Control NaCl ABA * * Control 4ºC 37ºC

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Patricia Blankenship
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1 Dul regultion of wter retention nd cell growth y stress-ssocited protein (SAP) gene in Prunus A. Lloret, A. Conejero, C. Leid, C. Petri, F. Gil-Muñoz, L. Burgos, M.L. Bdenes, nd G. Ríos.5 PpSAP Reltive expression.5 3 Control Desicction.5 PpSAP.5 Reltive expression c 5 4 Control NCl ABA PpSAP 3 Reltive expression 3 Control 4ºC 37ºC Figure S. PsSAP gene expression in detched leves nd lef discs under iotic stresses. In, detched leves were desiccted for one (white rs), three (light grey rs) nd seven dys (drk grey rs). In nd c, lef discs were treted for four (white rs) nd 4 hours (grey rs). An expression vlue of one is ssigned to the control. gene (pp865m) expression is included s positive control of stress response. An sterisk indictes significnt difference with the control with confidence level of 95 %.

2 pp553m GAGCTCGGACACCATTGACAATGTGAAGGCGAAGATCCAGGACAAGGAGGGAATCCCACCAGACCAGCAGAGGTTGATC TTTGCAGGGAAGCAGCTGGAGGACGGCCGTACTTTGGCTGACTACAATATTCAGAAGGAGTCTACCCTTCACCTTGTGC TGCGTCTGAGGGGTGGTATGCAGATCTTTGTGAAGACCTTGACTGGAAAGACCATCACGTTGGAGGTGGAGAGCTCGGA CACAATCGACAATGTTAAGGCCAAGATCCAGGATAAGGAAGGTATTCCCCCAGACCAGCAGAGGTTGATCTTTGCTGGT AAGCAGTTGGAGGATGGAAGGACTCTGGCCGACTATAACATCCAGAAGGAGTCTACCCTTCACCTTGTCCTCCGTCTCC GTGGTGGTTTCTAGGAAGTGGAAGTCTCTGGTGTCACCCTGCTTTTAATGAATGTTTCTGTGGTTCCCTTAAAACAATG TTTTTAAGTATGTTTTTTTAAGTTTCAAGTTTCATGGTACTGTTTCTGCCTCTTGGGGAGGCATTTATCTAGTTTGGTT TCCTGGTACTTTTTGTTTAATAAATAAATTTGTTCTTGAATAAAAAAAAAAAAAAAAAAAAAAAAAAAA pp96m GAAGACCCTTACAGGGAAGACAATAACTTTGGAGGTTGAGAGCTCTGACACTATTGACAATGTCAAAGCTAAGATTCAA GACAAGGAGGGGATCCCACCGGACCAGCAGAGGTTGATCTTTGCCGGGAAACAGCTTGAGGATGGTCGTACCCTTGCAG ATTATAATATCCAGAAAGAGTCTACTCTCCATCTTGTCCTTCGTCTGCGTGGTGGTGATTTCTGAGTTAGTCCGTTGGT TTTAGTTAGTTTGTTGGTTATAGTTTGTGTTCTCTAGTGATGGTGGTGAGATGATTATGGTGGTGTGTTGTGTTGTGTT GATTGGCCGGAGGGCCAATTTAGTA pp557m ACAAGGAGGGTATTCCCCCAGACCAGCAGAGGTTGATCTTTGCAGGCAAGCAGCTCGAGGATGGAAGGACCCTTGCTGA CTATAACATTCAAAAGGAGTCCACTCTGCACTTGGTGCTCCGGCTGCGTGGTGGTATGCAGATATTTGTGAAAACTCTG ACCGGGAAAACCATTACTCTGGAAGTGGAAAGCTCTGATACTATTGATAATGTTAAGGCCAAAATCCAGGACAAGGAGG GTATTCCCCCAGACCAGCAGAGGTTGATCTTTGCAGGCAAGCAGCTCGAGGATGGAAGGACCCTTGCTGACTACAACAT TCAAAAGGAGTCCACTCTGCACTTGGTGCTTCGACTGCGTGGTGGGATGCAGATCTTTGTGAAGACTCTGACTGGCAAG ACCATAACGTTGGAGGTGGAGAGTTCGGATACAATTGATAATGTGAAGGCCAAGATTCAGGACAAGGAGGGCATTCCTC CAGACCAGCAGAGGCTCATCTTTGCCGGTAAGCAGCTCGAGGATGGGAGGACTCTTGCAGATTACAACATTCAGAAGGA GTCTACCCTTCACCTTGTCCTTCGTCTCCGAGGTGGTCTCTGAATTCTGAATGATCCTTGTGGTTCCATCCTTTTCTGA TCAgTGAACAGTGGCTTTGTTAAGTATTTTTATTTTTGAATTTTCGTACTTGGCTGATGGATGATGGGTCACACTCAGA CTATCTAGTTAATAAATAAACATGCTTTGTATGTAAAAAAAAAAAAAAAA pp77m GAGTTCTGATACCATTGATAATGTGAAAGCCAAGATTCAGGACAAGGAGGGCATTCCTCCGGACCAGCAGAGGCTCATC TTTGCTGGGAAGCAGCTTGAGGATGGCCGCACCCTAGCTGATTACAATATCCAAAAGGAGTCAACCCTTCACTTAGTGC TTCGTCTTCGTGGGGGTATGCAAATCTTTGTCAAGACACTAACTGGGAAGACGATAACCTTGGAGGTTGAAAGCTCGGA TACCATAGATAATGTCAAGGCAAAAATTCAGGATAAGGAAGGCATTCCCCCTGATCAGCAGAGGCTAATCTTCGCTGGT AAGCAACTTGAGGATGGCCGCACCCTTGCCGATTATAACATCCAAAAGGAATCAACTCTGCACCTGGTGCTTCGGTTGC GTGGTGGTATGCAAATCTTTGTGAAAACTTTGACTGGGAAGACGATTACTTTGGAAGTGGAAAGCTCAGATACTATTGA CAATGTGAAGGCCAAAATCCAGGACAAGGAGGGTATCCCTCCAGACCAGCAGAGGTTGATATTTGCAGGCAAGCAGCTG GAGGATGGAAGGACCCTTGCCGACTACAATATTCAAAAGGAGTCGACCCTGCACTTGGTGCTCCGTCTGCGTGGTGGGA TGCAGATCTTTGTGAAGACTCTGACTGGGAAGACGATAACTTTGGAGGTGGAGAGTTCTGATACCATTGATAATGTGAA AGCCAAGATTCAGGACAAGGAGGGCATTCCTCCGGACCAGCAGAGGCTCATCTTTGCCGGTAAGCAGCTGGAGGACGGG AGGACTCTTGCAGATTACAACATTCAGAAGGAGTCTACCCTTCACCTTGTCCTTCGTCTCCGTGGCGGTTTCTGAAGTC TGAATGATGCTTGTGGTTCCATCCTTGTCGGAGCAGTGAGAAGTGACTTTTAAAAAAA Figure S. Sequence of yest two-hyrid clones.

3 Figure S3. Drought stressed plnts for seven dys. Two month old plnts of WT nd trnsgenic lines 35S::PpSAP #, #5 nd #6 re shown; scle r, 5 cm.

4 .8 GRF5-like.8 AN-like.4.4 AN3-like RPA-like RPT-like ARL-like Reltive expression CBLIPK-like AWPM9-like Reltive expression Dehydrin-like c c AFP-like GOLS-like c H-3-like.8 RDB9B-like.4.4 ATCP-like WT # #5 #6 35S::PpSAP.8.4 WT # #5 #6 35S::PpSAP Figure S4. Expression of severl genes ssyed in trnsgenic plum overexpressing PpSAP. An expression vlue of one is ssigned to the WT. Dt re mens from three iologicl smples with two technicl replictes ech, with error rs representing stndrd devition. Letters (-) indicte significnt difference etween smples.

5 Tle S. Primers used in this study. qrt-pcr in pech Forwrd Reverse PpSAP ACACAGGCTTCCTCTACTCCATCTTT GAACCCTCATTCCGAGACATTTATCAG TCATCTTCCGCTGCCTTTGTAGCCT GACACTGCCAAGAACACCAAGGACA Tuulin-like CAGATGCCCAGTGATGCCTCAG TGCTTGCCTGATCCAGTCTCAC AGL6-like ACCACCTGAAGTCCTCCAAGATTG GCTTCATACAAAGCAATGCCAACAC SAND-like TCGTGGGTACCAGGAAAACGACAT CCTGCTAGCTTGTGTTCATCTCCA Actin-like CTTCTTACTGAGGCACCCCTGAAT AGCATAGAGGGAGAGAACTGCTTG qrt-pcr in plum PpSAP TGATCTCAGAGGAGGACCTGCATAT CAAGTCCTTGTGGCATTTGGAACAC Plum SAP-like ACACAGGCTTCCTCTACTCCATCTTT PpSAP + plum SAP-like AACGTGTTGGTCTAACCGGCTTCAAT GAACCCTCATTCCGAGACATTTATCAG TTGTCCAGCTTTTCTGCCTTCACAAC CBLIPK-like ACCCCAATGTGGTTCAGCTCTTTGA AGTAATCAACAGCCCCAACCAACTG TIP-like AGTTGTTGCTTGCTTGCTACTCAAG GCACCAACAATCAAACCAATTGCGA GRF-like GGGGTTCTATCCATTTGGGATTCTC TTTTTGGTCAGGAACGGCATCTCTC GRF5-like GGAGGTGCAGAAGAACAGATGGTAA TTGAACGGTTTCTGCCTCTGTGCAT RPA-like GGGTTGCTAACAAGCAGTTTAAGAC GGGACCTAACATATCAACTGGAACA AN-like AAAGCCTGGTGAGCTGTTGAAGGAT AGTATCTAGGAACCAACCACCACCT AN3-like ATGTACCTGGCTGCCATTGCTGATT GCTTGTTGCTGCATGTAATGTGCTC TOR-like GCAGTACCAAAGAAGATTGGGCAGA GCAAATAACTCGCGCCCAACAAAT ARL-like TCGTCGAACACTATCACACGGTAG CAACAGAGTCGCCGTGAGACATATA RPT-like GTGGCAAACTCAACATCTGCAACT GTGAGAGGTCATCAGCAACCCTAA AWPM9-like CCCAGCCAATATGGCGAATATCAGAA CATAGTGAGCAGCAGTAAGTTTGTGCT Dehydrin-like GTACTCTCATGACACCCACAAAACTAC CCCGGCCCCACCGTAAGCTCCAGTT AFP-like TTCCGTTGGTGGTGGAGTGGATGCA TTACTAGCAGGGCTTCTTGCTTCAC H-3-like AAACCGCTGCTCATCCTCCATACT TGCTTCTCCTCCATGTACTTGGCT RD9B-like ATCTGCTAAGAACGTCGTCGCTTC GGCTTTGCTGTAACCTCCTGATGA ATCP-like AAGGTTATGGACAAGGACGGGGAT CATGGCCTTGATGTCTTCATCAGTG GOLS-like TGACCACCTGTTTGACTACCCAGA GCTGGCAGTACCCAATCTTGTACT Two-hyrid cloning prok cloning CCAGAATTCATGGAGCACAACGAGACAG CAGTCTAGATGGAGGAGCAGAAGCTGATCTC GCCGGATCCTCAGATTTTGTCCAGCTTTTC GCCGGATCCTCAGATTTTGTCCAGCTTTTC

6 Tle S. List of genes tested y qrt-pcr in trnsgenic plum nd pech. Nme Rice or Aridopsis Pech puttive gene/protein ortholog Protein similrity Reference CBLIPK-like LOC_Osg89 pp5365m CBL-intercting protein kinse () TIP-like LOC_Osg7445 pp364m Tonoplst intrinsic protein-3 () GRF-like LOC_Os3g597 pp963m Growth-regulting fctor () GRF5-like LOC_Osg5369 pp7593m Growth-regulting fctor 5 () RPA-like LOC_Os3g54 pp338m Repliction protein A suunit B () AN-like ATG5 pp39m Angustifoli () AN3-like AT5G864 pp39m Angustifoli3 (3) TOR-like ATG53 ppm Serine/threonine-protein kinse TOR (4) ARL-like ATG448 pp358m Argos-like (5) RPT-like AT4G94 Prupe.8G89 6S protesome regultory suunit (6) pp865m Lte emryogenesis undnt (7) AWPM9- like pp88m AWPM9 (7) Dehydrin-like Prupe.7G6 Dehydrin (7) AFP-like pp6974m ABI5 inding protein (7) H-3-like ATG85 pp94m Histone H-3 (8) RD9B-like AT5G53 pp989m Responsive to desicction 9B (8) ATCP-like AT5G4948 pp594m NCl-inducile clcium-inding protein (9) GOLS-like ATG566 pp894m Glctinol synthse (9) () Dnsn, P. K., Kothri, K. S., Vij, S. & Tygi, A. K. OsiSAP overexpression improves wter-deficit stress tolernce in trnsgenic rice y ffecting expression of endogenous stress-relted genes. Plnt Cell Rep. 33, (4). () Kim, G. T. et l. The ANGUSTIFOLIA gene of Aridopsis, plnt CtBP gene, regultes lef-cell expnsion, the rrngement of corticl microtuules in lef cells nd expression of gene involved in cell-wll formtion. EMBO J., (). (3) Kim, J. H. & Kende, H. A trnscriptionl coctivtor, AtGIF, is involved in regulting lef growth nd morphology in Aridopsis. Proc. Ntl. Acd. Sci. U. S. A., (4). (4) Mennd, B. et l. Expression nd disruption of the Aridopsis TOR (trget of rpmycin) gene. Proc. Ntl. Acd. Sci. U. S. A. 99, (). (5) Hu, Y., Poh, H. M. & Chu, N. H. The Aridopsis ARGOS-LIKE gene regultes cell expnsion during orgn growth. Plnt J. 47, 9 (6). (6) Kurep, J. et l. Loss of 6S protesome function leds to incresed cell size nd decresed cell numer in Aridopsis shoot orgns. Plnt Physiol. 5, (9). (7) Leid, C. et l. Histone modifictions nd expression of DAM6 gene in pech re modulted during ud dormncy relese in cultivrdependent mnner. New Phytol. 93, 67 8 (). (8) Giri, J. et l. Rice A/AN zinc-finger contining stress-ssocited proteins (SAP/) nd receptor-like cytoplsmic kinse (OsRLCK53) interct vi A zinc-finger nd confer iotic stress tolernce in trnsgenic Aridopsis plnts. New Phytol. 9, 7 73 (). (9) Kng, M. et l. Aridopsis SAP5 functions s positive regultor of stress responses nd exhiits E3 uiquitin ligse ctivity. Plnt Mol. Biol. 75, ().

7 Tle S3. Stility index of reference genes for rel-time experiments using three methods. NormFinder BestKeeper Ct Tissues Tuulin-like AGL6-like SAND-like Actin-like Reproductive uds Tuulin-like AGL6-like SAND-like.8.46 Actin-like Stress ssys Tuulin-like AGL6-like SAND-like.4..6 Actin-like

3.94 ± 0.50 (95% CI) Correlative inhibition index (slope)

3.94 ± 0.50 (95% CI) Correlative inhibition index (slope) Supplementl Tle S. Selected rchitecturl prmeters of phy nd phyphy grown under. Vlues re mens ± SE, except for predicted primry rosette rnches where the vlues re the men with the ssocited 9% confidence