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. The ability of apocarotenoids to rescue the lateral root capacity of D15-treated plants (normalized to D15 treatment). Abbreviations: dimethyl-b-cyclocitral (dmbc), b-ionone (bI), safranal (Saf), abscisic acid (ABA), dihydroactinidiolide (DHAD), b-cyclocitral (b-cyc). Data were analyzed using a one-way ANOVA (p < 0.0001) with Dunnett’s multiple comparisons test (n = 3 experiments). The symbols * and **** indicate p values ≤ 0.05 and 0.0001, respectively.

100 )

Area 80

60

40

(counts*min/g 20 Normalized Peak 0

-cyc β DHAD -ionone β

Figure S3. Gas chromatography – mass spectrometry analysis identified selected apocarotenoids in untreated plant tissue (n = 4 experiments).

15 *

*

10 Lateral Roots (#) 5

1 uM Control 250 nM 500 nM 750 nM β-cyc Concentration

Figure S4. Number of lateral roots per plant in Arabidopsis seedlings exposed to varying concentrations of b-cyclocitral diluted in media. Data were analyzed using a one-way ANOVA with Dunnett’s multiple comparison test (n = 3 experiments). The endogenous concentration of b-cyclocitral in leaves was previously calculated to be approximately 300 nM (13). The symbols * indicate p values ≤ 0.05.

A WOX5 * B EN7 1.5 * 1.5 *

a * * * * 1.0 1.0 Primordi Primordia + 0.5 + 0.5 7 EN

WOX5 0.0 0.0 Control β-cyc D15 D15 Control β-cyc D15 D15 +β-cyc +β-cyc

Figure S5. The number of lateral root primordia (normalized to control seedlings) identified using A) pWOX5:GFP, which marks initiated lateral root primordia (n = 4 experiments), and B) pEN7:GAL4; UAS:H2A-GFP, a marker line for the endodermis (n = 4 experiments). Images depict representative examples of lateral root primordia identified using pWOX5:GFP (A) and pEN7:GAL4; UAS:H2A-GFP (B). Scale bars are 50 µm. Data were analyzed using ratio paired t- tests. The symbols * indicate p values ≤ 0.05.

300 ns m) μ ( 200

100 Cell Length

0

-cyc β Control Figure S6. Quantification of the cell length of mature cortex cells in b-cyclocitral treated roots (n = 30). The abbreviation “ns” stands for non-significant (p = 0.27).

A Control D15 B

250 ****

200 m) μ ( 150

100 Cortex Cells Length of Mature 50

0

D15 Control

Figure S7. (A) Longitudinal cross-sections of mature root tissue stained with propidium iodide. Cortex cells are outlined in orange. (B) Quantification of the cell length of mature cortex cells in microns (n ≥ 27 plants). The symbols **** indicate p values ≤ 0.0001, calculated using an unpaired t-test.

ns

15

10

5

0 Dry Shoot Mass (mg)

-cyc β Control

Figure S8. Mass of dried rice shoots treated with b-cyclocitral (n = 8 plants). The abbreviation “ns” stands for non-significant (p = 0.93), calculated using an unpaired t-test.

Table S1. Gene ontology (GO) term enrichment analysis of Arabidopsis genes upregulated by b- cyclocitral. Gene function analysis was performed using the PANTHER Classification System (32) on previously published data (13).

GO Term Fold Enrichment p value

response to host immune response (GO:0052572) 16.32 4.38E-02

response to immune response of other organism involved in symbiotic interaction (GO:0052564) 16.32 4.38E-02

positive regulation by symbiont of host immune response (GO:0052556) 16.32 4.38E-02

positive regulation by organism of immune response of other organism involved in symbiotic interaction (GO:0052555) 16.32 4.38E-02

modulation by symbiont of host immune response (GO:0052553) 16.32 4.38E-02

modulation by organism of immune response of other organism involved in symbiotic interaction (GO:0052552) 16.32 4.38E-02

response to symbiotic fungus (GO:0009610) 16.32 4.38E-02

jasmonic acid biosynthetic process (GO:0009695) 13.63 5.11E-04

jasmonic acid metabolic process (GO:0009694) 11.19 2.21E-03

oxylipin metabolic process (GO:0031407) 10.97 1.22E-02

regulation of jasmonic acid mediated signaling pathway (GO:2000022) 9.79 2.52E-02

glutathione metabolic process (GO:0006749) 8.71 4.57E-06

toxin catabolic process (GO:0009407) 8.52 1.15E-03

secondary metabolite catabolic process (GO:0090487) 8.52 1.15E-03

response to toxic substance (GO:0009636) 8.13 4.29E-10

toxin metabolic process (GO:0009404) 7.97 1.68E-04 positive regulation of immune response (GO:0050778) 7.58 2.85E-04 positive regulation of immune system process (GO:0002684) 7.58 2.85E-04 response to chitin (GO:0010200) 6.83 4.10E-09 response to oomycetes (GO:0002239) 6.78 2.64E-02 regulation of programmed cell death (GO:0043067) 6.78 2.64E-02 response to endoplasmic reticulum stress (GO:0034976) 6.75 8.88E-03 positive regulation of innate immune response (GO:0045089) 6.64 1.03E-02 regulation of cell death (GO:0010941) 6.25 6.13E-03 regulation of immune response (GO:0050776) 5.97 6.21E-05 regulation of immune system process (GO:0002682) 5.69 4.52E-05 detoxification (GO:0098754) 5.6 4.44E-02 response to UV-B (GO:0010224) 5.6 4.44E-02 response to organonitrogen compound (GO:0010243) 5.57 5.13E-09 regulation of innate immune response (GO:0045088) 5.48 1.25E-03 response to virus (GO:0009615) 5.45 2.16E-02 response to wounding (GO:0009611) 5.36 1.95E-09 cellular modified amino acid metabolic process (GO:0006575) 5.33 1.15E-04

positive regulation of defense response (GO:0031349) 5.19 3.40E-02 organic acid catabolic process (GO:0016054) 5.07 1.32E-03 carboxylic acid catabolic process (GO:0046395) 5.07 1.32E-03 aging (GO:0007568) 4.66 7.46E-04 response to jasmonic acid (GO:0009753) 4.64 1.38E-06 response to karrikin (GO:0080167) 4.63 3.97E-03 response to cadmium ion (GO:0046686) 4.53 4.24E-11 response to salicylic acid (GO:0009751) 4.4 3.83E-05 small molecule catabolic process (GO:0044282) 4.32 2.04E-03 response to nitrogen compound (GO:1901698) 4.22 5.94E-08 response to metal ion (GO:0010038) 4.02 3.49E-12 response to bacterium (GO:0009617) 3.86 7.71E-10 response to reactive oxygen species (GO:0000302) 3.85 1.83E-02 positive regulation of response to stimulus (GO:0048584) 3.84 1.40E-03 response to water deprivation (GO:0009414) 3.69 2.93E-06 protein folding (GO:0006457) 3.67 8.29E-04 response to inorganic substance (GO:0010035) 3.66 1.48E-20 response to water (GO:0009415) 3.59 5.46E-06

regulation of defense response (GO:0031347) 3.48 6.08E-03 response to heat (GO:0009408) 3.45 3.81E-02 defense response to bacterium (GO:0042742) 3.42 5.09E-05 response to salt stress (GO:0009651) 3.38 3.11E-09 response to other organism (GO:0051707) 3.38 9.48E-23 response to external biotic stimulus (GO:0043207) 3.37 1.08E-22 innate immune response (GO:0045087) 3.37 1.09E-03 immune system process (GO:0002376) 3.36 1.26E-04 response to biotic stimulus (GO:0009607) 3.33 8.26E-23 immune response (GO:0006955) 3.3 1.58E-03 regulation of signal transduction (GO:0009966) 3.24 1.75E-02 response to osmotic stress (GO:0006970) 3.23 1.67E-09 regulation of signaling (GO:0023051) 3.19 2.21E-02 regulation of cell communication (GO:0010646) 3.15 2.63E-02 organic hydroxy compound metabolic process (GO:1901615) 3.15 2.63E-02 response to oxygen-containing compound (GO:1901700) 3.12 1.05E-24 sulfur compound metabolic process (GO:0006790) 3.11 3.64E-04 response to acid chemical (GO:0001101) 3.1 9.37E-18

secondary metabolic process (GO:0019748) 3.03 2.33E-05 response to oxidative stress (GO:0006979) 2.99 8.23E-05 response to external stimulus (GO:0009605) 2.98 4.45E-22 monocarboxylic acid metabolic process (GO:0032787) 2.97 1.54E-04 response to temperature stimulus (GO:0009266) 2.95 4.57E-06 response to chemical (GO:0042221) 2.91 1.79E-41 response to organic cyclic compound (GO:0014070) 2.88 4.10E-03 secondary metabolite biosynthetic process (GO:0044550) 2.84 4.50E-02 oxidation-reduction process (GO:0055114) 2.83 1.87E-17 response to fungus (GO:0009620) 2.81 2.55E-05 response to cold (GO:0009409) 2.72 1.79E-02 multi-organism process (GO:0051704) 2.63 9.97E-17 oxoacid metabolic process (GO:0043436) 2.62 1.58E-09 organic acid metabolic process (GO:0006082) 2.61 1.80E-09 defense response to other organism (GO:0098542) 2.56 5.73E-07 defense response (GO:0006952) 2.53 9.05E-13 response to organic substance (GO:0010033) 2.49 2.30E-17 regulation of response to stimulus (GO:0048583) 2.48 1.92E-03

carboxylic acid metabolic process (GO:0019752) 2.43 3.77E-06 response to stress (GO:0006950) 2.42 7.72E-31 response to abiotic stimulus (GO:0009628) 2.41 1.61E-15 homeostatic process (GO:0042592) 2.35 4.02E-02 response to stimulus (GO:0050896) 2.19 7.62E-47 cellular catabolic process (GO:0044248) 2.16 2.14E-04 small molecule metabolic process (GO:0044281) 2.13 4.74E-07 cellular response to stress (GO:0033554) 2.12 9.90E-03 response to hormone (GO:0009725) 2.09 4.66E-07 response to endogenous stimulus (GO:0009719) 2.08 5.92E-07 regulation of biological quality (GO:0065008) 2.05 7.54E-03 catabolic process (GO:0009056) 1.9 1.02E-03 cellular response to chemical stimulus (GO:0070887) 1.88 3.36E-02 cellular response to stimulus (GO:0051716) 1.79 2.58E-06 organonitrogen compound metabolic process (GO:1901564) 1.4 2.79E-02 biological regulation (GO:0065007) 1.39 1.22E-03 metabolic process (GO:0008152) 1.39 5.84E-11 cellular process (GO:0009987) 1.29 3.44E-06

organic substance metabolic process (GO:0071704) 1.25 1.93E-02 biological_process (GO:0008150) 1.09 6.27E-04

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