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1 Supplementary Materials for Coordinated Responses to Oxygen and Sugar Deficiency llow Rice Seedlings to Tolerate Flooding Kuo-ei Lee, Peng-en Chen, Chung-n Lu, Shu Chen, Tuan-Hua David Ho, Su-May Yu* *To whom correspondence should be addressed. This PDF file includes: Published 6 October 9, Sci. Signal. 2, ra61 ( DOI: /scisignal.333 Fig. S1. Dendrogram showing the evolutionary relationship of members of the rice CIPK family and other Snf1-related protein kinases from rice, mammals and yeast. Fig. S2. Genotyping identified two allelic Tos17-tagged CIPK homozygous ( / ) and heterozygous (+/ ) Nipponbare rice mutants. Fig. S3. mino acid sequence of CIPK1 closely resembles that of the rice SnRK1, mammalian MPKα1 and yeast Snf1 protein kinases. Fig. S4. mino acid sequence of CIPK1 resembles that of protein kinases upstream of SNF1 and MPK. Fig. S. Sucrose rescues underwater seedling shoot development in the cipk1 mutant. Fig. S6. CIPK1 controls underwater root development. Fig. S7. CIPK1 control mature plant growth under partially-flooded conditions. Fig. S8. Seedling hypoxia tolerance varies among rice varieties. Fig. S9. TP is consumed more slowly in T and cipk1 embryos germinated under water than in air. Table S1. The IRRI Germplasm Center (IRGC) accession number of rice germplasms used in the present study. Table S2. Primers used in the present study.

2 OsCIPK 22 SnRK3 OsCIPK 2 OsCIPK 29 OsCIPK 13 OsCIPK 16 OsCIPK 14 OsCIPK 18 OsCIPK 3 OsCIPK 1 OsCIPK OsCIPK 2 OsCIPK 19 OsCIPK 12 OsCIPK 1 OsCIPK 27 OsCIPK OsCIPK 26 OsCIPK 11 OsCIPK 6 OsCIPK 28 OsCIPK 9 OsCIPK 3 OsCIPK 23 OsCIPK 8 OsCIPK 24 OsSnRK1B OsCIPK 21 OsCIPK 17 OsCIPK 1 OsSnRK1 OsSPK 1 ScSnf1 OsSPK 3 OsSPK 4 OsSPK OsSPK 6 OsSPK 7 OsSPK 8 OsSPK 1 OsSPK 2 OsSPK 9 SnRK2 OsCIPK 4 OsCIPK 7 HuMPKα SnRK1/Snf1/MPKα HuCaMKKβ HuCaMKKα.1 Snf1/MPKα kinases Fig. S1. Dendrogram showing the evolutionary relationship of members of the rice CIPK family and other Snf1-related protein kinases from rice, mammals and yeast. Deduced amino acid sequences of the rice SnRK1, SnRK2 and SnRK3 (CIPK) families were identified by BLST search of the NCBI and Swiss-Prot/TrEMBL databases using sequences of the human MPK and yeast Snf1 upstream protein kinases: human LKB1 (Q183, human CaMKKα (Q8NS, human CaMKKβ (Q96RR4), yeast Elm1 (P328, yeast Tos3 (QCJ7, and yeast Sak1 (P3899). Sequences of the rice CIPK family have also been reported (. The amino acid sequence alignment and phylogenic analysis were carried out using lignx (Vector NTI, version 9.., Invitrogen), GeneScan (The New GENSCN eb Server at MIT) and MEG 3.1 (Center for Evolutionary Functional Genomics) programs. The scale value of.1 indicates.1 amino acid substitutions per site. Reference: 1. U. Kolukisaoglu, S. einl, D. Blazevic, O. Batistic, J. Kudla, Calcium sensors and their interacting protein kinases: genomics of the rabidopsis and rice CBL-CIPK signaling networks. Plant Physiol 134, 43-8 (4). 1

3 TG TF1 TF1 Tos17 Tos17 cipk1-1 cipk1-2 TG B CR2 CR1 Line no. Genotype Lane TNG67 Nipponbare cipk1-1 cipk /+ +/+ +/ / / +/+ +/ / CF1 Primer TF1+CR1 CF1+CR2 Fig. S2. Genotyping identified two allelic Tos17-tagged CIPK homozygous ( -/- ) and heterozygous ( +/- ) Nipponbare rice mutants. Only the homozygous mutant displays hypoxia sensitive phenotype while the heterozygous mutant was as normal as the segregated wild type. () Diagram shows positions of retrotransposon Tos17 insertions at positions 19 and 16 bp downstream of the translation initiation codon TG in coding regions of CIPK1 in mutants cipk1-1 (NE33 and cipk1-2 (NE34), respectively. Box indicates the single exon and lines indicate and 3 untranslated regions of CIPK1. Triangles indicate Tos17. TG indicates the translation initiation codon. rrows indicate positions of DN primers used for PCR analyses. (B) Genotyping of cipk1-1 and cipk1-2. PCR with DN primers CF1 and CR2 produced a product of 1623 bp from wild-type (+/+) rice genomic DN, and with DN primers TF1 and CR1 produced a product of 37 bp from the rice genomic DN-Tos17 junction region in cipk1 mutant. 2

4 MESRGKILMERYELGRLLGKGTFGKVHYRNLESNQSVIKMMDKQQILKVGLSEQ OsSnRK MEGGSDG-NPLGGYRIGKTLGIGSFGKVKIEHILTGHKVIKILNRR--KSMEMEEK HuMPK alpha MTEKQKHDGRVKIGHYILGDTLGVGTFGKVKVGKHELTGHKVVKILNRQKIRSLDVVGK ScSnf1 MSSNNNTNTPNNSSHHHHHHHHHHHHHGHGGSNSTLNNPKSSLDG-HIGNYQIVKTLGEGSFGKVKLYHTTTGQKVLKIINKKVLKSDMQGR I II III IV V VI ) IRREITTMRLVHKNIVQLHEVMTRNKIYFVMEYVKGGELFEKVKRGKLTEVVHKYFQQLISVDYCHSRGVYHRDLKPENLLLDENENLKVSDFGL OsSnRK17) VKREIKILRLFMHPHIIRLYEVIDTPDIYVVMEYVKSGELFDYDVEKGRLQEEERRFFQQIISGVEYCHRNMVVRRDLKPENLLLDSKCNVKIDFGL HuMPK alpha1 IRREIQNLKLFRHPHIIKLYQVISTPSDIFMVMEYVSGGELFDYICKNGRLDEKESRRLFQQILSGVDYCHRHMVVHRDLKPENVLLDHMNKIDFGL ScSnf1) IEREISYLRLLRHPHIIKLYDVIKSKDEIIMVIEYGN-ELFDYIVQRDKMSEQERRFFQQIISVEYCHRHKIVHRDLKPENLLLDEHLNVKIDFGL VII VIII IX X XI ) SLSESKRQDGLLHTTCGTPYVPEVISKIGYDGKSDISCGVILFVLVGYLRFQGPNLMEMYRKIQHGEFRCPGFSRKLQKLLYKIMDPNPSTRI * OsSnRK17) SNVMRDG---HFLKTSCGSPNYPEVISGKLYGPEVDVSCGVILYLLCGTLPFDDENIPNLFKNIKGGIYTLPSHLSPLGRDLIPRMLVVDPMKRI HuMPK alpha1 SNMMSDG---EFLRTSCGSPNYPEVISGRLYGPEVDISSGVILYLLCGTLPFDDDHVPTLFKKICDGIFYTPQYLNPSVISLLKHMLQVDPMKR ScSnf1 SNIMTDG---NFLKTSCGSPNYPEVISGKLYGPEVDVSCGVILYVMLCRRLPFDDESIPVLFKNISNGVYTLPKFLSPGGLIKRMLIVNPLNRI ) SIQKIKESTFRKGPEENRILKERTLNENTTKN VPVLGVRRKKNHEDVKPMSVTNLN----FEIISFSKG OsSnRK14) TIREIREHQFTVGLPRYLVPPPDTQQVKK LDDETQNDVINMGFDKNQLIESLHKR----LQNETVYYLLLDNR---LR HuMPK alpha1) SIKDIREHEFKQDLPKYLFPEDPSYSSTMID DELKEVCEKFECSEEEVLSCLYNRN----HQDPLVYHLIIDNRRIMNE ScSnf1 SIHEIMQDDFKVDLPEYLLPPDLKPHPEEENENNDSKKDGSSPDNDEIDDNLVNILSSTMGYEKDEIYESLESSEDTPFNEIRDYMLIKENKSLIKD FD--LSGMFIVK--ERN ER OsSnRK1 ) TTSGYLGEFHESMVSSLQVTPETPN STDHRQHGHMESPGFGLRHHF HuMPK alpha1 KDFYLTSPPDSFLDDHHLTRPHPERVPFLV-----ETPRRHTLDELNPQKSKHQG ScSnf1 MKNKSVSDELDTFLSQSPPTFQQQSKSHQKSQVDHETKQHRRMSITQQRTYHQSPFMDQYKEEDSTVSILPTSLPQIHRNMLQGSPSKISP FTSDKSSTIIS KLEDVKLNLRVRKKDNGVVKMQ GRKEGR--N---- OsSnRK1 ) ----DRKLGLQSRHPREIITEVLKLQELNVCKKIGHHNMKCR SPSFPSHESMMHNNHG HuMPK alpha1 ----VRKKHLGIRSQSRPNDIMEVCRIKQLDYEKVVNPYYLRVRRKNPVTSTYSKMSLQLYQVDSRTYLLDFRSIDDEITEKSGTTPQRSGSV ScSnf1 LVTKKSKTRHFGIRSRSYPLDVMGEIYILKNLGEKPSEEDLTIKLR KYDIGNKTNTNEKIPD G-VLQFDIEIFEVTTSYHIIEMKQTSGDSLEYRQLLEEGIR--PLKDIVL OsSnRK1 2) FGSMIETDDSEKSTHTVKFEIQLYKTRDEKYFLDLQRLSG---PQLLFLDLCSFLTQLRVL---- HuMPK alpha1 SNYRSCQRSDSDEQGKSSEVSLTSSVTSLDSSPVDLTPRPGS--HTIEFFEMCNLIKILQ----- ScSnf1 ) LMKMVIQLFQIETNNYLVDFKFDGESSYGDDTTVSNISEDEMSTFSYPFLHLTTKLIMELVNSQSN Fig. S3. mino acid sequence of CIPK1 closely resembles that of the rice SnRK1, mammalian MPKα1 and yeast Snf1 protein kinases. mino acid sequences of the rice CIPK1 (K12177, rice SnRK1 (B36298), human MPKα1 (P464), and yeast Snf1 (38) were compared. Gaps have been introduced to maximize the alignment. Roman numerals above sequences indicate the 11 conserved sub-domains of Ser/Thr protein kinases (2). Identical, conserved and similar amino acid residues are highlighted in yellow, blue, and green, respectively. The TP binding site is marked by a dot, the phosphorylation site (Thr) is marked by an asterisk, the catalytic center is marked by a dashed line, and the conserved sp-phe-gly (DFG) and la-pro-glu (PE) motifs that define the activation loop are marked by brackets. The NF domain is boxed. Reference: 2. S. K. Hanks,. M. Quinn. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol, (199. 3

5 MEGGPVCCQDPRELVERVIDVTHLEEDGGPEPTRNGVDPPPRRSVIPGSTSRLLPRPSLSRKL MSSCVSSQPSSNRPQDELGGRGSSSSESQKPCELRGLSSLSIHLGMESFIVVTECEPGCVDLGLRDRPLEDGQEVPLDSSGS-QRPHLSGRKL MSPRQLIPTLIPEPLSQQSC MMITKGGDYSEY MDRSDKKVNVEEVNVPSNLQIELEKSGTSSSVSLRSPTKSSTNLGMEGRDNSISSSVDSLN MESRGKILMERYELGRLLGKGTFGKVHYRNLESNQSVIKMMDKQ MEVVDPQQLGMFTEGELMSVGMDTFIHRIDSTEVIYQPRRKRKLIGKYLMGDLLGEGSYGKVKEVLDSETLCRRVKILKKK 4) SLQERPG SYLEQGPYTGPSHISPRRRPTIESHHVISDEDCVQLNQYKLQSEIGKGYGVVRLYNESEDRHYMKVLSKK ) SLQERSQGGLGGSLDMNGRCICPSLPYSPVSSPQSSPRLPRRPTVESHHVSITGMQDCVQLNQYTLKDEIGKGSYGVVKLYNENDNTYYMKVLSKK IREDELDSPPITPTSQTSSFGSSFSQQKPTYSTIIGENIHTILDEIRPYVKKITVSDQDKKTINQYTLGVSGSGQFGYVRKYSSTLGKVVVKIIPKK SEIHTQYIEKYKDCDEECVELRKSVREYLEKENKTNLYFSEKKSYLINKEFIDIQVQLNSQLCPKVKLKQIEEPNSKEFSKLKNNPTQHLFIKIYSKP 8) MLLERQRVRQLNHPQHQQHISSSLKTPTTTSSFCSSGSSKNKVKETNRISLTYDPVSKRKVLNTYEIIKELGHGQHGKVKLRDILSKQLVIKIVDRH III IV V ) QILKVGLS EQIRREITTMRLVHKNIVQLHEVMT------RNKIYFVMEYVKGGELFEKVKRGKL 4) KLRRIPNGE NVKKEIQLLRRLRHKNVIQLVDVLYNE----EKQKMYMVMEYCVCGMQEMLDSVPEKR KLLKQYGFPRRPPPRGSQQGG PKQLLPLERVYQEIILKKLDHVNVVKLIEVLDDP----EDNLYLVFDLLRKGPVMEVPCDKPFS ) KLIRQGFPRRPPPRGTRPPGG CIQPRGPIEQVYQEIILKKLDHPNVVKLVEVLDDP----NEDHLYMVFELVNQGPVMEVPTLKPLS PNQQYSVNQVMRQIQLKSKGKITTNMSGNEMRLMNIEKCREIFSRLRNNVHIVRLIECLDSP----FSESIIVTNCSLGELQKRDDDEDI QLIRRNIVYSSDGKIRTQMHRVIN EIKILQFLKINPTYYYIKLHKVLESSPSTLSSGKLYLIFDFFPIGPTMPDTGRCE 8) EKKQRKFFTFIKSSK ISENDKIKREIIMKKCHHKHVVQLIEVLDDL----KSRKIYLVLEYCSRGEVKCPPDCMES VI ) TEVV HKYFQQLISVDYCHSRGVYHRDLKPENLLLDENENLKVSDFGLSLSESK---RQDGL LHTTCGT 8) FPVCQ HGYFCQLIDGLEYLHSQGIVHKDIKPGNLLLTTGGTLKISDLGVELHPF---DDT CRTSQGS EEQ RLYLRDVILGLEYLHCQKIVHRDIKPSNLLLGDDGHVKIDFGVSNQFEG-----NDQ LSSTGT EDQ RFYFQDLIKGIEYLHYQKIIHRDIKPSNLLVGEDGHIKIDFGVSNEFKG-----SDL LSNTVGT LPQKKIV ISNCSVSTFKKILEDMTKGLEYLHSQGCIHRDIKPSNILLDEEEKVKLSDFGSCIFTPQSLPFSDNFEDCFQRELNKIVGT RRSGFEKNLLVYPSPLERTEVSIRRMYCIGVLSRLHELGIVHRDVKPDNIMLHTGDFLNDFNSEFLIQG KYVQGTEGT 2) DKGPSLL SF-----QETREILRGVVLGLEYLHYQGIIHRDIKPNLLISGDGTVKISDFGVSLSST---NSSDSSESLDELELKTVGT VII I II * VIII IX X PYVPEVISKIG YDGKSDISCGVILFVLVGYLRFQGPNLMEMYRKIQHGEFRCPG--FS ) PFQPPEINGLDT FSGFKVDISGVTLYNITTGLYPFEGDNIYKLFENIGKGSYIPGD--CG ) PFMPEISDSGQS FSGKLDVTGVTLYCFVYGKCPFIDDFILLHRKIKNEPVVFPEEPEIS PFMPESLSETRKI FSGKLDVMGVTLYCFVFGQCPFMDERIMCLHSKIKSQLEFPDQPDI PFIPELCHLGNSK R---DFVTDGFKLDISLGVTLYCLLYNELPFFGENEFETYHKIIEVSLSSKINGNTLN ) YFFPPEHCNINNNSNKIVTERNESSEINEQEECPQSTLQLGCPDMLGVTICFYGTLPFQGNSILELFDNITKCKIEFPRQPTLSN PFFPEMCLGEDFT RYNLTKENLFRGSCISFMIDIVGVTLYCLLFGMLPFFSDFELKLFEKIVNDPLKFPTFKEIQSNKVSKVSCE XI ) ---RKLQKLLYKIMDPNPSTRISIQKIKESTFRKGPEENRILKERTLNENTTKNVPVLGVRRKKNHEDVKPMSVTNLNFEIISFSKGFDLSGMFIV ) ---PPLSDLLKGMLEYEPKRFSIRQIRQHSFRKKHPPEPVPIPPSP DTKDRRSMTVVPYLEDLHGDED ) ---EELKDLILKMLDKNPETRIGVPDIKLHPVTKNGEEPLPSEE----E HCSVVEVTEEEVKNSVRLI 7) ---EDLKDLITRMLDKNPESRIVVPEIKLHPVTRHGEPLPSEDEN CTLVEVTEEEVENSVKHI 2) DLVIKRLLEKDVTLRISIQDLVKVLSRDQPIDSRNHSQISSSSVNPVRNEGPVRRFFGRLLTKKGKK--KTSGKGKDKVLVSTSKVTPSIHID ----ECINILHLLDPEPNSRTRQFLESDFSKN EEYEMKDLLLKLLEKNPQKRMTIPIKKHPFVSDFDHVPENDEKLLSSVLEQKLRFQCN QTDQFEPISISKHELKNVSGV ) KERNERFTSDKSSTIISKLEDVKLNLRVRKKDNGVVKMQGRKEGRNGVLQFDIEIFEVTTSYHIIEMKQTSGDSLEYRQLLEEGIRPLKDIVL EDLFDIEDDIIYTQDFTVPGQVPE----EESHNGQRRGLPKVCMNGTEQLSTKSREGRPNPRKCSSSKIRRLSCKQQ ) PSTTVILVKSMLRKRSFGNPFEPQRREERSMSPGNLLVKEGFGEGGKSPELPGVQEDES PSLTVILVKTMIRKRSFGNPFEG-SRREERSLSPGNLLTKKPTRECESLSELKERQRRQPPGHRPPRGGGGSLVRGSPCVESCPPGSPRMH 4) EEPDKECFSTTVLRSSPDSSDYCS----SLGEEIQVTDFLDTFCRSNESLPNLTVNNDKQNSDMKTDRSESSSHSSLKIPTPIKMIRLKSSPKENGNR ) GKKIKESVLKSIPLKDPSDLSNKNYLHPTETTRGRGDNVIVSEGSVLSNIKELSNDGCLNTDSDTNININDDDHYSGDDNDGHLTKRELERELNKFDD ) 4) 8) PLRPEEMEPE ) THINCSQDKPSSPLMDRTVGKRTVNNSGRKLHSSNILNFKYINSEDSDIRETVEDVKTYLNFDNGQI ) KHEGNMVNLPINSSFSLDSFYIDNFMRMGMSSPEGDSVSSVPNLPSPSSTRLGRSPVFSGVTNQPSPIRPVLPQQKSSFCTGRYDKSHNSLLR Fig. S4. mino acid sequence of CIPK1 resembles that of protein kinases upstream of SNF1 and MPK. mino acid sequence comparison of the rice CIPK1 (K12177, human LKB1 (Q183, human CaMKKα (Q8NS, human CaMKKβ (Q96RR4), yeast Elm1 (P328, yeast Tos3 (QCJ7, and yeast Sak1 (P3899). Gaps have been introduced to maximize the alignment. Conserved domains are described in the legend to fig. S3. The NF domain is boxed. 4

6 14 1 ir T ( ) Shoot length (mm) T+Suc ( ) cipk1 ( ) cipk1 +Suc ( ) B Shoot length (mm) Days after imbibition ater T +Suc ( ) cipk1 +Suc ( ) T ( ) cipk1 ( ) Days after imbibition Fig. S. Sucrose rescues underwater seedling shoot development in the cipk1 mutant. () T and cipk1 (-/-) seeds were germinated in air, by placing on filter papers containing water or 9 mm sucrose (Suc) solution for various lengths of time. Shoot elongation of cipk1 was slightly retarded compared to T. (B) T and cipk1 (-/-) seeds were germinated under water or 9 mm sucrose solution for various lengths of time. cipk1 seeds germinated but both shoot and root growth were severely retarded; however, sucrose recovered shoot growth to the same extent as in T. Shoot length of seedlings at each time points was measured daily. Error bars indicate the S.E. of shoot length (n = 3). (NOV; p<.

7 Root length (cm) ir T ( ) cipk1 ( ) B Days after imbibition ater Root length (cm) T ( ) cipk1 ( ) C Root length (cm) Days after imbibition ater + Suc T ( ) cipk1 ( ) Days after imbibition Fig. S6. CIPK1 controls underwater root development. T and cipk1 (-/-) seeds were germinated on the surface of -cm high.3% agarose gel in a ml glass bottle, and seedlings were grown at 28 C under a 16-hr light / 8-hr dark condition for 7 days. Some seedlings were then submerged by filling up the glass bottle with water or 9 mm sucrose (Suc) solution, and the root length was measured daily up to 2 days after imbibition. () Roots growth in air. (B) Roots growth under water. (C) Roots growth under 9 mm sucrose solution. Error bars indicate the S.E. of root lengths (n = ). rrow indicates the date starting treatments. (NOV; p<. 6

8 3 cm water 1 cm soil cm B T cipk1 T cipk1 Flooded 3 cm above soil Flooded 1 cm above soil Fig. S7. CIPK1 control mature plant growth under partially-flooded conditions. () Diagram showing how rice plants were flooded with water. Two-week-old rice seedlings were grown in pot soil (approximately -cm deep) and flooded with water, either 3-cm (left) or 1-cm (right) high above soil surface. (B) The growth of T and cipk1 (-/-) plants was similar when flooded with 3 cm water but slower in cipk1 (-/-) than in the T when flooded with 1 cm water. Rice plants were grown in the greenhouse and the photo was taken 9 weeks after flooding. 7

9 Shoot length (cm) Shoot length (cm) Shoot length (cm) C DI E G +S +S M2 +S Nipponbare +S DI +S Haboganj aman +S DI 3 Kurkaruppan B Shoot lenght (cm) D Shoot length (cm) F Shoot length (cm) DI Goda Heenati +S 2 1 +S DI 3 3 +S Swarna +S FR13 2 +S 1 +S DI Shoot length (cm) S +S DI Fig. S8. Seedling hypoxia tolerance varies among rice varieties. Rice seeds were germinated in air or in water with or without 9 mm sucrose for various lengths of time. Shoot length of seedlings were measured daily. Error bars indicate the S.E. of shoot length (n = 3). (NOV; p<. (-D) seedlings of varieties tolerant to hypoxia. (E-G) seedlings of varieties intolerant to hypoxia., in air; +S, in air with sucrose;, under water;, under water with sucrose. Sucrose improved seedling development in all rice varieties. DI, days after inhibition. These rice varieties correspond to those shown in Table 1. 8

10 TP content (nmole.embryo Day T cipk1 T cipk1 ir ater Fig. S9. TP is consumed more slowly in T and cipk1 embryos germinated under water than in air. 9

11 Table S1. The IRRI Germplasm Center (IRGC) accession number of rice germplasms used in the present study. Germplasm name IRGC accession number Oryza sativa cv. FR Oryza sativa cv. Kurkaruppan 1449 Oryza sativa cv. Goda Heenati Oryza sativa cv. Habiganj man Oryza sativa cv. Swarna Oryza sativa cv. M

12 Table S2. Primers used in the present study. Target gene Primers nnealing temperature ( C) Fragment size (bp) CIPK1 1CF1: -CGGGTCCCGTGTCTCCTGCTCTCC (T genotyping) 1CR2: -CGGGTCCCGCTCCTCCCGTCTGCC-3 CIPK1 (Mutant 1TF1: -TTGTTGGTTGCGTTGTTG-3 37 genotyping) 1CR1: -GCGTGGGCTTCGGG-3 CIPK1 (RT-PCR) 1F2: -TGCCTTCTTCTTCG R3: -TTCCCGGCTC-3 CIPK1 (ORF) 1OF1: -TCCCGTCTGCCTTGG OR1: -TGTCTCCTGCTCTCC-3 CIPK11 (ORF) 11OF1: -GCG GTTCGGCCGTTTGGGTTGGG OR1: -GCG GTTCCTTTCTTTGTTTGTTCTGC-3 CIPK21 (ORF) 21OF1: -GCG GTTCCGGTGGGGGTCGG OR1: -GCG GTTC TCGCTCTGTTTTCG-3 αmy3 (RT-PCR) 3RTF1: -CGGCTCTCTGCCTCTGG RTR1: -CCTGCTTTTGCCC-3 αmy3 (Promoter) 3PF1: -CTCTCCCTGCTCCTGG PR1: -GCGTCCGGTCGTGGCG-3 αmy3 (ORF) 3OF1: -GGCCGTCGCGCTCG OR1: -CCCCGGTTCCTGC-3 αmy7 7RT1: -GCGGCTCTCGTTTGC RT2: -TGCTCCGTTTCGGTCG-3 αmy8 8RT1: -GGTTCCTGCCGGTGGC RTB: -GGTCGCGCGTGC-3 SnRK1 SnRK1aF1: -CCGGGTCGGCGG SnRK1aR1: -CTCTGTCGGTGCGG-3 MYB S1 S1RTF1: -TGGCGGCTGGCGC S1RTR1: -GCTTTCCCGGGTGTCG-3 dh1 dh1f1: '- CCGTTCGCGGTCTTGC -3' dh1r1: '- CGGTCCGGCCG -3' dh2 dh2f1: '- CCGGGTGCTGTTGG -3' 4 83 dh2r1: '- CGTCGCCTGGG -3' Sub1C Sub1CF1: '- CGTGGTGTGCGTGCGTC -3' 6 3 Sub1CR1: '- CGTTTTGTTGTCCTGTCTGC -3' EXP7 Exp7F1: '- TCTCCTCCGCCGGGTGC -3' Exp7R1: '- CCCCCTGTGCGTTCC -3' α-tubulin TubF1: -GGGGTCGTCGTCGTCTGG TubR1: -GCCCGGTTCCCG-3 18S rrn 18SF1: -CCTTCCTTTCGTGGTGGT SR1: -CGTTGGGTTTGTTGTCTCTT-3 Gene accession number: CIPK1 (LOC_Os11g224), CIPK11 (LOC_Os1g691), CIPK21 (LOC_Os7g4429), αmy3 (LOC_Os8g3691), αmy7 (LOC_Os2g271), αmy8 (LOC_Os8g369), SnRK1 (F62479., MYBS1 (Y1142), dh1 (LOC_Os11g148), dh2 (LOC_Os11g11), Sub1C (DQ4396), Exp7 (LOC_Os3g67), α-tubulin (LOC_Os7g3873), 18S rrn (LOC_Os3g6846). 11