1Running title：MRW, HO1/CO and Ca2+ in adventitious rooting

2 3Title: Methane-rich water induces cucumber adventitious rooting 4through heme oxygenase1/carbon monoxide and Ca2+ pathways 5 6Weiti Cui · Fang Qi · Yihua Zhang · Hong Cao · Jing Zhang · Ren Wang · 7Wenbiao Shen 8

9

10W. Cui · F. Qi · Y. Zhang · H. Cao · J. Zhang · W. Shen ()

11College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural

12University, Nanjing 210095, China

13e-mail: [email protected]

14

15R. Wang

16Jiangsu Province Key Laboratory for Plant Ex-situ Conservation, Institute of Botany,

17Jiangsu Province and the Chinese Academy of Sciences, Nanjing 210014, China

1 1 18Supplementary data

19

20Materials and methods

21

22Preparation of nitrogen-rich water (NRW) and air-rich water (ARW)

23

24A 100% saturated nitrogen-rich water (NRW) prepared by bubbled N2 (99.9%,

25Nanjing Special Gases Factory Co., Ltd, China) into 500 mL distilled water, at a rate

26of 160 mL min-1 for up to 1 h. Meanwhile, a 100% saturate of air-rich water (ARW)

27was also obtained by bubbled air into 500 mL distilled water, at a rate of 160 mL min-1

28for up to 1 h. Then, the corresponding saturated stock solutions were rapidly diluted to

2980% (v/v) for further experiment in IAA-depleted cucumber explants.

30

31Plant materials and growth conditions

32

33Commercial available soybean (Glycine max ‘Sudou 5’) and mung bean (Phaseolus

34radiatus ‘Mingguang’) seeds were used for the universal test of MRW-induced AR

35formation. Identical seeds were selected and surface-sterilized followed by rinsing

36extensively in distilled water. After germinated in distilled water on filter papers for 2

37d, the seedlings were transferred to a plastic box with vermiculite and quarter-strength

38Hoagland’s solution in illuminating incubator and maintained at 25±1°C for another 5

39d with a 14/10 h photoperiod, light intensity of 200 μmol m-2 s-1. Then, corresponding

2 2 40seedlings were used as explants by removing primary root. Explants were maintained

41under the same conditions of temperature and photoperiod for another 5 d in the

42presence of different concentrations of MRW.

3 3 43Supplementary Figure S1

a

Con MRW CO 0.1 mM 0.1 mM pH 5.8 pH 6.1

0.1 mM 0.1 mM 0.1 mM 0.2 mM 0.5 mM pH 6.4 pH 6.7 pH 7.0 pH 6.4 pH 6.4 b 30 9 Root number Root length 1

- a t

n aA A a l )

p 20 m x e

6 m

( r

b e

B h t b

bc g m bc bc bc BC n u c bc e l n BC BC BC BC t t c BC 10

C o o 3 o o R R

0 0 5.8 6.1 6.4 6.7 7.0 6.4 6.4 pH 0.1 0.2 0.5 mM

Na HPO -NaH PO 2 4 2 4

44Fig. S1 Effects of MRW, CO, and different buffer solutions on the formation of

45adventitious root (AR) in IAA-depleted cucumber. Explants with or without auxin

46depletion pretreatments were further incubated in water (Con), 80% concentrations of

47MRW, 20% concentrations of CO, 0.1 mM Na2HPO4-NaH2PO4 buffer solutions

48(pH5.8, 6.1, 6.4, 6.7, and 7.0), 0.2 mM and 0.5 mM Na2HPO4-NaH2PO4 buffer

49solutions (pH 6.4) for 4 d. Photographs were then taken (a). Bar = 0.5 cm.

50Meanwhile, the root number and length per explant were recorded (b). Mean and SE

51values were calculated from at least three independent experiments (n = 16). Within

4 4 52each set of experiments, bars with different letters were significantly different in

53comparison with Con at P < 0.05 according to Duncan’s multiple test

5 5 54Supplementary Figure S2

55Fig. S2 Effects of MRW, nitrogen-rich water (NRW), air-rich water (ARW), and

Root number A 1 10 30 -

t a

Root length ) n a m l 25

p 8

b m

x B (

e

h

r 20 t e 6 g b n e l m

15 u t n o

t 4 c c C o

o C c C 10 R o R 2 5 0 0 on W W W in C R R R em M N A H % % % 80 80 80

56hemin on cucumber AR formation. IAA-depleted cucumber explants were incubated

57with water (Con), MRW (80% concentration), NRW (80% concentration), ARW (80%

58concentration), and hemin (10 μM) for 4 d. Then, the root number and length per

59explant were recorded. Mean and SE were calculated from at least three independent

60experiments (n = 16). Bars with different letters were significantly different in

61comparison with Con at P < 0.05 according to Duncan’s multiple test

6 6 62Supplementary Figure S3 63

a b 10 55 Root number

1 A - Root length t 8 44 ) n a a l m p

ab m x (

e 6 B 33

h r t

e bc c g

b c n e m

4 C 22 l

u t

n C o C t o o R

o 2 11 R 0 0 0 1 10 50 100 0 1 10 50 100 MRW (%) MRW(%) c d 10 30 Root number 1 - t Root length A ) n a 25 a l 8 m p m

x ab (

e

h

r AB

20 t e g b

n e

m bc 6 bc l

BC

u c t

n 15 o C C t o o R o

R 4 10 0 0 0 1 10 50 100 0 1 10 50 100 MRW (%) MRW(%)

64Fig. S3 Effects of MRW on adventitious root formation in soybean and mung bean.

65Explants of soybean (a, b) and mung bean (c, d) were incubated in distilled water

66(Con, 0%), 1%, 10%, 50% and 100% saturated MRW for 5 d. Photographs were then

67taken (a, c). Bar = 1 cm. Meanwhile, the root number and length per explant were

68recorded (b, d). Mean and SE were calculated from at least three independent

69experiments (n = 16). Within each set of experiments, bars with different letters were

70significantly different in comparison with Con at P < 0.05 according to Duncan’s

71multiple test

7 7