advances.sciencemag.org/cgi/content/full/5/11/eaaw6787/DC1

Supplementary Materials for

Anchorene is a carotenoid-derived regulatory metabolite required

for anchor root formation in Arabidopsis

Kun-Peng Jia, Alexandra J. Dickinson, Jianing Mi, Guoxin Cui, Ting Ting Xiao, Najeh M. Kharbatia, Xiujie Guo, Erli Sugiono, Manuel Aranda, Ikram Blilou, Magnus Rueping, Philip N. Benfey, Salim Al-Babili*

*Corresponding author. Email: salim.babili@kaust.edu.sa

Published 27 November 2019, Sci. Adv. 5, eaaw6787 (2019)

DOI: 10.1126/sciadv.aaw6787

The PDF file includes:

Supplementary Text Fig. S1. Structures of diapocarotenoids and supposed precursors for anchorene. Fig. S2. Effects of diapocarotenoid and its derivatives on Arabidopsis root development. Fig. S3. Characterization of ANR development by different DR5 marker lines. Fig. S4. Root formation under different treatments and in various mutants. Fig. S5. Anchorene effects on DR5 expression in ANR primordia. Fig. S6. Anchorene isomer identification and anchorene quantification. Fig. S7. Conversion of OH-Apo12′ into anchorene in plants. Fig. S8. Involvement of auxin signaling and distribution on ANR development. Fig. S9. Transcriptomic change analysis of collet tissues upon different treatments by RNA-seq. Fig. S10. Effect of anchorene on plant growth. Fig. S11. The synthesis route and derivatization for anchorene. Table S1. The nutrient element composition in Argo soil and Silver sand. Table S2. Mutants and marker lines used in this study. Legends for datasets S1 and S2 References (61–64)

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/5/11/eaaw6787/DC1)

Dataset S1 (Microsoft Excel format). Gene list (1.5-fold change) for different treatments in RNA-seq. Dataset S2 (Microsoft Excel format). BP enrichment for different treatments in RNA-seq.

Supplementary Text

Synthesis D6-anchorene and anchorene derivatives

Unless otherwise noted, all commercially available compounds were used as provided without

further purification. CH2Cl2 and THF used for the reactions were purified by an MBraun solvent

purification system (SPS). Solvents for chromatography were technical grade and freshly

distilled prior to use. Analytical thin-layer chromatography (TLC) was performed on Merck

silica gel aluminium plates with F-254 indicator, visualized by irradiation with UV light. Column

chromatography was performed using silica gel (Macherey Nagel, particle size 0.040-0.063 mm).

Solvent mixtures are understood as volume/volume. 1H-NMR and

13C-NMR were recorded on a

Varian AV400 or AV600 spectrometer. Data are reported in the following order: chemical shift

(δ) in ppm and coupling constants (J) are in Hertz (Hz). IR spectra were recorded on a Perkin

Elmer-100 spectrometer and are reported in terms of frequency of absorption (cm-1

). Mass

spectra (EI-MS, 70 eV) were conducted on a Finnigan SSQ 7000 spectrometer.

The synthetic route for isotopic labeled anchorene and anchorene derivatives as shown in fig.

S11A. 4a, 4b (AR diethyl ester): (2E, 4E, 6E)-diethyl-2,7-dimethylocta-2,4,6-trienedioate (61,

62): But-2-ene-1,4-diol 1 (1 equiv) dissolved in CH2Cl2 was added to a solution of MnO2 (18

equiv) in CH2Cl2 at 0 °C. Phosphorane 2 (2.4 equiv) dissolved in DCM was then added. The

reaction mixture was stirred at room temperature until the TLC showed the full consumption of

the starting material. MnO2 was removed by filtration over celite and the filtrate concentrated in

vacuo. Purification by column chromatography (hexane: EtOAc 5:1) gave the product 3 as white

crystalline solid. Ester 3 (1 equiv) in dry THF was added to a suspension of LiAlH4 (2.4 equiv)

in dry THF at 0 °C. The reaction mixture was stirred at this temperature for 1 h. The reaction was

quenched by slow addition of water and 20% NaOH solution. The organic phase was separated,

and the aqueous phase washed with EtOAc. The combined organic phase was dried over MgSO4

and concentrated leading to the desired diol 4.

4a: 1H NMR (400 MHz, CDCl3): δ (ppm): 1.31 (t, J = 7.2 Hz, 6 H), 2.0 (d, J = 7.2 Hz, 6 H),

4.22 (q, J = 7.2 Hz, 4H), 6.79 (dd, J = 7.6 Hz/2.8 Hz, 2 H), 7.28 (dd, J = 8.0 Hz/1.6 Hz, 2H).

4b: 1H NMR (600 MHz, CDCl3): δ (ppm): 1.32 (t, J = 7.2 Hz, 6 H), 4.23 (q, J = 7.2 Hz, 4H),

6.79 (dd, J = 7.8 Hz/3.0 Hz, 2 H), 7.29 (dd, J = 7.8 Hz/3.0 Hz, 2H). 13

C NMR (150.9 MHz,

CDCl3): δ (ppm) :14.3, 60.8, 130.1, 133.6, 137.2, 168.0. MS (EI) m/z (%): 258.2 [M+.] (100). IR

(ATR): ṽ = 2114, 1694, 1615, 1475, 1368, 1284, 1225, 1101, 995 cm-1

.

5a (AR dialcohol): (2E, 4E, 6E)-2,7-dimethylocta-2,4,6-triene-1,8-diol

6a, 6b (anchorene) (2E,4E,6E)-2,7-dimethylocta-2,4,6-trienedial (63): To the suspension of

LiAlH4 (2.4 equiv) in dry THF at 0 °C was added the ester 4 (1 equiv) in dry THF. The reaction

mixture was stirred at this temperature for 1 h. The reaction was quenched by slow addition of

water and 20% NaOH solution. The organic phase was separated and the aqueous phase washed

with EtOAc. The combined organic phase dried over MgSO4 and concentrated. The residue 5

was oxidized without further purification. To a cooled solution of the crude diol 5 in acetone was

added MnO2 (18 equiv). The reaction mixture was allowed to warm up to room temperature and

stirred for 24 h. The solid was removed by filtration over a pad of celite and washed with CH2Cl2.

The solvent was removed in vacuo and the residue was purified by column chromatography

(SiO2, hexane:EtOAc 5:1). The dialdehyde 6 was isolated as yellow solid.

5a: 1H NMR (600 MHz, CDCl3): δ (ppm) = 1,61 (s, 2H), 1.81 (s, 6H), 4,10 (s, 4H), 6.16 (d, J =

7.8 Hz, 2H), 6.45 (dd, J = 7.2 Hz/3.0 Hz, 2 H).

6a: 1H NMR (600 MHz, CDCl3): δ (ppm) = 1.93 (s, 6H), 6.96 - 7.02 (m, 2H), 7.07 (dd, J = 7.8

Hz/ 3.0 Hz, 2H), 9.53 (s, 2 H).

6b: 1H NMR (400 MHz, CDCl3): δ (ppm) = 6.99 (dd, J = 8.4 Hz/ 2.8 Hz,2H), 7.07 (dd, J = 8.0

Hz/ 3.2 Hz, 2H), 9.55 (s, 2H). 13

C NMR (100.5 MHz, CDCl3): δ (ppm) = 134.4, 140.9, 146.0,

194, 4. MS (EI) m/z (%) = 170.1 [M+.] (100). IR (ATR): ṽ = 2078, 1718, 1662, 1369, 1270, 1171,

1032, 979 cm-1

.

7 (AR diacid): (2E, 4E, 6E)-2,7-dimethylocta-2,4,6-trienedioic acid (64): A mixture of dial 6a (6

mmol, 1 equiv), Ag2O (9.1 mmol, 1.4 equiv), 3 mL NaOH (10% solution) and 20 mL H2O was

refluxed for 24h. The reaction mixture was diluted with 50 mL H2O, and the solid was separated

by filtration. The filtrate was acidified with diluted HNO3 and the resulting solid filtered up and

recrystallized from ethanol to give the product as off-white solid.

7:1H NMR (600 MHz, DMSO-d6): δ (ppm) = 1.92 (s, 6H), 7.12 - 7.32 (m, 4H), 12.4 (bs, 2H).

Supplemental Figures:

Fig. S1. Structures of diapocarotenoids and supposed precursors for anchorene. (A) Structures of

diapocarotenoids used in this study. Diapo1 [10,14’-diapocarotene-10,14’-dial; (2E,4E,6E)-4-methylocta-

2,4,6-trienedial]; Diapo2 [8,15-diapocarotene-8,15-dial; (2E,4E,6E)-2,6-dimethylocta-2,4,6-trienedial]; Diapo3

[12,12’-diapocarotene-12,12’-dial; (2E,4E,6E)-2,7-dimethylocta-2,4,6-trienedial]; Diapo4 [8,14’-

diapocarotene-8,14’-dial; (2E,4E,6E,8E)-2,6-dimethyldeca-2,4,6,8-tetraenedial]; Diapo5 [8,12’-diapocarotene-

8,12’-dial; (2E,4E,6E,8E,10E)-2,6,11-trimethyldodeca-2,4,6,8,10-pentaenedial]; Diapo6 [10,10’-

diapocarotene-10,10’-dial; (2E,4E,6E,8E,10E)-4,9-dimethyldodeca-2,4,6,8,10-pentaenedial]. (B) Supposed

precursors for anchorene in plant carotenoid biosynthesis pathway. Enzyme names are shown in blue; red

dashed lines in carotenoids indicate the position that could be oxidatively cleaved to produce anchorene. (C)

Structures of AR derivatives: AR dialcohol [(2E,4E,6E)-2,7-dimethylocta-2,4,6-triene-1,8-diol]; AR diacid

[(2E,4E,6E)-2,7-dimethylocta-2,4,6-trienedioic acid]; AR diethyl ester [diethyl (2E,4E,6E)-2,7-dimethylocta-

2,4,6-trienedioate].

Fig. S2. Effects of diapocarotenoid and its derivatives on Arabidopsis root development. (A)

Representative pictures (left) and quantification data (right) to show the primary root length of Col-0 seedlings

treated by different diapocarotenoids. Red arrows indicate emerged anchor roots (ANRs); n=26, 26, 27, 25, 12,

26, 24, 28, 21, 28, 15, 28, 22; 7 dps seedlings grown in half MS agar plates supplied with 5 µM or 25 µM of

the indicated chemicals. (B) Representative pictures (left) and quantification data (right) to show the ANR

formation of Col-0 seedlings after RAM excision. Red arrows indicate collet positions; ANR quantification

was shown as percentage of seedlings with 0, 1 and 2 ANRs; n=61, 53. (C) The dose response of AR effect on

ANR formation under normal and RAM excision conditions (n=3 biological replicates). (D) Anchorene (AR)

inhibits primary root length. (E) AR effects on lateral root formation. n=45 for each treatment. (F) The vertical

plate design depicts the local application of AR on seedlings. The right panels show a close up of the seedlings;

3 dps seedlings are shown. (G) Representative seedlings treated with AR applied locally in shoots and collets

(shoot), or root tips (root). 3 dps seedlings were transferred to different combinations of 1/2MS media with

acetone (Mock) or 20 µM AR (AR) for another 4 days. Quantification of ANR (H) and primary root length (I)

of seedlings grown in G. (J) Representative pictures (left) and ANR quantification data (right) of Col-0

seedlings treated by AR analog and different derivatives. In H, data are presented as mean ± SD (three

independent replicates); in D (n=19, 20, 22, 23), I (n=19, 18, 19, 16) and J (n=48, 60, 52, 49, 51, 52, 48, 48, 54,

53, 55), data are presented as mean ± SEM from one representative experiment; in D, H, I and I, different

letters denote significant differences (one-way ANOVA with Tukey multiple-comparison test, P < 0.05). Photo

Credit: K.J. and S.A., KAUST (1)

Fig. S3. Characterization of ANR development by different DR5 marker lines. (A) 3 dps pDR5::LUC

seedlings indicate the ANR initiation. (B) 5 dps pDR5::LUC seedlings treated by NF and anchorene. Arrows in

A and B indicate collets. (C) Effects of anchorene on 3 dps to 6 dps pDR5rev::GFP seedlings. White arrows

indicate ANR initiation sites, and the red arrow indicates emerged ANR. Photo Credit: K.J. and S.A., KAUST

(1)

Fig. S4. Root formation under different treatments and in various mutants. (A) The effect of anchorene

(AR) and D15 on LR No. after RAM excision. Data were presented as mean ± SD (one-way ANOVA with

Tukey multiple-comparison test, P < 0.05, n=26, 24, 25, 30); different letters denote significant differences. 20

µM AR and 100 µM D15 were used. Quantification of ANR formation after RAM excision (ANR-RE) from

-branch carotenoids biosynthesis mutants (B), strigolactone biosynthesis deficient mutants (C), ABA

biosynthesis deficient mutants (E) and carotenoids cleavage dioxygenase ccd/nced mutants (G). Effects of

strigolactone analog GR24 (D) and ABA (F) on ANR-RE. In B-G, two-tailed Student’s t-test, *P < 0.05, **P <

0.01. In B, C, E and G, data are presented as mean ± SD from 3 independent replicates. In D and F, data are

presented as mean ± SE from 1 representative experiment; in D, n= 25, 28 respectively, 1 µM GR24 was used;

in F, n=46, 45, 30 respectively, 0.1 and 0.5 µM ABA were used.

Fig. S5. Anchorene effects on DR5 expression in ANR primordia. (A) pDR5rev::GFP expression in ANR

primordia and its abutting endodermis region of seedlings under different treatments. White arrows indicate

ANR primordia, and the red arrows indicate endodermis cells. (B) GFP fluorescence intensity quantification in

ANR primordia of seedlings under different treatments. data were presented as mean ± SD (two-tailed Student

t-test, *P <0.05 **P <0.01); n=11, 12, 7, 8 respectively from two independent experiments. 1 µM NF and 20

µM anchorene (AR) were used. Photo Credit: A.J.D. and P.N.B., Duke University (2)

Fig. S6. Anchorene isomer identification and anchorene quantification. (A) EICs of AR (peak II) and its

isomers (peak I and peak III) from Arabidopsis shoot tissue (upper) and root tissue (middle), and EIC of

authentic AR standard (AS, bottom). (B) Product ion spectra of endogenous AR isomers including peak I and

peak III from Arabidopsis extracts, and authentic AS. (C) Quantification of tissue-specified endogenous AR

content in Arabidopsis. Relative AR content in mock and short term (D) or continuous (E) NF treated

Arabidopsis seedlings. Two-tailed Student’s t-test, in C and E, n=4; in D, n=3; **P <0.01; ***P <0.001. 12-

day-old seedlings were used for AR identification and quantification; in D, 11 day-old seedlings were treated

by NF for another 24 hours. 2 µM NF was used.

Fig. S7. Conversion of OH-Apo12′ into anchorene in plants. (A) Proposed formation of anchorene from 9-

cis-zeaxanthin. (B) Structures of all-trans-3-OH-β-apo-10’-carotenal (OH-Apo10’) and all-trans-3-OH-β-apo-

12’-carotenal (OH-Apo12’). (C) Anchorene quantification in mock, OH-Apo10’ and OH-Apo12’ fed

Arabidopsis seedlings. (D) ANR No. quantification in mock, OH-Apo10’ and OH-Apo12’ fed seedlings. Two-

tailed Student’s t-test, **P <0.01, ***P <0.001; in C, n=4 for each treatment; in D, data are presented as mean ±

SD from 3 independent replicates; in C, 12-day-old seedlings were incubated with indicated chemicals for 6

hours, and 20 µM OH-Apo10’ and OH-Apo12’ were used; in D, 10 µM OH-Apo10’ and OH-Apo12’ were

used, and 10-day-old seedlings were used for ANR No. counting.

Fig. S8. Involvement of auxin signaling and distribution on ANR development. (A) ANR and ANR-RE of

representative Col-0 and arf7arf19 seedlings treated with AR. (B) ANR and ANR-RE of representative Col-0

seedlings treated by auxin analog NAA and auxin transport inhibitor NPA. (C) ANR and ANR-RE of

representative Col-0 seedlings treated by AR and NPA. (D) The gravitropism phenotype of representative Col-

0 seedlings treated by AR and NPA. 5 dps seedlings were used for gravitropism analysis. (E) AR partially

rescued root gravitropism loss in NPA treated seedlings (n=27, 43, 41, 45 individually). The orientation of root

growth was measured and then was assigned to one of twelve 30° sectors; the length of each bar represents the

percentage of seedlings which displayed root growth within that sector. (E) PIN3-GFP expression in the ANR

primordia of mock and AR-treated pPIN3::PIN3-GFP seedlings shown by confocal microscopy imaging. 0.1

µM NAA, 1 µM NPA and 20 µM AR were used. (F) Quantification of AR effect on PIN3-GFP fluorescent

intensity in ANR primordia. Data are presented as mean ± SD (***P <0.001 by two tailed Student’s t-test; n=15,

13) from two independent experiments. (H) ANR-RE of auxin efflux transporter mutant pin3-4 has no

significant difference from corresponding wild-type seedlings. Two-tailed Student’s t-test, n=26, 22

respectively. Photo Credit: G.C. and M.A., KAUST (3)

Fig. S9. Transcriptomic change analysis of collet tissues upon different treatments by RNA-seq. The

Venn diagrams show the numbers of the down-regulated (Dn) and up-regulated (Up) genes that overlap

between AR treatment and NPA treatment (A), and RAM excision (-RAM) (B). Heatmap clustering shows

that the majority of AR and NPA overlapping genes are regulated in an oppositional expression pattern (C),

while the majority of AR and -RAM overlapping genes are regulated in a similar pattern (D). (E) Biological

Processes (BP) analysis of overlapping genes of AR treatment with NPA treatment, and with -RAM. (F)

Partial KEGG of Arabidopsis tryptophan metabolism pathway (KEGG id: ath00380) shows the tryptophan-

auxin biosynthesis pathway regulated by AR. The up-regulated genes and down-regulated genes are marked as

red and green colors, respectively; tryptophan and indole acetate are marked by orange stars; TAA1 (2.6.1.99,

AT1G70560) and YUC7 (11413168, AT2G33230) are marked by red stars. Photo Credit: A-D: K.J. and S.A.,

KAUST (1). Photo Credit: A.J.D. and P.N.B., Duke University (2)

Fig. S10. Effect of anchorene on plant growth. (A) 17-day-old seedlings grown in half MS agar plates with

or without AR pretreatment. (B) 10-day-old hydroponic grown Nipponbare rice plants upon AR treatment.

Quantification of the primary root (C) and shoot (D) length of Nipponbare rice plants upon AR treatment. 20

µM AR was used. In C and D, two-tailed Student’s t-test, ***P <0.001, n=8 (Mock), 8 (AR) respectively.

Photo Credit: K.J. and S.A., KAUST (1)

Fig. S11. The synthesis route and derivatization for anchorene. (A) The synthetic route for anchorene, D6-

anchorene and anchorene derivatives. (B) The derivatization reaction for anchorene.

Table S1. The nutrient element composition in Argo soil and Silver sand.

Element mg/Kg

Argo soil Silver sand

Fe 780.53±2.19 1890.26±6.28

K 4001.53±82.19 758.83±24.47

Mg 11139.33±138.88 214.63±6.89

Mn 51.81±0.62 15.13±1.30

P 1468.06±25.08 25.57±2.98

Zn 58.20±1.75 17.77±0.42

g/Kg

C 439.93±8.22 3.56±0.34

N 10.69±0.54 ND

S ND ND

H 53.07±0.58 0.33±0.01

“ND” indicates non-detected.

Table S2. Mutants and marker lines used in this study.

Alleles name Gene locus Description References

psy-1 (Salk_054288) AT5g17230 Knock out mutant 25

ispH-1 AT4g34350 Knock out mutant 25

lut1 AT3G53130 Knock out mutant 25

lut2 AT5G57030 Knock out mutant 25

ccd1-1 AT3g63520 Knock out mutant 25

ccd4-1 AT4g19170 Knock out mutant 25

ccd7 (max3-11) AT2g44990 Knock out mutant 25

ccd8 (max4-6) AT4g32810 Knock out mutant 25

nced2-3 (Salk_090937) AT4g18350 Knock out mutant 25

nced3 (N3KO-6620) AT3g14440 Knock out mutant 25

nced5 (N5KO-4250) AT1g30100 Knock out mutant 25

nced6 (WISC.DSLox471G6) AT3g24220 Knock out mutant 25

nced9 (Salk_051969) AT1g78390 Knock out mutant 25

aba1-6 (CS3772) AT5g67030 Knock out mutant 25

aba3-1 (CS157) AT1g16540 Knock out mutant 25

pin3-4 (SALK_038609) AT1G70940 Knock out mutant

Identified by

SALK

arf7arf19

AT5G20730 &

AT1G19220 Knock out mutant 30

pDR5::nlsYFP - Synthetic auxin marker line 34

pWOX5::GFP AT3G11260 WOX5 promoter marker line 43

pDR5::LUC - Synthetic auxin marker line 24

pDR5Rev::GFP - Synthetic auxin marker line 49

pPIN3::PIN3-GFP AT1G70940 PIN3 marker line 50

pLAX3::LAX3-YFP AT1G77690 LAX3 marker line 44

Dataset S1. Gene list (1.5-fold change) for different treatments in RNA-seq.

Dataset S2. BP enrichment for different treatments in RNA-seq.