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1 /FP18046_AC CSIRO 2018 Supplementry Mteril: Funtionl Plnt Biology, 2018, 45(11), Supplementry Mteril Hordeum vulgre nd Hordeum mritimum respond to extended slinity stress displying different temporl umultion pttern of metolites Selm Ferhihi A, Kmel Hessini A,B, Emili Dell Aversn C, Luis D Ameli C, Psqulin Woodrow C, Loredn F. Cirmiello C, Amodio Fuggi C nd Petroni Crillo C,D A Lortory of Extremophile Plnts, Center of Biotehnology of Borj Cedri, University of Elmnr, B.P. 901, Hmmm-Lif 2050, Tunisi. B Biology Deprtment, Fulty of Siene, Tif University, Tif, P.O. Box 888, Sudi Ari. C Deprtment of Environmentl, Biologil nd Phrmeutil Sienes nd Tehnologies, University of Cmpni Luigi Vnvitelli, Vi Vivldi 43, Csert, Itly. D Corresponding uthor. Emil: petroni.rillo@unimpni.it

2 Tle S1. Min physiologil prmeters, inorgni ions, hydrogen peroxide (H2O2), mlondildehyde (MDA), pigments, rohydrtes, glyine etine, proteins, totl nd free mino ids ontent in leves of H. mritimum nd H. vulgre under ontrol (nonsline ondition), slt stress (200 mm NCl) nd slt removl tretment Slt ws grdully dded to slt treted plnts strting from 15 dys fter sowing (DAS). Slt removl tretment strted from 30 DAS. Hrvests were done t 30, 33, 36, 42 nd 48 DAS. Vlues re men s.d. (n = 3)

3 Hordeum mritimum Hordeum vulgre Dy Control Sl stress Slt removl Control Slt stress Slt removl Fresh weight (g per plnt) ± ± ± ± ± ± ± ± ± ± ± 0.09 d 1.30 ± ± ± ± ± ± ± ± ± ± ± 0.60 d 0.86 ± 0.11 e 2.72 ± ± ± ± ± ± 0.04 d 3.45 ± 0.45 e Reltive wter ontent (%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.8 Lef wter potentil (Mp) ± ± ± ± ± ± ± ± ± ± 0.08 d ± ± 0.10 d ± ± ± ± ± ± ± ± ± ± ± 0.06 d ± ± ± ± ± ± 0.06 d ± 0.10 Chloride (μmol g 1 FW) ± ± ± ± ± ± ± ± ± ± 4.6 d 277 ± 4.0 e 205 ± ± ± ± ± 4.2 d 274 ± 3.8 e 195 ± ± ± ± ± ± 6.9 d 127 ± 7.1 e ± ± ± ± ± 17.8 d 109 ± 3.5 e Nitrte (μmol g 1 FW) ± ± ± ± ± 1.7 d 22.4 ± 1.7 d ± ± ± ± ± 1.3 d 21.8 ± 4.9 d ± ± ± ± ± 1.1 d 28.8 ± 2.3 e ± ± ± ± 5.3 d 17.3 ± 1.1 e 62.3 ± ± ± ± ± 10.3 e 15.9 ± 0.7 d 74.3 ± 2.8 e Potssium (μmol g 1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.5 d 57.5 ± ± ± ± ± ± ± 2.8 Sodium (μmol g 1 FW) ± ± ± ± ± 9.4 d ± 9.4 d ± ± ± ± ± 5.4 d ± 9.4 e ± ± ± ± 1.7 d ± 15.1 e ± 6.7 f ± ± ± ± ± 16.2 d 84.7 ± ± ± ± ± ± 26.3 d 65.5 ± 8.3 Potssium:Sodium ± ± ± ± ± 0.05 d 0.41 ± 0.05 d ± ± ± ± ± 0.05 d 0.41 ± 0.05 d ± ± ± ± 0.61 d 0.26 ± 0.03 e 0.51 ± 0.06 f ± ± ± ± 0.46 d 0.19 ± 0.03 e 0.68 ± 0.08 f ± ± ± ± ± 0.02 d 0.89 ± 0.10 e Sp osmollity ((mosmol kg 1 ) ± ± ± ± ± 235 d 1170 ± 235 d ± ± ± ± ± 22 d 1077 ± 212 d ± ± ± ± ± ± ± ± ± ± ± 58 d 553 ± ± ± ± ± ± 45 d 524 ± 3 H 2 O 2 (μmol g 1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.22 MDA (nmol g 1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.3 d 27.0 ± ± 7.0 d 38.6 ± ± ± ± ± ± ± ± ± ± ± ± ± 2.1

4 Hordeum mritimum Hordeum vulgre Dy Control Sl stress Slt removl Control Slt stress Slt removl Totl hlorophyll (mg g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± 0.33 d 4.05 ± ± ± 0.57 d 2.22 ± ± 0.32 d ± ± ± ± ± ± ± ± ± ± ± ± 0.21 Chlorophylle /Chlorophylle ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.27 Crotenoids (mg g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 d 0.24 ± ± ± ± ± 0.02 d ± ± ± ± ± ± ± ± ± ± ± ± 0.01 Strh (µmol gluose eq g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.3 Gluose (µmol g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.48 d ± ± 3.82 d ± ± ± ± ± ± 3.34 Frutose (µmol g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.39 d ± ± ± ± ± ± 0.82 Surose (µmol gluoseeq g -1 FW) ± ± ± ± ± ± ± ± 0.68 d 4.38 ± ± ± ± 0.68 d ± ± ± ± ± ± ± ± ± ± ± 1.69 d 5.92 ± ± ± ± ± ± ± 0.31 Glyine etine (µmol g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.51 Totl proteins (mg g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.6 Totl free mino ids (µmol g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.4 d 56.1 ± ± 3.8 d ± ± ± ± ± ± 1.6

5 Hordeum mritimum Hordeum vulgre Dy Control Sl stress Slt removl Control Slt stress Slt removl Alnine (mino ids re expressed s µmol g -1 FW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.28 d 2.45 ± ± ± ± 0.08 d 0.91 ± ± ± ± ± ± ± 0.48 Arginine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06 Asprgine ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.66 d 4.77 ± ± ± ± 0.16 d ± ± ± ± ± ± ± ± ± ± ± ± 1.30 Asprtte ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.22 Glutmte ± ± ± ± ± ± ± ± ± ± ± 0.28 d 2.15 ± 0.31 d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.40 Glutmine ± ± ± ± ± 0.36 d 7.12 ± 0.36 d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.29 Glyine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 Histidine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.11 Isoleuine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06 Leuine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 d ± ± ± ± ± ± 0.06 Lysine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06

6 Hordeum mritimum Hordeum vulgre Control Sl stress Slt removl Control Slt stress Dy Slt removl Methionine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 Ornithine ± ± ± ± ± 0.02 d 0.39 ± 0.02 d ± ± ± ± ± 0.03 d 0.31 ± ± ± ± ± ± 0.02 d 0.26 ± 0.11 d ± ± ± ± ± ± ± ± ± ± ± ± 0.04 Phenyllnine ± ± ± ± ± ± ± ± ± ± ± 0.11 d 0.53 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.18 Proline ± ± ± ± ± 3.3 d 20.6 ± 3.3 d ± ± ± ± ± 1.4 d 7.8 ± 0.1 e ± ± ± ± 0.1 d 26.5 ± 0.3 e 7.5 ± ± ± ± ± ± 0.4 d 2.6 ± ± ± ± ± ± ± 0.3 Serine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.48 Threonine ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.19 Tryptophn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06 Tyrosine ± ± ± ± ± ± ± ± ± ± ± ± 0.08 d ± ± ± ± ± ± ± ± ± ± 0.09 d 0.64 ± ± 0.14 d ± ± ± ± ± ± 0.24 Vline ± ± ± ± ± ± ± ± ± ± ± ± 0.16 d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06

7 Tle S2. Sodium, hloride nd potssium ontent of entire shoot t 30 nd 48 dys fter sowing (DAS), older lef tissues lredy present efore 30 DAS nd still present t 48 DAS nd lef younger tissues developed in the period DAS fter the esstion of slinity tretment. H. mritimum nd H. vulgre plnts of seond independent experiment run with sme experimentl design s in Tle S1 nd Figure 1. Hrvests were done t 30 nd 48 DAS. Vlues re men s.d. (n = 3) Hordeum mritimum Hordeum vulgre Dy Control Sl stress Slt removl Control Slt stress Slt removl Chloride (mmol g 1 DW) ± ± ± ± ± 0.07 d 1.93 ± 0.07 d ± ± ± ± ± 0.03 d 1.61 ± 0.02 e ± ± ± ± ± 0.03 d 1.70 ± 0.12 e ± ± ± ± ± ± ± ± ± ± ± ± 0.03 d Nitrte (mmol g 1 DW) ± ± ± ± ± 0.01 d 0.17 ± 0.01 d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 d 0.60 ± ± ± ± ± ± 0.00 d 0.70 ± 0.05 Potssium (mmol g 1 DW) ± ± ± ± ± 0.01 d 0.42 ± 0.01 d ± ± ± ± ± 0.01 d 0.41 ± 0.02 d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 d 0.55 ± 0.04 Sodium (mmol g 1 DW) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.10 d 0.82 ± 0.05 e ± ± ± ± ± 0.15 d 0.62 ± 0.08 Potssium:Sodium ± ± ± ± ± 0.05 d 0.41 ± 0.05 d ± ± ± ± ± 0.05 d 0.41 ± 0.05 d ± ± ± ± 0.61 d 0.26 ± 0.03 e 0.51 ± 0.06 f ± ± ± ± 0.46 d 0.19 ± 0.03 e 0.68 ± 0.08 f ± ± ± ± ± 0.02 d 0.89 ± 0.10 e

8 Tle S3. Sodium, hloride nd potssium ontent of entire shoot t 30 nd 48 dys fter sowing (DAS), older lef tissues lredy present efore 30 DAS nd still present t 48 DAS nd lef younger tissues developed in the period DAS fter the esstion of slinity tretment nd expressed on fresh nd dry weight ses The H. mritimum nd H. vulgre plnt mteril used ws from seond independent experiment run with sme experimentl design s in Tle S1 nd Figure 1. Hrvests were done t 30 nd 48 DAS. Vlues re men s.d. (n = 3) Dy Chloride (μmol g 1 FW) ± ± ± ± ± ± ± ± 3.5 Sodium (μmol g 1 FW) ± ± ± ± ± ± ± ± 8.3 Potssium (μmol g 1 FW) ± ± ± ± ± ± ± ± 2.8 Chloride (mmol g 1 DW) Hordeum mritimum ± ± ± ± ± ± ± ± 0.05 Sodium (mmol g 1 DW) ± ± ± ± ± ± ± ± 0.12 Potssium (mmol g 1 DW) ± ± 0.11 Hordeum vulgre Entire shoot Older tissue Younger tissue Entire shoot Older tissue Younger tissue ± ± ± ± ± ± 0.04

9 Fig. S1. Photogrphs showing representtive H. vulgre plnts t 30, 36, 42, 48 DAS. Fig. S2. Photogrphs showing representtive H. mritimum plnts t 30, 36, 48 DAS.

10 Fig. S3. Shoot fresh (A) nd dry (B) weights of H. mritimum (Hm) nd H. vulgre (Hv) plnts under ontrol, stress (200 mm NCl) nd slt removl tretments. Slt ws grdully dded to slinity tretments strting from 15 DAS. Slt removl tretment strted from 30 DAS. Hrvest ws done t 30, 33, 36, 42 nd 48 DAS. Metolites were expressed s µmol g -1 FW. Vlues re men s.d. (n=3).