<p> 1Running title：MRW, HO1/CO and Ca2+ in adventitious rooting</p><p>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</p><p>9</p><p>10W. Cui · F. Qi · Y. Zhang · H. Cao · J. Zhang · W. Shen ()</p><p>11College of Life Sciences, Laboratory Center of Life Sciences, Nanjing Agricultural</p><p>12University, Nanjing 210095, China </p><p>13e-mail: [email protected] </p><p>14</p><p>15R. Wang</p><p>16Jiangsu Province Key Laboratory for Plant Ex-situ Conservation, Institute of Botany,</p><p>17Jiangsu Province and the Chinese Academy of Sciences, Nanjing 210014, China</p><p>1 1 18Supplementary data</p><p>19</p><p>20Materials and methods</p><p>21</p><p>22Preparation of nitrogen-rich water (NRW) and air-rich water (ARW) </p><p>23</p><p>24A 100% saturated nitrogen-rich water (NRW) prepared by bubbled N2 (99.9%,</p><p>25Nanjing Special Gases Factory Co., Ltd, China) into 500 mL distilled water, at a rate</p><p>26of 160 mL min-1 for up to 1 h. Meanwhile, a 100% saturate of air-rich water (ARW)</p><p>27was also obtained by bubbled air into 500 mL distilled water, at a rate of 160 mL min-1</p><p>28for up to 1 h. Then, the corresponding saturated stock solutions were rapidly diluted to</p><p>2980% (v/v) for further experiment in IAA-depleted cucumber explants.</p><p>30</p><p>31Plant materials and growth conditions</p><p>32</p><p>33Commercial available soybean (Glycine max ‘Sudou 5’) and mung bean (Phaseolus</p><p>34radiatus ‘Mingguang’) seeds were used for the universal test of MRW-induced AR</p><p>35formation. Identical seeds were selected and surface-sterilized followed by rinsing</p><p>36extensively in distilled water. After germinated in distilled water on filter papers for 2</p><p>37d, the seedlings were transferred to a plastic box with vermiculite and quarter-strength</p><p>38Hoagland’s solution in illuminating incubator and maintained at 25±1°C for another 5</p><p>39d with a 14/10 h photoperiod, light intensity of 200 μmol m-2 s-1. Then, corresponding</p><p>2 2 40seedlings were used as explants by removing primary root. Explants were maintained</p><p>41under the same conditions of temperature and photoperiod for another 5 d in the</p><p>42presence of different concentrations of MRW.</p><p>3 3 43Supplementary Figure S1</p><p> a</p><p>Con MRW CO 0.1 mM 0.1 mM pH 5.8 pH 6.1</p><p>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</p><p>- a t</p><p> n aA A a l )</p><p> p 20 m x e</p><p>6 m</p><p>( r</p><p> b e</p><p>B h t b</p><p> bc g m bc bc bc BC n u c bc e l n BC BC BC BC t t c BC 10</p><p>C o o 3 o o R R</p><p>0 0 5.8 6.1 6.4 6.7 7.0 6.4 6.4 pH 0.1 0.2 0.5 mM</p><p>Na HPO -NaH PO 2 4 2 4</p><p>44Fig. S1 Effects of MRW, CO, and different buffer solutions on the formation of</p><p>45adventitious root (AR) in IAA-depleted cucumber. Explants with or without auxin</p><p>46depletion pretreatments were further incubated in water (Con), 80% concentrations of</p><p>47MRW, 20% concentrations of CO, 0.1 mM Na2HPO4-NaH2PO4 buffer solutions</p><p>48(pH5.8, 6.1, 6.4, 6.7, and 7.0), 0.2 mM and 0.5 mM Na2HPO4-NaH2PO4 buffer</p><p>49solutions (pH 6.4) for 4 d. Photographs were then taken (a). Bar = 0.5 cm.</p><p>50Meanwhile, the root number and length per explant were recorded (b). Mean and SE</p><p>51values were calculated from at least three independent experiments (n = 16). Within</p><p>4 4 52each set of experiments, bars with different letters were significantly different in</p><p>53comparison with Con at P < 0.05 according to Duncan’s multiple test</p><p>5 5 54Supplementary Figure S2</p><p>55Fig. S2 Effects of MRW, nitrogen-rich water (NRW), air-rich water (ARW), and</p><p>Root number A 1 10 30 -</p><p> t a</p><p>Root length ) n a m l 25</p><p> p 8</p><p> b m</p><p> x B (</p><p> e</p><p> h</p><p> r 20 t e 6 g b n e l m</p><p>15 u t n o</p><p> t 4 c c C o</p><p> 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</p><p>56hemin on cucumber AR formation. IAA-depleted cucumber explants were incubated</p><p>57with water (Con), MRW (80% concentration), NRW (80% concentration), ARW (80%</p><p>58concentration), and hemin (10 μM) for 4 d. Then, the root number and length per</p><p>59explant were recorded. Mean and SE were calculated from at least three independent</p><p>60experiments (n = 16). Bars with different letters were significantly different in</p><p>61comparison with Con at P < 0.05 according to Duncan’s multiple test</p><p>6 6 62Supplementary Figure S3 63</p><p> a b 10 55 Root number </p><p>1 A - Root length t 8 44 ) n a a l m p</p><p> ab m x (</p><p> e 6 B 33</p><p> h r t</p><p> e bc c g</p><p> b c n e m</p><p>4 C 22 l</p><p> u t</p><p> n C o C t o o R</p><p> 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</p><p> x ab (</p><p> e</p><p> h</p><p> r AB</p><p>20 t e g b</p><p> n e</p><p> m bc 6 bc l</p><p>BC </p><p> u c t</p><p> n 15 o C C t o o R o</p><p>R 4 10 0 0 0 1 10 50 100 0 1 10 50 100 MRW (%) MRW(%)</p><p>64Fig. S3 Effects of MRW on adventitious root formation in soybean and mung bean.</p><p>65Explants of soybean (a, b) and mung bean (c, d) were incubated in distilled water</p><p>66(Con, 0%), 1%, 10%, 50% and 100% saturated MRW for 5 d. Photographs were then</p><p>67taken (a, c). Bar = 1 cm. Meanwhile, the root number and length per explant were</p><p>68recorded (b, d). Mean and SE were calculated from at least three independent</p><p>69experiments (n = 16). Within each set of experiments, bars with different letters were</p><p>70significantly different in comparison with Con at P < 0.05 according to Duncan’s</p><p>71multiple test</p><p>7 7</p>