Jarin-1, an inhibitor of JA-Ile biosynthesis in Arabidopsis thaliana, acts differently in other plant species

ABSTRACT Jasmonates (JAs), including jasmonic acid (JA) and its biologically active derivative JA-Ile, are lipid-derived plant signaling molecules. They govern plant responses to stresses, such as wounding and insect herbivory. Wounding elicits a rapid increase of JA and JA-Ile levels as well as the expression of JAR1, coding for the enzyme involved in JA-Ile biosynthesis. Endogenous increase and application of JAs, such as MeJA, a JA methylester, result in increased defense levels, often accompanied by diminished growth. A JA-Ile biosynthesis inhibitor, jarin-1, was shown to exclusively inhibit the JA-conjugating enzyme JAR1 in Arabidopsis thaliana. To investigate whether jarin-1 does function similarly in other plants, we tested this in Medicago truncatula, Solanum lycopersicum, and Brassica nigra seedlings in a root growth inhibition assay. Application of jarin-1 alleviated the inhibition of root growth after MeJA application in M. truncatula seedlings, proving that jarin-1 is biologically active in M. truncatula. Jarin-1 did not show, however, a similar effect in S. lycopersicum and B. nigra seedlings treated with MeJA. Even JA-Ile levels were not affected by application of jarin-1 in wounded leaf disks from S. lycopersicum. Based on these results, we conclude that the effect of jarin-1 is highly species-specific. Researchers intending to use jarin-1 for studying the function of JAR1 or JA-Ile in their model plants, must test its functionality before use.

The signaling hormone jasmonic acid (JA) regulates up to 85% of wound-and herbivore-induced genes in Arabidopsis thaliana leaves. 1 Besides regulating the response to herbivore attacks, JAs are also involved in regulating plant growth processes, such as primary root growth, reproductive development, and leaf senescence. 2,3The amino acid conjugate JA-Ile has been identified as the main endogenous bioactive JA molecule. 4However, recent studies report other endogenous bioactive JA molecules, such as JA conjugated with other amino acids or hydroxylated JA-Ile. 5he JA signaling pathway can integrate primary and specialized metabolism via the regulation of repressor-transcription factor complexes, which may be associated with re-allocation of carbon and recalibration of growth rates. 6Therefore, the activation of inducible plant defenses often results in reduced growth and development. 7This is commonly referred to as the growth-defense trade-off. 8The mechanisms of growth-defense trade-offs have been widely studied in plants against insect herbivory (reviewed in). 8,9Even knowledge about how to uncouple this mechanism has still been less explored, researchers are always searching for an effective and efficient approaches to solve this scientific question.
A previously described JA-Ile biosynthesis inhibitor, jarin-1, was firstly shown to exclusively inhibit the JA-conjugating enzyme JAR1 in A. thaliana and Cardamine hirsuta.
Chemically, jarin-1 is a derivative of the alkaloid cytisine and harbors a 3-aminocytisine residue linked to two different carboxylic acid residues. 103][14] However, the effect of jarin-1 in inhibiting JA-Ile biosynthesis was not always evidenced as done for A. thaliana. 10This leads to the hypothesis that the function of this chemical inhibitor is highly dependent on the plant species used.
In this study, we investigated whether jarin-1 does act on relieving root growth inhibition by jasmonates in other plant species, such as Medicago truncatula, Solanum lycopersicum and Brassica nigra.We performed the well-established root growth inhibition assay by treatment of seedlings from M. truncatula, S. lycopersicum and B. nigra with MeJA and applied jarin-1 simultaneously with MeJA to check, whether jarin-1 prevents the root growth inhibition or not.Our results show clearly that jarin-1 did not rescue the inhibitory effect of MeJA in all plant species tested.
First, to test whether jarin-1 counteracts MeJA function in M. truncatula, we used seedlings grown in vitro and treated them with different concentrations of jarin-1 (0, 5, 10 or 30 µM).Half of the plants were simultaneously treated with 10 µM MeJA, a treatment leading to significantly decreased root growth in various plant species. 15Root length was determined eight days after treatment (Figure 1).Treatment with 30 µM jarin-1 alone had a clear negative effect on root growth, which was also observed on seedlings treated with 10 µM jarin-1, although to a smaller extent (Figure 1).Simultaneous application of 5 or 10 µM jarin-1 partially alleviated the root growth inhibition by MeJA.This demonstrated that, like in A. thaliana, 10 jarin-1 can partially mitigate MeJA-induced root growth inhibition in M. truncatula.Based on our results and the results from the previous report in A. thaliana, we selected the 10 µM jarin-1 treatment to test its effect on MeJAroot growth in seedlings of S. lycopersicum and B. nigra.Application of 10 µM jarin-1 had, however, no effect on the MeJA-induced root growth inhibition in seedlings of S. lycopersicum and B. nigra.In other words, seedlings treated with 10 µM MeJA and 10 µM jarin-1 simultaneously, had similar root lengths as seedlings treated with 10 µM MeJA alone (Figure 2, Table 1).This suggests that the function of jarin-1 might dependent on the plant species.To check the effect of jarin-1 on the endogenous rise of JA-Ile in tomato leaves upon wounding, leaf disks were treated with 30 µM jarin-1 for one hour followed by wounding with forceps.This concentration was selected since 10 µM jarin-1 did not rescue MeJAinduced root growth inhibition of S. lycopersicum seedlings and 30 µM jarin-1 was most effective in inhibiting JA-Ile production in A. thaliana plants. 10Determination of JA-Ile levels at one hour after wounding revealed, however, that jarin-1 had .Four days after germination on agar plates in the dark, seedlings were placed into aluminium-wrapped 50 ml falcon tubes (without cover) filled with half-strength Hoagland's solution supplemented or not with MeJA (10 µM) and/or jarin-1 (5, 10, 30 µM).There were eight treatments (tubes) in total, including mock, 10 µM MeJA, 5 µM jarin-1, 10 µM jarin-1, 30 µM jarin-1, 5 µM jarin-1 plus 10 µM MeJA, 10 µM jarin-1 plus 10 µM MeJA, 30 µM jarin-1 plus 10 µM MeJA.Seedlings were incubated for 8 days in a growth room at 24°C and 16 h light/8 h dark.Subsequently, seedlings were carefully removed from falcon tubes and photographed.Root lengths were determined using a ruler.Different letters indicate significant differences between treatments according to two-way ANOVA (p<0.05).

Mock
Jarin-1 MeJA MeJA+jarin-1  no effect on the wound-induced biosynthesis of JA-Ile in tomato leaf disks (Figure 3).Based on our results, we propose that the function of jarin-1 as inhibitor of formation of active jasmonates depends on the plant species.To use it, researchers should test the activity of jarin-1 in their model plants before addressing specific scientific questions related to the function of JAR1 or JA-Ile.
In conclusion, jarin-1 is a small-molecule inhibitor of jasmonate responses that is active in affecting the root growth inhibition by MeJA in A. thaliana and M. truncatula.However, in S. lycopersicum and B. nigra, we did not find this phenomenon under our experimental conditions.It still serves as an effective chemical tool in dissecting the complex jasmonate signaling networks to avoid the time-consuming genetic knockout for non-model plants.Its usability has, however, to be tested for other plant species.

Figure 3 .
Figure 3. Application of jarin-1 did not affect wound-induced accumulation of JA-Ile in tomato leaf disks.Leaf disks of 6-week-old S. lycopersicum plants were placed on solutions containing DMSO (mock) or 30 µM jarin-1.After one hour, half of the disks were wounded using forceps and harvested one hour later.JA-Ile levels were determined according to Balcke et al. 16 .Bars represent mean ± SD (n=3).Different letters indicate statistically significant differences according to ANOVA with Tukey's HSD test (p<0.01).

Table 1 .
Effect of jarin-1 on root length of S. lycopersicum and B. nigra seedlings 12 days after chemical treatment.Root lengths were determined using a ruler.Data are mean ± SD (n ≥ 7).DMSO (mock), dimethyl sulfoxide.Different letters indicate significant differences between treatments according to two-way ANOVA (p < 0.05).