Diverse allyl glucosinolate catabolites independently influence root growth and 1 development 2

27 Glucosinolates (GSLs) are sulfur-containing defense metabolites produced in the Brassicales, 28 including the model plant Arabidopsis ( Arabidopsis thaliana ). Previous work suggests that 29 specific GSLs may function as signals to provide direct feedback regulation within the plant to 30 calibrate defense and growth, including allyl-GSL, a defense metabolite and one of the most 31 widespread GSLs in Brassicaceae that has also been associated with growth inhibition. Here we 32 show that at least three separate potential catabolic products of allyl-GSL or closely related 33 compounds affect growth and development by altering different mechanisms influencing plant 34 development. Two of the catabolites, raphanusamic acid and 3-butenoic acid, differentially affect 35 processes downstream of the auxin signaling cascade. Another catabolite, acrylic acid, affects 36 meristem development by influencing the progression of the cell cycle. These independent 37 signaling events propagated by the different catabolites enable the plant to execute a specific 38 response that is optimal to any given environment. 39 40 41 42 43 44 45 46 47 48 49 50


INTRODUCTION
to a response to endogenous allyl-GSL, Col-0 plants growing on exogenous allyl-GSL will 146 accumulate allyl-GSL and show a typical responsiveness to allyl-GSL in comparison to other 147 accessions (Francisco et al. 2016a, Jeschke et al. 2019 (Table 1). In our experiments, we used 148 the Arabidopsis Col-0 accession, as it provides a clean background wherein there is no 149 endogenous compound to confound these experiments. Based on the multiple lines of evidence 150 presented in Table 1, we decided to work with a concentration of 50 μM, which is lower than the 151 endogenous allyl-GSL concentration produced by different accessions and lower than the 152 endogenous concentration produced by AOP2::Col-0 plants, and hence can be considered as 153 physiologically relevant. As a control, we first tested if allyl-GSL was taken up and accumulated 154 in the plants under our experiments (Francisco et al. 2016a). We grew Col-0 seedlings on a 155 medium supplemented with or without allyl-GSL, and after 14 days we measured the GSL 156 content in the seedlings. As expected, accumulation of allyl-GSL was detected only in plants that (Francisco et al. 2016a). As expected, accumulation of raphanusamic acid was detected in plants 233 grown on a medium supplemented with this molecule (Fig. S1B). 234 The separation and quantification of acrylic acid and butenoic acid is difficult, due to the generic 235 structure and low mass of these molecules, and data on the endogenous concentrations of these 236 catabolites do not exist. Given the accepted biosynthetic pathway for raphanusamic acid 237 production from an isothiocyanate, this predicts that that there will be a 1:1 equivalency in the 238 production of raphanusamic acid and carboxy acid precursor from every isothiocyanate 239 (Bednarek et al. 2009, Piślewska-Bednarek et al. 2018, Wittstock & Burow 2010 2016). Therefore, we compromised on a uniform concentration of 50 μM for all of the 241 catabolites.

242
To test the effect of these catabolites on root length, we grew seedlings on MS media with 243 different concentrations of IAA and each compound. In contrast to allyl-isothiocyanate and allyl-244 nitrile, all three compounds strongly inhibited root length, and their effect was greater than with 245 either allyl-GSL, nitrile or isothiocyanate (Fig. 2C). Further, there were two distinct inhibition Based on the root growth assays, we conclude that these 251 three catabolites of allyl-GSL are all bioactive, and because of the stronger activity, are probably 252 closer to the actual active compounds responsible for the effects we observed when adding allyl-253 GSL. The presence of two different activities suggests that these molecules function via at least 254 two mechanisms: butenoic acid and acrylic acid may affect root growth through a similar 255 pathway, while raphanusamic acid works through a different one. 256 To determine whether allyl-GSL or one of its catabolites produce toxic compounds that lead to 257 root inhibition by causing cell death, seedlings were grown on allyl-GSL or each catabolite for 258 opposite medium, or to a new medium with the same supplementation. After an additional seven 263 days the primary roots length of the seedlings was measured. Roots of seedlings grown on a 264 media with allyl-GSL and IAA and then transferred to a clean media were significantly longer 265 than the roots of seedlings grown on allyl-GSL and IAA for the entire experiment (Fig. 2D). This 266 indicates that the seedlings were able to recover from the allyl-GSL treatment. We then 267 conducted a more direct method to test if allyl-GSL or any of its catabolites cause cell death.

268
Seedlings were grown on each of the catabolites and stained with trypan blue, which selectively 269 stains dead cells. Surveying roots from 10 seedlings grown in each of the treatments showed no 270 evidence of cell death (Fig. 2E). As a positive control for the staining method, seedlings were 271 treated with 500 mM of NaCl for 24 hours, then imaged. These seedlings showed a strong blue 272 coloration, indicating massive cell death (Fig. S3). These results show that the application of 273 allyl-GSL or its catabolites did not lead to cell death.

274
Allyl-GSL catabolites affect GSL content. To further characterize how allyl-GSL and each of 275 its catabolites affect roots, we checked whether they affect endogenous GSL accumulation in the 276 plants. Endogenous GSL levels were measured from seedlings grown for 14 days on medium 277 with each catabolite. We found that allyl-GSL and butenoic acid affected the aliphatic 4-carbon 278 GSL pathway (Fig. 3). This phenotype was defined by calculating the ratio between 4-279 methylsulfinylbutyl (4MSB) to 4MSB and 4-methylthiobutyl (4MTB) (Fig. 3). The amount of 280 indolic GSL, synthesized from tryptophan, was also impacted, hence presenting an effect on a 281 parallel GSL pathway. Interestingly, only acrylic acid affected this phenotype, as seedlings that 282 were grown on a medium with acrylic acid had a higher amount of indolic GSL (Fig. 3, detailed 283 statistic in Sup.T1).

284
Allyl-GSL catabolites have different effects on the root meristem. To dissect the 285 developmental process by which the allyl-GSL catabolites influence root growth, we measured 286 their effects on root morphology. Inhibition of primary root growth can arise from either a 287 reduction in the number of cells (as a result of an inhibition in cell divisions), or because each 288 cell is smaller (Beemster & Baskin 1998). As the root growth assays suggest that the catabolites 289 are closer to the actual active compounds, we focused on testing their effect on roots 290 morphology. Seedlings were grown on MS medium supplemented with each compound, and the 291 meristem size and the distance from the tips to the elongation zone of the seedlings were measured. The distance from the tip to the elongation zone was defined as the distance from the 293 root tip to the first root hair. Seedlings treated with raphanusamic acid, butenoic acid and acrylic 294 acid had shorter elongation zones in comparison to seedlings grown on control media (Fig. 4A,295 detailed statistic in Sup.T1). We then measured the meristem size as the number of cells between 296 the quiescent center and the first elongated cell. Only acrylic acid caused a significant reduction 297 in the number of cells in the meristem in comparison to the control roots (Fig. 4B, detailed 298 statistic in Sup.T1). To test how these catabolites may interact with IAA signaling processes, we 299 assayed how these catabolites affect auxin signaling in the meristems. For this purpose, we used 300 plants expressing the interaction domain II of Aux/IAA attached to a VENUS marker (DII-301 VENUS), which is sensitive to the presence of auxin in a dose-dependent manner (Brunoud et al. 302 2012). DII-VENUS seedlings were grown on MS medium and treated with one of the allyl-GSL 303 catabolites. The seedlings were imaged using confocal microscopy, and the mean density of the 304 VENUS fluorescence in the root was measured. The root meristem requires a specific 305 concentration of auxin for proper development (Sabatini et al. 1999), hence we quantified the 306 mean fluorescence intensity only in the meristem area, as indicated in Figure 4C. Two hours 307 following treatment with raphanusamic acid or butenoic acid, the DII-VENUS fluorescence 308 intensity in the meristems was significantly lower in comparison to the control (Fig. 4D, detailed 309 statistic in Sup.T1). In contrast, acrylic acid did not have a significant effect on the DII-VENUS 310 fluorescence intensity. This indicates that the short treatment with raphanusamic acid or butenoic 311 acid increased auxin-related signaling in the root meristem. In opposition to our hypothesis, these 312 results indicate that even though acrylic acid and butenoic acid have similar root inhibition 313 phenotypes, this likely happens via different mechanisms. This opens the door for a potential 314 third mechanism that affects root length and demonstrates that each allyl-GSL catabolite may 315 have a different mechanism to affect root inhibition. We then continued to dissect the 316 involvement of the catabolites on each one of these processes.  (Bush et al. 2015). These seedlings were grown on MS medium, treated with or without 324 acrylic acid, butenoic acid, or raphanusamic acid for two hours and then imaged using confocal 325 microscopy. We counted the number of cells in each root that were in late G2 phase, and  Using seedlings expressing the pPIN7::PIN7-GFP auxin transporter marker, we found that the following the treatment (Fig. 5A, S4A). These experiments show that raphanusamic acid affects 355 auxin signaling and transporters at around 60 minutes following the treatment.

356
Butenoic acid affects the auxin machinery. As butenoic acid also had a rapid effect on auxin 357 signaling in the meristem, we tested how this molecule affects each of the auxin marker and 358 receptor lines. Treatment of 30 minutes with butenoic acid treatment resulted in a significant 359 reduction in the DII-VENUS intensity compared to the untreated seedlings ( Fig. 5B,C). We also 360 found a significant reduction in the intensity of the PIN1 transporter 60 minutes following the 361 treatment ( Fig. 5B, S4B), and a significant reduction in the intensity of the PIN7 transporter in 362 the root cap 90 minutes following the treatment (Fig. 5B, S4C). Lastly, we found that treatment 363 with butenoic acid significantly reduced the activity of TIR1-VENUS reporter 90 minutes 364 following the treatment (Fig. 5B, S4A). Using these marker lines, we conclude that butenoic acid 365 affects the auxin machinery in a specific order. Butenoic acid first affects auxin signaling, then 366 auxin transporters PIN1 and PIN7, and finally the auxin receptor TIR1.

367
Our results clearly show that both raphanusamic acid and butenoic acid affect the auxin 368 machinery, but the timing of the effects of each one of the catabolites on the different 369 components of the auxin machinery is different. In combination with their different chemical 370 structure, this suggests that raphanusamic and butenoic acid may have different molecular 371 targets. To test whether they have different molecular targets, we analyzed if they interact to 372 modulate root growth. In pharmacological assays an interaction between two compounds 373 suggests that they work through the same target (Jia et al. 2009). Seedlings were grown on 374 medium supplemented with or without butenoic acid, raphanusamic acid, or both, and root 375 lengths were measured. ANOVA was used to test the effect of each defense catabolite 376 individually as well as their interaction in respect to root length (Fig. 5E). Within these 377 conditions, there was no detectable interaction between the two catabolites. In agreement with 378 the auxin-related marker genes, this suggests that the two catabolites function through different 379 targets. Hence, we conclude that although butenoic acid and raphanusamic acid both affect the 380 auxin signaling machinery, this is via independent mechanisms.

402
To genetically test the in planta interaction of the catabolites with the auxin machinery and its 403 receptors, we measured their effect on the root growth of the tir1-1 afb2-1 afb3-1 triple mutant.

404
This mutant controls for the partial redundancy between TIR1 and the AFBs (Dharmasiri et al.

405
2005). We grew Col-0 and the triple mutant seedlings on medium with or without allyl-GSL, 406 raphanusamic acid, and acrylic acid, measured their root length at day seven, then calculated the 407 percentage of elongation compared to the untreated seedlings. The auxin triple mutant affected 408 the sensitivity to allyl-GSL, but in contrast, had no effect on the sensitivity to raphanusamic acid 409 and acrylic acid (Fig. S6). This supports the observation that acrylic acid does not work through 410 the auxin pathway, and it suggests that allyl-GSL might have some additional auxin-related 411 pathways that are yet to be identified. them on a medium with or without raphanusamic acid, acrylic acid, and butenoic acid, and 419 measured their root length after five days. All three allyl-GSL catabolites had an effect on the 420 tested species, but there was specificity to the effects for each catabolite. Acrylic acid inhibited 421 the root growth of dill, lettuce and basil with no effect on tomato. In contrast, butenoic acid 422 inhibited the root growth of dill and lettuce while stimulating basil and having no influence on 423 tomato. Finally raphanusamic acid also inhibited dill and lettuce while dramatically inducing 424 root growth in tomato (Fig. 6). These results show that the allyl-GSL catabolites can influence 425 growth across a wide range of species (probably since it works through conserved mechanisms), 426 and that similar to Arabidopsis, it is likely that there are three different mechanisms being 427 targeted.

429
Allyl-GSL catabolites work through different mechanisms. In this work we found that the 430 defense metabolite allyl-GSL affects Arabidopsis root growth and development by three 431 different catabolic products, with each compound having a unique regulatory effect on root 432 development (summarized in Fig. 7). Acrylic acid has the most unique mechanism out of the 433 catabolites that were tested. Acrylic acid was the only molecule that affected meristem 434 development by influencing the cell cycle in the root tips. In contrast to acrylic acid, both 435 butenoic acid and raphanusamic acid manipulated several steps of the auxin machinery.

487
The fact that the effect of allyl-GSL is dependent upon environmental conditions suggests that 488 future studies aiming to dissect GSL mechanisms will have to consider a precise description of 489 the behavior of each molecule under different environments and different conditions. Adding to 490 this complexity is the fact that some of the catabolites can be synthesized by more than one 491 precursor, rather than only from allyl-GSL. This is the case for butenoic acid, which can also be 492 synthesized from but-3-enyl GSL (which is not produced in Col-0 plants), and raphanusamic 493 acid, whichcan be synthesized from any GSL-derived isothiocyanate (Bednarek et al. 2009).  The complexity of the role of defense metabolites in coordinating defense and growth is known.

501
In this work, we present another layer to this complexity by showing that one metabolite can 502 have multiple mechanisms to affect development under different environments. We show that allyl-GSL, which can regulate its catabolic products according to the changing biotic conditions, 504 has multiple ways to affect plant growth and development. We propose that this allows the plant 505 to sense the specific processes influencing defense metabolism, and then enable a specific 506 response that is optimal to any given environment. This may apply to other plant species and 507 different defense metabolites also known to have different developmental effects. To test root curliness, seedlings were grown vertically on clean MS medium for four days, then 535 transferred to medium supplemented with different concentrations of IAA, allyl-GSL or both.

536
After one additional day the plates were tilted to a 45° angle against the gravity vector, and after 537 3 days of growth the number of root curls within 1 cm from the tip was counted (Mochizuki et al.  removed, and the seedlings were rinsed with distilled water before placing on microscope slides.

546
For positive control: six-day-old seedlings were treated with 500 mM NaCl and stained with 547 trypan blue after 24 hours.

549
Seedlings were submerged in 0.005 mg ml -1 propidium iodide in distilled water, placed on 550 microscope slides, and imaged using a Zeiss LSM700 laser scanning microscope with ×20/NA 551 0.8. All pictures were taken with the exact settings. Rainbow spectrum was applied for 552 PIN1:GFP pictures. YFP/GFP fluorescence in the root tips was quantified using ImageJ software 553 (https://imagej.nih.gov/ij/). The mean fluorescence was compared to the mean fluorescence of 554 the untreated plants (control). Definition of meristem area (Fig. 4C): the meristematic area was 555 defined with a deltoid shape: 1 row under the quiescent center + 8 rows above the quiescent 556 center, minus 2 rows on each side.

558
GSLs were measured as previously described (Kliebenstein et al. 2001a-c). Briefly, 4-6 14-day-559 old seedlings from the same Petri plate were pooled, weighed and harvested in 400 μL of 90% methanol. Tissues were homogenized for 3 min in a paint shaker, centrifuged, and the 561 supernatants were transferred to a 96-well filter plate with DEAE sephadex. The filter plate with 562 DEAE sephadex was washed once with water, 90% methanol, and water again. The sephadex-563 bound GSLs were eluted after an overnight incubation with 110μL of sulfatase. Individual 564 desulfo-GSLs within each sample were separated and detected by HPLC-DAD, identified, 565 quantified by comparison to standard curves from purified compounds, and further normalized to 566 the fresh weight.

Yeast-two hybrid assays 572
Yeast-two hybrid experiments were performed as described previously (Prigge et al. 2010).

577
In vitro pull-down assays 578 In vitro pull-down assays were performed as described previously (Parry et al. 2009). The

585
Statistical analyses were conducted using R software (https://www.R-project.org/) with the 586 RStudio interface (http://www.rstudio.com/). Significance was tested via two-way ANOVA 587 using "stat" package. Specific models are listed in Supplemental Table 1, but they followed the 588 following general format: in each experiment, Treatment (application of allyl-GSL or associated 589 catabolites) and Auxin (the auxin concentrations ranging 0-0.5 µM) were considered as fixed 590 effects. Experiment and Plate were treated as random effects in the linear model.     Table S1). (D) Arabidopsis seedlings were grown vertically on clean 651 experiment. Significance was tested by t-Test, P < 0.0001, Error bars represent standard errors.

656
(E) Seedlings were grown on MS medium with or without 50 µM allyl-GSL. Seven-day-old 657 seedlings were stained with trypan blue and photographed. Two experiments were conducted, 658 with 5-10 replicates in every experiment per each treatment. Bar = 0.5 mm. For all panels significance was tested via two-way ANOVA (* < 0.05, ***<0.0001, relative to control untreated seedlings.