, with two to three replicates per condition per experiment. Significance was tested via two-way ANOVA, followed by Dunnett’s test (*P < 0.05, **P < 0.01, and ***P <0.0001, relative to untreated control seedlings; for detailed statistics, see Supplemental Table S1). Error bars represent means ± se.

Figure 4.

Effect of allyl-GSL breakdown products on meristem development. A, Arabidopsis seedlings were grown on MS medium with allyl-GSL breakdown products. At 5 d old, the seedlings were photographed, and the length from the root tips to the first root hair was measured. B, At 7 d, cell walls were stained using propidium iodide and imaged using confocal microscopy, and the numbers of cells from the quiescent center to the first elongated cell were counted. C, Confocal images of 7-d-old DII-VENUS seedlings stained with propidium iodide. The meristematic area as measured is defined with a deltoid shape (one row under the quiescent center + eight rows above the quiescent center − two rows on each side). D, Seven-day-old DII-VENUS seedlings grown on MS medium and treated for 2 h with allyl-GSL breakdown products in liquid MS medium. The seedlings were imaged using confocal microscopy. The relative integrated density of the VENUS fluorescence in the meristem area was quantified. E, A cell expressing stronger cyclin B1-1::GFP expression in the nucleus then in the cytoplasm. F, Percentage of G2 cells. Seven-day-old seedlings expressing cyclin B1-1::GFP were grown on MS medium, treated for 2 h with allyl-GSL catabolites in liquid MS medium, and imaged using confocal microscopy. Cells expressing stronger nuclear GFP intensity compared with the cytoplasm were counted, and their percentage with respect to the total number of cells expressing GFP was calculated. Results are least-squared means over two independent experimental replicates, with 5 to 10 seedlings per condition per experiment. Error bars represent means ± se. Significance was tested via two-way ANOVA (*P < 0.05 and ***P < 0.0001, relative to untreated control seedlings; for detailed statistics, see Supplemental Table S1).

Figure 5.

Effect of raphanusamic acid and butenoic acid on auxin responses. A and B, Arabidopsis seedlings expressing the different auxin markers (indicated in the inset key) were grown on MS medium. At 7 d old, they were either left in clear medium or treated with 50 μm raphanusamic acid (A) or butenoic acid (B) and imaged using confocal microscopy. The integrated density of the fluorescence markers in the root tips was measured for each treatment at every time point, and the intensity was calculated relative to that of control seedlings (untreated). Results are least-squared means over two independent experimental replicates, with 5 to 10 seedlings per condition per experiment. Error bars represent means ± se. Significance was tested via two-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.0001, relative to control untreated seedlings). C, Images of 7-d-old DII-VENUS seedlings treated with 50 μm of raphanusamic acid for 60 min or 50 μm of butenoic acid for 30 min or not treated (control). D, Yeasts expressing TIR1/AFBs and IAA protein were grown on a medium containing auxin (20 μm), or auxin and raphanusamic acid or butenoic acid (150 μm). The interaction between the proteins was analyzed after overnight incubation at 30°C. E, Arabidopsis seedlings were grown vertically on clear MS medium or MS medium supplemented with 50 μm of raphanusamic acid and butenoic acid. At 14 d old, the seedlings were photographed and their roots were measured. Results are least-squared means over two independent experimental replicates, with 15 to 18 seedlings per condition per experiment. Error bars represent means ± se.

Figure 6.

Effect of allyl-GSL catabolites on root length of different species. Basil, dill, lettuce and tomato seedlings were grown vertically on MS medium with or without 50 μm of the different allyl-GSL catabolites. At 5 d old their root lengths were measured. Results are least-squared means over two independent experimental replicates, with 12 to 24 seedlings per species per treatment. Error bars represent means ± se. Lowercase letters represent statistically different values within species according to ANOVA followed by Tukey’s honestly significant (HSD) mean-separation test (P < 0.05).

Figure 7.

Allyl-GSL catabolites inhibit root growth through different pathways. Raphanusamic acid and butanoic acid manipulate several steps of the auxin machinery, while acrylic acid influences the cell cycle in the root tips.