Glucosinolate-Derived Isothiocyanates Inhibit Arabidopsis Growth and the Potency Depends on Their Side Chain Structure

Isothiocyanates (ITCs), the biologically important glucosinolate breakdown products, can present health-promoting effects, play an important role in plant defense and affect plant cellular mechanisms. Here, we evaluated the biological effects of ITCs on Arabidopsis thaliana by assessing growth parameters after long-term exposure to low concentrations of aliphatic and aromatic ITCs, ranging from 1 to 1000 µM. Treatment with the aliphatic allylisothiocyanate (allyl-ITC) led to a significant reduction of root length and fresh weight in a dose-dependent manner and affected the formation of lateral roots. To assess the importance of a hormonal crosstalk in the allyl-ITC-mediated growth reduction, the response of auxin and ethylene mutants was investigated, but our results did not allow us to confirm a role for these hormones. Aromatic ITCs generally led to a more severe growth inhibition than the aliphatic allyl-ITC. Interestingly, we observed a correlation between the length of their side chain and the effect these aromatic ITCs caused on Arabidopsis thaliana, with the greatest inhibitory effect seen for 2-phenylethyl-ITC. Root growth recovered when seedlings were removed from exposure to ITCs.


Introduction
Naturally-occurring isothiocyanates (ITCs) can be generated by plants belonging to the Brassicales order when a particular group of nitrogen-and sulfur-containing secondary metabolites called glucosinolates are degraded. This usually happens when plant tissue is disrupted upon mechanical damage or herbivory, bringing together the otherwise spatially separated glucosinolate substrates and β-thioglucosidases called myrosinases which catalyze their hydrolysis into various biologically important phytochemicals [1,2]. The diversity of degradation products is mainly determined by the variable structures of the glucosinolate side chain, which originate from their precursor amino acids. More than 130 different glucosinolates have been identified so far [3]. The outcome of glucosinolate hydrolysis strongly depends on the reaction conditions, taking into consideration the presence or absence of specific proteins and cofactors, and thus hydrolysis products such as nitriles and epithionitriles, can be produced instead of ITCs [4][5][6].
While intact glucosinolates are considered biologically inactive, their degradation products have many documented biological effects. In human and animal cells ITCs affect for example, phase I and phase II enzymes, modulate the cell cycle, and lead to programmed cell death [7,8]. ITCs play also an important role in plant defense against pests and pathogens [9,10]. Glucosinolate-containing plants have also been used in biofumigation to control soil pests and weeds [11], and several ITCs have therefore grown on solid in vitro medium supplemented with allyl-ITC ( Figure 1) at a range of concentrations exerting no to almost complete root growth inhibition. This range, from 50 to 1000 µM, showed that allyl-ITC inhibited growth of the main root in a dose-dependent manner (Figure 2A). Root length inhibition ranged from 10% at 50 µM allyl-ITC to an almost complete inhibition (88%) at 1000 µM allyl-ITC ( Figure 2B).  This range, from 50 to 1000 µM, showed that allyl-ITC inhibited growth of the main root in a dose-dependent manner (Figure 2A). Root length inhibition ranged from 10% at 50 µM allyl-ITC to an almost complete inhibition (88%) at 1000 µM allyl-ITC ( Figure 2B).

Allyl-ITC Reduced the Total Biomass of Arabidopsis thaliana In Vitro
Allyl-ITC showed also a dose-dependent inhibitory effect on the overall plant biomass ( Figure 2C). Concentrations of 700 and 1000 µM allyl-ITC reduced the biomass values, respectively, by about 71% and 90% of the control treatment. The lower concentrations of allyl-ITC did not have a significant inhibitory effect on the total plant biomass ( Figure 2C).

Allyl-ITC Induced Other Root Morphogenic Changes
Roots and shoots of 10 day old seedlings exposed to allyl-ITC were also analyzed for other morphogenic changes. When monitoring the number of lateral roots and the region they extended from, we observed that allyl-ITC concentrations of 300, 500, and 700 µM led to a higher number of lateral roots extending from the hypocotyl-root junction, compared to the control treatment. In contrast, the number of lateral roots extending from the main root decreased, leading to a reduction in lateral root density (Figure 3).

A Role for Auxin or Ethylene in Root Growth Inhibition Triggered by Allyl-ITC Could Not Be Established
Root development is regulated by many processes, including a crosstalk between auxin and ethylene [37,44], and the expression of auxin-and ethylene-related genes is affected after short time exposure to allyl-ITC [20]. Auxin marker lines were therefore used to assess if also a long-term exposure to allyl-ITC affected the auxin response ( Figure 4). Seedlings of the DR5::GUS [45] and DII-VENUS [46] reporter lines responded, as expected, to allyl-ITC by a reduced root elongation similar to Col-0 wild type seedlings. But no changes in GUS staining or YFP fluorescence which would indicate an altered auxin response due to the allyl-ITC treatment, were observed ( Figure 4). In addition, none of the auxin response mutant lines that we tested (Table 1) showed a difference to the wild type in the allyl-ITC-induced effect on root length after 10 days of exposure, besides the morphological differences inherent to some of the auxin mutants. Root length inhibition by allyl-ITC of the two ethylene insensitive mutants etr1-3 (ethylene response 1-3) and ein2-1 (ethylene insensitive 2-1) ( Table 1) after ten days was not different to that of wild type seedlings either.

Effects on Arabidopsis Growth Depended on ITC Side Chain Structure
To assess if the structure of the side chain had an impact on the growth inhibitory effect of the compound, four ITCs with an aromatic side chain of varying length ( Figure 1) were also tested. Similar to the allyl-ITC exposure assay described above, concentrations of each ITC were chosen to cover a range giving no to almost complete root growth inhibition. Phenyl-ITC concentrations of 50 and 100 µM reduced root length by about 11% and 18%, respectively, compared to the control treatment ( Figure 5A,E). While 300 µM phenyl-ITC inhibited root growth by more than 85%, concentrations of 500 µM or higher led to a complete inhibition. Benzyl-ITC ( Figure 5C,F) and 4-phenylbutyl-ITC ( Figure 5C,H) had a clear inhibitory effect on the main root length already at 25 µM. Moreover, benzyl-ITC treatment led to stronger inhibition than 4-phenylbutyl-ITC. On concentrations of benzyl-ITC and 4-phenylbutyl-ITC higher than 25 µM, seeds germinated, but

A Role for Auxin or Ethylene in Root Growth Inhibition Triggered by Allyl-ITC Could Not Be Established
Root development is regulated by many processes, including a crosstalk between auxin and ethylene [37,44], and the expression of auxin-and ethylene-related genes is affected after short time exposure to allyl-ITC [20]. Auxin marker lines were therefore used to assess if also a long-term exposure to allyl-ITC affected the auxin response ( Figure 4). Seedlings of the DR5::GUS [45] and DII-VENUS [46] reporter lines responded, as expected, to allyl-ITC by a reduced root elongation similar to Col-0 wild type seedlings. But no changes in GUS staining or YFP fluorescence which would indicate an altered auxin response due to the allyl-ITC treatment, were observed ( Figure 4). In addition, none of the auxin response mutant lines that we tested (Table 1) showed a difference to the wild type in the allyl-ITC-induced effect on root length after 10 days of exposure, besides the morphological differences inherent to some of the auxin mutants. Root length inhibition by allyl-ITC of the two ethylene insensitive mutants etr1-3 (ethylene response 1-3) and ein2-1 (ethylene insensitive 2-1) ( Table 1) after ten days was not different to that of wild type seedlings either.

Effects on Arabidopsis Growth Depended on ITC Side Chain Structure
To assess if the structure of the side chain had an impact on the growth inhibitory effect of the compound, four ITCs with an aromatic side chain of varying length ( Figure 1) were also tested. Similar to the allyl-ITC exposure assay described above, concentrations of each ITC were chosen to cover a range giving no to almost complete root growth inhibition. Phenyl-ITC concentrations of 50 and 100 µM reduced root length by about 11% and 18%, respectively, compared to the control treatment ( Figure 5A,E). While 300 µM phenyl-ITC inhibited root growth by more than 85%, concentrations of 500 µM or higher led to a complete inhibition. Benzyl-ITC ( Figure 5C,F) and 4-phenylbutyl-ITC ( Figure 5C,H) had a clear inhibitory effect on the main root length already at 25 µM. Moreover, benzyl-ITC treatment led to stronger inhibition than 4-phenylbutyl-ITC. On concentrations of benzyl-ITC and 4-phenylbutyl-ITC higher than 25 µM, seeds germinated, but root growth was quickly arrested. At the two lowest concentrations (i.e., 1 and 5 µM) that were tested, no root growth inhibition was observed. 2-phenylethyl-ITC had the strongest effect of all tested ITCs on root growth ( Figure 5C,G), with a dramatic reduction of root length occurring when increasing the dose from 5 to 25 µM.
While seed germination was inhibited or slightly delayed at some of the highest ITC concentrations that were tested ( Figure 5; Figure S1), the ITC-triggered reduction of primary root length measured after ten days was due to a post-germination inhibition of root growth, and not due to a delayed germination.
As to the effect of aromatic ITCs on plant biomass, phenyl-ITC showed again a clear effect at concentrations of 300 µM and higher, inhibiting biomass by more than 75% ( Figure 5B). For the three other aromatic ITCs (benzyl-ITC, 2-phenylethyl-ITC and 4-phenylbutyl-ITC), seedlings hardly developed at this dose ( Figure 5F-H), and they showed a significant inhibition of biomass already at a ten times lower concentration ( Figure 5D). Similar to root growth, 2-phenylethyl-ITC exerted the highest inhibition, reducing biomass by more than 85% at 25 µM ( Figure 5D). Table 1. List of auxin and ethylene mutants used to investigate the role of these hormones in allyl-ITCtriggered growth inhibition. Root length of seedlings grown on 1 /2 Murashige & Skoog (MS) medium or 1 /2 MS medium supplemented with allyl-ITC was measured on day 10. Allyl-ITC concentrations tested were 50, 100, 300, and 500 µM, except for ein2-1 and etr1-3, where 500 µM allyl-ITC was used.

Mutant Name
Gene Name (ID) Description eir1-1 (pin2-1) EIR1/PIN2 (At5g57090) auxin efflux carrier pin3-4 PIN3 (At1g70940) regulator of auxin efflux and transport pin4-3 PIN4 (At2g01420) putative auxin efflux carrier As to the effect of aromatic ITCs on plant biomass, phenyl-ITC showed again a clear effect at concentrations of 300 µM and higher, inhibiting biomass by more than 75% ( Figure 5B). For the three other aromatic ITCs (benzyl-ITC, 2-phenylethyl-ITC and 4-phenylbutyl-ITC), seedlings hardly developed at this dose ( Figures 5F-H), and they showed a significant inhibition of biomass already at a ten times lower concentration ( Figure 5D). Similar to root growth, 2-phenylethyl-ITC exerted the highest inhibition, reducing biomass by more than 85% at 25 µM ( Figure 5D).    . Scale bars = 1 cm. Each root growth inhibition percentage was generated using the average root length of at least 48 seedlings growing on a given ITC-supplemented medium divided by the average root length of at least 48 seedlings growing on the control medium (n > 48). Each percentage of fresh weight reduction was calculated from the pooled biomass of 15 seedlings per plate (n = 4) for the given ITC treatments compared to the control treatment.

Root Growth Recovered When Seedlings Were Removed from Exposure to ITCs
To assess if plants were able to recover from the ITC-triggered inhibition of root growth, seedlings were first grown on medium supplemented with either 500 µM allyl-ITC or 15 µM 2-phenylethyl-ITC, before being transferred to new control medium ( Figure 6). After this transfer, roots grew more rapidly (6.80 ± 0.75 mm/day when transferred from allyl-ITC and 6.21 ± 1.39 mm/day when transferred from 2-phenylethyl-ITC) than those of seedlings still exposed to the ITC . Scale bars = 1 cm. Each root growth inhibition percentage was generated using the average root length of at least 48 seedlings growing on a given ITC-supplemented medium divided by the average root length of at least 48 seedlings growing on the control medium (n > 48). Each percentage of fresh weight reduction was calculated from the pooled biomass of 15 seedlings per plate (n = 4) for the given ITC treatments compared to the control treatment.

Root Growth Recovered When Seedlings Were Removed from Exposure to ITCs
To assess if plants were able to recover from the ITC-triggered inhibition of root growth, seedlings were first grown on medium supplemented with either 500 µM allyl-ITC or 15 µM 2-phenylethyl-ITC, before being transferred to new control medium ( Figure 6). After this transfer, roots grew more rapidly (6.80 ± 0.75 mm/day when transferred from allyl-ITC and 6.21 ± 1.39 mm/day when transferred from 2-phenylethyl-ITC) than those of seedlings still exposed to the ITC (4.50 ± 0.55 mm/day on allyl-ITC and 4.96 ± 0.99 mm/day on 2-phenylethyl-ITC), and reached growth rates similar to seedlings never exposed to ITC (i.e., transferred from control plates to new control plates; 7.53 ± 1.05 mm/day and 6.92 ± 1.32 mm/day). This was observed for both 2-phenylethyl-ITC ( Figure 6A) and allyl-ITC ( Figure 6B), showing that root growth can recover when the exogenously applied ITCs are removed. , before being transferred to new control medium and grown for another ten days. Two Way ANOVA and a post hoc Holm-Sidak test was used to determine significant differences at each time point. For the purpose of readability, only significant differences in root length between seedlings transferred to new control plates and those remaining on ITC-supplemented plates (marked by "a") or those remaining on control plates (marked by "b") are indicated (n = 48; * p < 0.001; error bars indicate SDs).

Discussion
The aim of the present study was to assess the growth inhibiting effect of a range of ITCs on the model plant A. thaliana under standardized in vitro conditions. Three of the five ITCs used in this study are glucosinolate-derived compounds that occur naturally. Allyl-ITC is generated by the hydrolysis of the aliphatic glucosinolate sinigrin. Allyl-ITC was chosen as it can be generated by Figure 6. Root growth recovery upon removal of ITC treatment. Root length of seedlings that were grown for five days on solid in vitro control medium, or medium supplemented with (A) 2-phenylethyl-ITC (15 µM; PEITC) or (B) allyl-ITC (500 µM; AITC), before being transferred to new control medium and grown for another ten days. Two Way ANOVA and a post hoc Holm-Sidak test was used to determine significant differences at each time point. For the purpose of readability, only significant differences in root length between seedlings transferred to new control plates and those remaining on ITC-supplemented plates (marked by "a") or those remaining on control plates (marked by "b") are indicated (n = 48; * p < 0.001; error bars indicate SDs).

Discussion
The aim of the present study was to assess the growth inhibiting effect of a range of ITCs on the model plant A. thaliana under standardized in vitro conditions. Three of the five ITCs used in this study are glucosinolate-derived compounds that occur naturally. Allyl-ITC is generated by the hydrolysis of the aliphatic glucosinolate sinigrin. Allyl-ITC was chosen as it can be generated by A. thaliana [47,48], although not by the Col-0 accession, and had been used in some earlier studies to assess the effects of ITCs on different plants. Benzyl-ITC and 2-phenylethyl-ITC are generated by the hydrolysis of the aromatic glucosinolates glucotropaeolin and gluconasturtiin, respectively. These glucosinolates are present at very low concentrations in some A. thaliana accessions only [49,50], but are readily produced in Brassicales species like Tropaeolum majus and Nasturtium officinale, respectively [51]. Glucosinolates that generate phenyl-ITC or 4-phenylbutyl-ITC probably do not occur naturally, despite early reports on their existence [3]. Using this series of five ITCs allowed us to assess if the structure and the length of their side chain has an impact on their effect on A. thaliana growth.
All five ITCs that were tested had a negative effect on plant growth, measured in our assays as a reduction in primary root length and total biomass. Our results confirmed earlier studies by showing that some glucosinolate-derived ITCs can be phytotoxic at high concentrations. By exposing in vitro-grown A. thaliana plants for 1 h to ITCs in the vapor phase, it was recently shown that allyl-ITC at the molar range led to leaf bleaching, reduced subsequent plant growth, and some lethality [22]. It had previously been shown that spraying vermiculite-grown A. thaliana plants with allyl-ITC at concentrations higher than 10 mM inhibited fresh weight, led to bleaching of rosette leaves and reduced chlorophyll content, while no effect was observed at a 1 mM concentration [15]. Our assays revealed that ITCs exert growth inhibitory effects at concentrations that are much lower than those previously used, and that root growth recovered from an exposure to ITCs. Concentrations of 100 µM allyl-ITC and phenyl-ITC, and 25 µM for three other aromatic ITCs (benzyl-ITC, 2-phenylethyl-ITC, and 4-phenylbutyl-ITC) showed a clear inhibitory effect on plant growth. Extrapolation of root growth inhibition data shown in Figure 5C also suggests that the aromatic ITCs benzyl-ITC and 2-phenylethyl-ITC will exert effects at doses lower than 25 µM. This was indeed confirmed by using 2-phenylethyl-ITC at 15 µM during the recovery assay ( Figure 6).
Roots of A. thaliana Col-0 seedlings contain approximately 3.2 µmol of total aliphatic glucosinolates per g of dry weight, which would correspond to a concentration of approximately 320 µM [52]. The concentrations we tested could therefore be within the amount of ITCs generated by hydrolysis of root aliphatic glucosinolates upon damage, and even higher amounts of ITCs could be generated locally. Indeed, glucosinolates are not evenly distributed in A. thaliana tissues, and cells containing an estimated amount of glucosinolates that exceeds 100 mM have been identified [53][54][55][56]. The quantity and nature of glucosinolates present in plant tissue is affected by numerous parameters, including the plant species and cultivar, the organ and the growth stage of a plant, and environmental conditions [41,42,57,58]. A. thaliana accessions show large qualitative differences in glucosinolate profiles and up to 20-fold changes in the total amount of aliphatic glucosinolates in rosette leaves [49]. Between different organs of the accession Col-0, the total glucosinolate concentration varied nearly 100-fold [41]. The amount of ITCs that will be formed by hydrolysis of glucosinolates upon plant tissue disruption will therefore be influenced by these parameters, as well as the extent of the damage and the capacity of the plant to divert glucosinolate hydrolysis from ITC towards the formation of other products (e.g., nitriles and epithionitriles) [40]. Glucosinolates and their hydrolysis products have also been identified in exudates of healthy roots, but the reported amounts of ITCs were much lower than those having an inhibitory effect in our assays [59][60][61][62][63][64].
Although the five ITCs that we tested negatively affected plant growth, they exhibited differences in their potency to generate the observed growth inhibition, with the aromatic ITCs showing a greater effect than the aliphatic allyl-ITC. Based on the phenotype caused by the tested ITCs, they can be ranged as follows: 2-phenylethyl-ITC > benzyl-ITC > 4-phenylbutyl-ITC > phenyl-ITC > allyl-ITC (from the most potent to the least potent). Quantitative differences in the effects triggered by different ITCs have been reported earlier, although with variations between studies. Benzyl-ITC was more potent than three aliphatic ITCs (sulforaphane, butyl-ITC, isopropyl-ITC) in inducing electrolyte leakage, indicative of cell death, when syringe-infiltrated into soil-grown A. thaliana leaves [14]. Growth inhibition of A. thaliana seedlings was more severe when exposed for a short time to vapors of allyl-ITC than to those of 2-phenylethyl-ITC or benzyl-ITC [22], which is different from what we observed in the current study where seedlings were grown on medium supplemented with these ITCs. However, Hara et al. [15] also observed that 2-phenylethyl-ITC had stronger effects than allyl-ITC at equal doses when spraying vermiculite-grown A. thaliana plants with different ITCs, while the other tested compound (methyl-ITC) was much less potent. Effects of ITCs on the growth of plant species that do not produce glucosinolates have also been reported. Wheat coleoptile and root length was, for example, strongly inhibited when seeds were placed on filter paper wetted with 2-phenylethyl-ITC. Less inhibition was observed for allyl-ITC and benzyl-ITC, and phenyl-ITC had hardly any effect [65].
ITCs with different chemical structures and side chains differ in their physicochemical properties such as volatility, solubility, stability, reactivity, and ability to cross the cell membrane [66][67][68][69]. ITCs are reactive and unstable compounds, and allyl-ITC is known to decompose to allyl allyldithiocarbamate and to react with water to form allylamine, which then further reacts with residual allyl-ITC to form N,N -diallylthiourea in aqueous solution, with parameters such as pH and temperature affecting allyl-ITC decomposition [69][70][71][72][73]. Under certain conditions, 2-phenylethyl-ITC and benzyl-ITC can also decompose into their respective amines phenethylamine and benzylamine [74,75]. ITCs are volatile compounds, but differ in their vapor pressure [76,77]. These differences might then be exacerbated by parameters of the experimental setup such as the mode of ITC application, the nature of the solvent, the properties of the plant growth substrate or the environmental conditions during testing. Also, the growth stage of the plant might affect susceptibility to the compound, the processes that are affected, and the responses that are triggered. In addition, some of the differences between the effects of different ITCs might be explained by the use of different plant species [78,79]. The above-mentioned and other studies illustrate the importance of experimental conditions when investigating the effect of ITCs on plants, hence our choice of testing the effects of ITCs on the model organism A. thaliana under a standard in vitro assay that is widely used for studying many other abiotic and chemical stressors. Nevertheless, the differences we observed with the tested ITCs as to their effects on growth parameters of A. thaliana seedlings must be interpreted taking into account differences in their physicochemical properties under our experimental conditions. Measurements were done 10 days after starting exposure to the indicated concentrations, and although the effects of ITCs can be observed at earlier time points (Figure 6), differential breakdown, reaction with other compounds in the medium and volatility of the chosen ITCs will have affected the exposure of seedlings during the treatment period.
The nature of the plant parameter(s) that are assessed might also affect the outcome of a study, as different ITCs might affect them differently [15,65]. In the current study, we measured the main root length and total biomass production and we observed major differences between ITCs but a good correlation between the effects of each ITC on these two parameters. The assessment of other parameters might have given a different ranking of the tested ITCs.
When comparing the four aromatic ITCs, we observed an increase in potency with the chain length from phenyl-ITC to benzyl-ITC and 2-phenylethyl-ITC. As 4-phenylbutyl-ITC was less potent than the latter two, our study revealed that there is an optimum of the side chain length as to the inhibitory effect of aromatic ITCs on A. thaliana growth. The side chain length of aromatic ITCs also had an impact on the germination and growth of wheat [65] and on the emergence of Palmer amaranth (Amaranthus palmeri) [78], but not on other plant species [13,78]. Interestingly, studies on the tumor-inhibiting effects of aromatic ITCs have also shown an increased effect with an increase in the side chain length up to 6-phenylhexyl-ITC (i.e., a C 6 chain length) and a decline in the effect of ITCs with longer chains [80][81][82]. An increase in chain length might affect the delivery of ITCs to the target tissue, due to an increase in lipophilicity and a decrease in reactivity, and favor binding to their target enzyme [82].
The molecular mechanisms underlying the observed effects of ITCs on A. thaliana are not yet understood, but are currently investigated. It was recently shown that short-term exposure of A. thaliana to high allyl-ITC concentrations leads to disintegration of microtubules, inhibits actin-mediated intracellular transport, affects the cell cycle, and depletes the glutathione pool [17,[21][22][23]. As mentioned before, ITC treatments lead to stomatal closure [19], and it had been previously proposed that ITCs might lead to the inhibition of inward K + channels in guard cells to avoid water loss by stomatal closure in response to abiotic stress [83,84]. It has been shown that exogenous treatment with sinigrin, likely through the generation of allyl-ITC, affects aquaporins and regulates water transport under salt stress in Brassica oleracea [85]. That short-chain aliphatic glucosinolates may contribute to water saving under salt stress was also indicated by the double A. thaliana mutant myb28myb29, which lacks aliphatic glucosinolates and exhibited a greater reduction in the hydraulic conductivity under salt stress than wild type plants [86]. Whether any of these processes is involved in the longer-term growth inhibition observed here and whether differences between ITCs are due to a differential modulation of these processes needs further investigation. Here, we attempted to assess the involvement of the phytohormones auxin and ethylene in the ITC-triggered effects. Using the auxin marker lines DR5::GUS and DII-VENUS we did not find any indications that auxin levels changed after allyl-ITC treatment (Figure 4), and mutants affected in the auxin response did not exhibit a different response than wild type seedlings to allyl-ITC under the tested conditions. Similarly, ethylene insensitive mutants did not behave differently from wild type seedlings on ITC-supplemented medium. Hence, our results so far do not allow us to conclude if auxin and/or ethylene play a role in the allyl-ITC-triggered root growth inhibition. However, as previous transcriptional profiling has shown that the expression of many auxin responsive genes (indole-3-acetic acid inducible (IAA) and small auxin upregulated (SAUR)) were repressed and that many genes encoding ethylene response factors (ERFs) were affected by a short-term allyl-ITC treatment [20], it might be worth investigating these processes more thoroughly in the future. Interestingly, it was recently reported that treatment with the glucosinolate hydrolysis product indole-3-carbinol inhibited A. thaliana root elongation by interfering with auxin perception [87]. Considering that ITCs with different structures vary in their effect, as was observed in our study and has been reported in the literature, it can be expected that different glucosinolate hydrolysis products affect plant growth and development by different mechanisms. These remain, however, largely uncharacterized.
In the current study, we investigated the effect of different ITCs on the root growth and biomass of A. thaliana Col-0 under standard in vitro growth conditions that have been used for studying many other abiotic and chemical stress situations. This is part of our ongoing effort to characterize the morphogenic responses induced by ITCs and other glucosinolate hydrolysis products in more detail, to investigate further which molecular mechanisms underlie the observed effects (e.g., by using omics approaches, by assessing the response of mutant plants), and to test the antagonistic or synergistic effect with other environmental factors (e.g., light, temperature) or chemical compounds (e.g., hormones, abiotic stress inducers).

Isothiocyanates
The following ITCs were used during the course of this study: 2-propenyl-ITC (allyl-ITC;

Plant Material and Growth Conditions
A. thaliana accession Col-0 seeds were surface sterilized with chlorine gas for 4 h as described [88], and stratified for 3 days at 4 • C in water. Seeds were subsequently sown on square plates (120 × 120 × 17 mm, Greiner Bio One, Kremsmünster, Austria) containing 75

Treatment with Isothiocyanates and Assessment of Growth Parameters
To assess the effect of ITCs on plant growth, seeds were in parallel sown on 1 /2 MS medium as described above (control) and sown on 1 /2 MS medium supplemented with one of the above-mentioned ITCs at the concentration(s) indicated in the text (ranging from 50 to 1000 µM for allyl-ITC and phenyl-ITC and from 1 to 300 µM for benzyl-ITC, 2-phenylethyl-ITC and 4-phenylbutyl-ITC). A 1 M solution of each ITC was freshly prepared in dimethyl sulfoxide (DMSO) and further diluted in DMSO, so that for each of the ITC concentrations, an equal volume of solution was added to the medium (0.1% v/v DMSO in final). The control plates consisted of medium supplemented with DMSO to 0.1%. The ITC and DMSO-mock solutions were added to the autoclaved medium just before pouring the plates. Four replicate plates were used for each treatment. Root growth was monitored by taking pictures of the plates after 10 days, and primary root length was measured using the ImageJ software (https://imagej.nih.gov/ij/) [89]. The root length of at least 12 seedlings from each of the four replicate plates (n = 4) was measured. The number of lateral roots was monitored under a Zeiss Axio Zoom.V16 stereo zoom microscope. The lateral root density was calculated as ratio between the number of lateral roots and the primary root length (n > 35). In addition, the average seedling biomass was assessed at day 10 by pooling 15 seedlings from each of the four plates (n = 4), and dividing that biomass by the number of seedlings. Significant differences in root growth, biomass and lateral root density between the control and treatment groups were tested with ANOVA (One-or Two-Way ANOVA, for further details see the figure legends) using the SigmaPlot (Systat Software; ver. 13.0, https://systatsoftware.com/) software.

Recovery of Root Growth from Isothiocyanate Exposure
To assess whether the root growth was able to recover from ITC-triggered inhibition, seedlings were grown as described above on medium supplemented with ITC (allyl-ITC at 500 µM or 2-phenylethyl-ITC at 15 µM) or control medium (4 replicate plates per treatment). On the fifth day, half of the seedlings from each plate were moved to new control plates. Root length was measured at the time of transfer (day 5) and at different time points after transfer (days 7, 10, 12 and 15).

Histochemical Analysis of GUS Activity
Detection of β-glucuronidase (GUS) activity was carried out as previously described [90], with minor modifications. Briefly, 10 day old DR5::GUS seedlings were incubated in 90% (v/v) pre-chilled acetone at −20 • C for 1 h. Seedlings were then washed twice for 5 min with 100 mM sodium phosphate buffer (pH 7.8) before GUS reaction buffer (100 mM sodium phosphate buffer, pH 7.8; 10 mM EDTA; 1 mM potassium ferrocyanide; 1 mM potassium ferricyanide; 1% Triton X-100) supplemented with 1 mM X-GlcA cyclohexylammonium salt (Duchefa Biochemie X1405) was added. After vacuum infiltration for 3 min, samples were incubated for 3 h in the dark at 37 • C. The enzymatic reaction was stopped by replacing the staining solution with a mixture of ethanol and acetic acid (3:1), and stored at room temperature overnight. Samples were kept in 70% (v/v) ethanol at 4 • C prior to further investigations. Seedlings were mounted onto glass slides and analyzed by using a Nikon Eclipse (E800) upright microscope equipped with DS-Ri1 camera head and Plan Fluor 40x/0.75 (working distance 0.72 mm) objective. Images were further processed by using the NIS-Elements (F 3.0), Adobe Illustrator CC and Adobe Photoshop CC software (http://www.adobe.com/products/catalog.html).

Confocal Laser Scanning Microscopy (CLSM) Analysis
Seedlings of the auxin marker line DII-VENUS and the control line mDII-VENUS, grown on allyl-ITC-supplemented medium for 10 days, were rinsed in sterile water and mounted onto glass slides using water as a medium. Samples were investigated under a Leica (DMI 6000 CS Bino inverted microscope with Adaptive Focus Control) True Confocal Scanner (TCS) SP8 instrument equipped with single molecule detection (SMD) and multiphoton laser (MP) platforms. HCX IRAPO L 25x/0.95 water immersion objective (working distance 2.4 mm) was used with the pinhole size set to 1 airy unit (AU). The yellow fluorescent protein (YFP) variant VENUS was excited by a white light laser (WLL) at 514 nm and detected at 524-540 nm using sensitive hybrid detector (HyD). Images were processed by Leica Application Suite X (LAS X 2.0), Adobe Photoshop CC and Adobe Illustrator CC software.