Supplementary Materials for b-cyclocitral is a natural root growth regulator Alexandra J. Dickinson, Kevin Lehner, Medhavinee Mijar, Philip N. Benfey Correspondence to: [email protected] This PDF file includes: Materials and Methods Figs. S1 to S8 Tables S1 References 29-32 1 Materials and Methods Plant Growth and Treatment Conditions Arabidopsis Thaliana: All seeds were in the Columbia-0 background. Seeds were sterilized with chlorine gas, incubated in 4°C water for 2 days, and plated on 1% (wt/vol) Murashige and Skoog (1% MS) media with 1% agar. pEN7:GAL4; UAS:H2A-GFP seeds were acquired from the DeVeylder lab. pWOX5:GFP (29) and pDR5:LUC (5) lines have been published previously. Seedlings were exposed to 100-130 µmol/(m2s1) illumination and grown vertically under long- day conditions at 22°C. Plants were analyzed at 7 days after stratification (das), unless noted otherwise. D15-treated media was made using D15 (N-(4-fluorobenzyl)-methoxycinnamic hydroxamic acid) synthesized by LeadGen Labs, LLC solubilized in DMSO. 100 mM D15 stock solution was diluted directly into 1% MS media for final concentrations ranging from 1 µM to 100 µM. To perform the chemical screen testing the ability of apocarotenoids to rescue D15, plants were seeded on 1% MS media containing 30 µM D15 and either 100 nM, 1 µM, or 25 µM apocarotenoid (diluted from 1 M stock solutions dissolved in DMSO). For Supplemental Figure 3, the concentration of each apocarotenoid that showed the most beneficial effect on lateral root branching is displayed. These concentrations are: 100 nM of dimethyl-b-cyclocitral (dmBc), b- ionone (BI), safranal (Saf), dihyro-b-ionone (dHBI), abscisic acid (ABA), GR24, 25 µM dihydroactinidiolide (DHAD), and 1 µM b-cyclocitral. For all experiments, including those performed on tomato and rice, plants were never exposed to DMSO concentrations that exceeded 0.1%. Unless otherwise stated, for subsequent experiments characterizing the effect of b-cyclocitral, plants were exposed to volatile analytical grade b-cyclocitral (≥ 97%, Sigma Aldrich) using 100 x 15 mm partitioned petri dishes. One side of the partition was filled with 25 µM b-cyclocitral diluted in 1% MS media. Seeds were plated on the other side of the partition, on 1% MS media, either with or without D15 treatment. This media did not contact b-cyclocitral, so seedlings were exposed to only volatile b-cyclocitral. To determine the minimal working concentration of b- cyclocitral, b-cyclocitral was diluted into media at concentrations of either 250 nM, 500 nM, 750 nM, or 1 µM (Supplemental Figure 5). In this experiment, roots were in direct contact with the b-cyclocitral media. Tomato: Seeds of Heinz tomato (Solanum lycopersicum) were used for all tomato experiments. Seeds were sterilized using 40% liquid bleach for 30 min, then plated on 1% MS media in 120 x 120 x 17 mm square petri dishes. Seeds were kept in the dark at room temperature for 3 days, and then exposed to 100-130 µmol/(m2s1) illumination and grown vertically under long-day conditions at 22°C. For treating tomato seedlings, b-cyclocitral was added directly to the media for a final concentration of 100 µM. Tomato phenotypes were analyzed at 6 das. Rice: Rice variety, 9311 seeds were dehulled, sterilized with bleach, rinsed with sterile water, and germinated in the dark for 2 days at 30°C. Germinated seeds were transferred to Yoshida’s nutrient solution, solidified with 0.25% gelzan, in 2 L glass cylinders, as described previously (27). For chemical treatments, Yoshida’s nutrient solution was supplemented with 10 µM b- cyclocitral, 50 mM NaCl, or 100 mM NaCl, depending on the treatment. Unless otherwise stated, rice roots were imaged at 6 days after transfer to solution. Analysis of root system architecture traits was performed using GiA roots (28) and analysis of individual primary and crown roots was done using SmartRoot (30), as described previously. Crown root growth angle was calculated by measuring the angle between the crown root and the primary root. Root Phenotyping To measure lateral root capacity, defined as the number of emerged lateral roots after removal of the primary root apical meristem, the primary root apical meristem of each seedling was sterilely excised at 7 das. Plants were given 3 days to grow out lateral roots, and then each emerged lateral root was counted using a dissection microscope, as described previously (7). Luciferase activity was measured as previously described, using exposure times of 7 min (5). Maximum oscillation intensities of the lateral root clock were identified by imaging roots every 7 minutes over the course of 18 hours. The maximum oscillation intensity during that time course was identified and quantified manually using ImageJ software (National Institutes of Health). The background intensity of the root in a non-oscillating zone was subtracted for each measurement. WOX5+ and EN7+ primordia were visually counted using a stereo zoom microscope (Zeiss). Root meristems and elongation zones were stained using propidium iodide (PI) and imaged using confocal microscopy (Zeiss). Lateral and primary root lengths for Arabidopsis and tomato were measured using ImageJ (National Institutes of Health) software. 3D images of rice root system architecture were captured using a 360° revolving platform and images were analyzed using GiA Roots (www.giaroots.org) and SmartRoot (https://smartroot.github.io/) (28, 30). Sample preparation for GC-MS was performed as detailed in Tikunov et al. with minor modifications (31). Briefly, frozen sample powder (1 g) was weighed in a 20 mL headspace vial containing 1 mL of EDTA-NaOH (pH of 7.5, 50 mM EDTA) that was spiked with 38 ug of linalool as internal standard. Solid CaCl2 (1 g) was immediately added to the sample, which was then incubated at 70°C for 15 min with agitation. Volatile compounds were extracted by a 75 µm DVB/PDMS autosampler fiber (Sigma-Aldrich) at 70°C for 20 min with agitation, and then desorbed at 250°C for 3 min into a DB-WAXUI column (30 m x 0.25 mm x 0.25 µm, Agilent) in a Trace1310 GC (Thermo) coupled to a Thermo ISQ-LT MS. The inlet temperature was operated at 250°C in splitless mode during desorption. A constant flow rate of the carrier gas (He) was set at 1.2 mL/min. The oven temperature program started at 100°C held for 1 min, then increased to 250°C at 10°C/min and held for 3 min. Detection was completed under electron impact mode, with a scan range of 50-550 amu and scan rate of 10 scans/second. Transfer line and source temperatures were 240° and 250°C, respectively. Statistical Analysis In all box and whisker plots, the box extends from the 25th to 75th percentile and the whiskers extend to the minimum and maximum values of the data set. In bar graphs and xy plots, the bars or points represent mean values. The error bars represent standard deviations of the mean. All relative values are normalized to the control. Figure 1A: Lateral root capacity data was fit with a log inhibitor vs. normalized response (IC50 curve) using a variable slope in GraphPad Prism. Figure 1C: Lateral root capacity was analyzed using a one-way ANOVA with Tukey’s multiple comparison test (n = 4 experiments, p = 0.005). Figure 1E: pDR5:LUC LR clock oscillation intensity was analyzed using a one-way ANOVA with Tukey’s multiple comparison test (n ≥ 14 plants, p < 0.0001). Figure 2B: Primary root length was analyzed using an unpaired t-test (n ≥ 50 plants). Figure 2D: The number of meristematic cortex cells per plant was counted by hand and analyzed using an unpaired t-test (n ≥ 30 plants). Figure 2F: Primary root length of tomato plants was analyzed using an unpaired t-test (n ≥ 35 plants). Figure 2G: Lateral root length was analyzed using an unpaired t-test (n ≥ 74 roots from 35 plants). Figure 3B: For each day after transferring germinated rice seedlings to media, root system depth between treatments was compared using an unpaired t-test (n ≥ 5 plants). Figure 3C-H: All graphs show analysis of root system architecture traits measured on day 6 after transfer to media. Unpaired t-tests were used to analyze significance (n ≥ 7 plants). Figure 4 B: Root depth was analyzed using a 2-way ANOVA with Tukey’s multiple comparison test (n ≥ 16 plants). Data was collected 7 days after planting. The effects of b-cyclocitral treatment and salt concentration were both statistically significant (p < 0.0001). The interaction between these treatments was also significant (p < 0.0001). Figure 4 C: Network solidity was analyzed using a 2-way ANOVA with Tukey’s multiple comparison test (n ≥ 16 plants). Data was collected 7 days after planting. The effect of b- cyclocitral treatment and salt concentration were both statistically significant (p < 0.0001). The interaction between these treatments was also significant (p < 0.01). Supplemental Figures A Carotenes cis-phytoene pseudoionone tri-cis-carotene strigolactone all-trans-lycopene DHAD β-ionone α-carotene β-carotene dihydro β-ionone Xanthopyll β-cyclocitral Carotenoids zeinoxanthin zeaxanthin dimethyl-β-cyclocitral lutein violaxanthin safranal neoxanthin abscisic acid B pseudoionone GR24 (strigolactone) β-ionone O O O O CH3 O O O β-cyclocitral DHAD dihydro β-ionone dimethyl-β-cyclocitral O O O H CH H O 3 O safranal abscisic acid O H OH O O OH Figure S1. The carotenoid pathway (A) and the structures of b-carotene derived apocarotenoids (B). 100 **** EN7 * 50 0 -50 -100 -150 % Change in Lateral Root Capacity (Relative to D15) c I I β β β D15 ABA -cyc β Control D15 + D15 + Saf D15 + dHD15 + D15 + dm D15 + GR24D15 + DHADD15 + Figure S2.