expression of oscpk4 in rice leaves in response to salt stress. … · 2014-05-01 · expression of...
TRANSCRIPT
SUPPLEMENTAL DATA
Overexpression of a Calcium-dependent protein kinase confers salt and drought tolerance in rice by
preventing membrane lipid peroxidation
Sonia Campo, Patricia Baldrich, Joaquima Messeguer, Eric Lalanne, María Coca and Blanca San Segundo
Supplemental Figure S1. Expression of OsCPK4 in rice leaves in response to salt stress.
Supplemental Figure S2. Amino acid sequence of OsCPK4 and alignment of the N-terminal
variable domain of rice CPKs.
Supplemental Figure S3. Overexpression of OsCPK4 in transgenic rice.
Supplemental Figure S4. Expression of salt-associated genes in roots of rice plants
overexpressing OsCPK4.
Supplemental Table SI. cis-related motifs identified in the 2000 bp upstream region of the
OsCPK4 promoter.
Supplemental Table SII. Genes misregulated in leaves of plants overexpressing OsCPK4 relative
to vector control plants, identified by microarray analysis.
Supplemental Table SIII. List of primers used for OsCPK4 cloning purposes and mutant analysis.
Supplemental Table SIV. List of primers used for expression analysis of rice genes by qRT-PCR.
Supplemental Methods
Supplemental Literature Cited
0.0000#
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0.0020#
0.0030#
0.0040#
0.0050#
0'# 2h# 4h#
Supplemental Figure S1
*!
CONTROL!NaCl!
Rel
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l!(x
102 )!
0.5!
0.4!
0.3!
0.2!
0.1!
0’! 2h! 4h!
OsCPK4!
0.0!
*!
Supplemental Figure S1. Expression of OsCPK4 in rice leaves in response to salt stress. Transcript levels were determined by qRT-PCR and normalized to OsCYP and OsUbi mRNAs. OsCPK4 expression was not affected by salt treatment at time points earlier than 2h. Asterisks denote significant differences (*P < 0.05)
Supplemental Figure S2
A 1 MGACFSSHTA TAAADGGSGK RQQRKGDHKG KLPDGGGGEK EKEAARVEFG YERDFEGRYQ 61 VGRLLGHGQF GYTFAATDRA SGDRVAVKRI DKAKMVRPVA VEDVKREVKI LKELKGHENI 121 VHFYNAFEDD SYVYIVMELC EGGELLDRIL AKKNSRYSEK DAAVVVRQML KVAAECHLHG 181 LVHRDMKPEN FLFKSTKEDS PLKATDFGLS DFIKPGKKFH DIVGSAYYVA PEVLKRRSGP 241 ESDVWSIGVI TYILLCGRRP FWNKTEDGIF REVLRNKPDF RKKPWPGISS GAKDFVKKLL 301 VKNPRARLTA AQALSHPWVR EGGEASEIPV DISVLSNMRQ FVKYSRFKQF ALRALASTLK 361 EEELADLKDQ FDAIDVDKSG SISIEEMRHA LAKDLPWRLK GPRVLEIIQA IDSNTDGLVD 421 FEEFVAATLH IHQMAELDSE RWGLRCQAAF SKFDLDGDGY ITPDELRMVQ HTGLKGSIEP 481 LLEEADIDKD GRISLSEFRK LLRTASMSNL PSPRGPPNPQ PL
N-terminal domain!Kinase Domain!Junction Domain!Calmodulin-like domain (CaM)!C-terminal domain !
B 10 20 30 40 50 60 70 80 90 100 110 120 130 140
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|OsCPK3 MGNCCRSPAAAAREDVKSS--H-----------------------------FPASAG------------KKKPHQARNGGVGGGGGGGGGGGGGGGAGQK-------------RLPVLGEEGCELIGGI--DD------ OsCPK16 MGNCCRSPAAAAREDVKTS--H-----------------------------FPASTGG----------GKKKPHQARNG------GGGGGGGGGGGWEKK-------------RLSVLGEEGSEVNGGI--EE------ OsCPK1 MGNRTSRHHRAAPEQ-------------------------------------PPPQ--------------PKPKPQPQQQQQQW---PRPQQPTPP---P-------------AAAPDAAMGRVLGRPM--EDVRAT-- OsCPK15 MGARASR-HRQSPDQSQSQ--------------------------------SPSPHH--KHHHHHQTTRAPKPKPKPQPPPPQQ---PRSQPPPPPRHQPQQAP--------QQAAAEDGVGRVLGRPM--EDVRAT-- OsCPK2 MGNCCP------GSG---D--------------------------------AEPAS-------------SDASTGNGSSSFKAG---ASPSSAPAQNKPP------------------APIGPVLGRPM--EDVRSI-- OsCPK14 MGNCCPP-----GSSSEPD--------------------------------PPPAS-------------SGSSRPAGSAGAAASPATISPSAAPAPAKPP------------------APIGPVLGRPM--EDVKSI-- OsCPK12 MGNCFTKTYEIPITSGTMR--------------------------------RPAST-------------AERSKARGGDEPGTWRRPSFPRHGAPPHRPPTGSSSAAGALSRRASGGGGEMGPVLQRAM--VSVRSL-- OsCPK8 MGNCCGTPATAEEGGKRRR--------------------------------RGKQK-------------KANPFTVAYNRAPSSAGAAAGRPGLMVLRDPTG----------RDLGARYELGGELGRGE--FGIT---- OsCPK20 MGNCCVTPEGSGRGRKKQQQEQ-----------------------------KQKQKEPKQQQQQQKKGKKPNPFSIEYNRSSAPSG---HR--LVVLREPTG----------RDIAARYELGGELGRGE--FGVT---- OsCPK11 MGNNCVGP-SAAGQNGFFANV---ALWRP--RPADAAP--------------PALPPP----SSAPSDQAPEPVTIP-PSEHSSHHSSRSTDPSTPTSAAEQPANKAAPKVKRVQSAGLLADSVLKRDVNTARLKDL-- OsCPK17 MGNTCVGPSSAADRHGFFHSVSLAVLWRPGGRAEPSQP--------------PGYPPRESSHSSVTSSTAPERVTIA-DSDLSS---------STPNKGGNK------PKVRRVQSAGLLADSVLKRDS--ERLKDL-- OsCPK10 MGNTCVGP--SISKNGFFQSVS-TVLWKARQDGDDALPGANGAPDGGGQGRLPAPPPPTSDAPLAVQNKPPEHVKIVSTTDTASAEQDASKSSAGSDSGEAARPRPRVPPVKRVSSAGLLVGSVLKRKT--ESLKD--- OsCPK5 MGNTCG-VTLRSKYFASFRGAS------------------------------QRHDEAGYAPVATSAAAAAAADEPAGKKAPRGSAAAADAPHAASMKRGAP-------------APAELTANVLGHPT--PSLSE--- OsCPK13 MGNACG-GSLRSKYLSFKQTAS------------------------------QRHDTDDNNNAAAADSPKKPSRPPAAAKT-DDHPVSASAP-AAAMRRGQ--------------APADLGS-VLGHPT--PNLRDL-- OsCPK7 MGNQCQNGTLGSDYHN--RFPR------------------------------EHAVG--YVQGDSYLDLKKFDDTWPEVNN-------FKPTAASILRRGL----------------DPTSINVLGRKT--ADLREH-- OsCPK23 MGNSCQNGTYGNNYQNSNRFQN------------------------------DRFASR-YVDGN------DTEDCYSGSS---------RASLAGALRQGL----------------NLKSP-VLGYKT--PNVREL-- OsCPK24 MQPDPS~GSGGDGNAN--~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~--AKAKLAPPPVTAA----------~~~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~---GGRPVSVLPHKT--ANVRD--- OsCPK4 MGACFSSHTATAAADG-------------------------------------GSG--------------KRQQRKGDHKG------KLPDGGGGEKEKE-------------------AARVEFGYER---DFEGR-- OsCPK18 MGLCSS------------------------------------------------SS-------------ARRDAGTPGGGN-------GAGNKDNAGRKG---------------IVACGKRTDFGYDK---DFEA--- OsCPK19 MGSCCSRATSPDSGRGGANGYG-----------------------------YSHQTKP----------AQTTPSYNHPQPPPPAEVRYTPSAMNPPVVPP------------VVAPPKPTPDTILGKPY--DDVRSV-- OsCPK6 MGNYYSCGASSTSSPTS-----------------------------------PSLVDY-------YYCYHRYPSSCSSTST------ATSSGGRMPIRSHQ--------------QRLSSPTAVLGHET--PALREV-- OsCPK9 MGNTCCVAPATTDEVGAPPRDHHHAAKK------------------------SPAPSATTTTATRQRHGQEPKPKPKPRARAKPNPYDWAPPRVLPARGGAAAS------------AVRVLEGVVPHHPR--LRVTD---OsCPK21 MGGCYSAYASSRKLRGRIS----------------------------------KIS-----------LVIPDPVPDAEAASPRKDGVDGDGDDVRGGGGG---------------CDDGGDVVAIATTT-ADEFAR--- OsCPK22 MGGCSSAFAVSTRMIRFSRGR------------------------------VPAAILP------VTSNDEPCCSCSPENNNKNNDGGGGGCDGGEHQKGKSWRRWQYRRCGGGGGGGGGRKNAILGDAADVKTAAGFAE OsCPK29 MGNCCVSRPSGADKRRRCGSST-----------------------------APHTRG------------GRRVIGAANMRCLSTVSSVSDAARAVMSNEPAT-------------VLGNSGSSGNGGVMAAEEMLR---
Supplemental Figure S2. Amino acid sequence of OsCPK4 (A) and alignment of the N-terminal variable domain of rice CPKs (B). The arrow in A indicates the N-terminal region used for obtaining polyclonal antibodies. Underlined, consensus sequence (MGACFSSHTATAAADGGSGKRQQR) for N-myristoylation (http://plantsp.genomics.purdue.edu/myrist.html). A potential palmitoylation site (Cys4) was predicted (http://csspalm.biocuckoo.org/) (B) Multiple sequence alignment of extracted aminoacid sequences for the N-terminal domain of rice CPK proteins (ClustalW algorithm). Identical aminoacids are shaded in black (threshold 30%) while similar aminoacids are shaded in light grey. For a phylogenetic analysis of rice CPKs we refer to Campos-Soriano et al. (2011) and Asano et al. (2005).
13!
OsCPK4!
1! 3! 7! 11! 10! 14! 17! 43! 45! 48! WT!
BrEt!
Supplemental Figure S3
OsCPK4
A!
B!
75!
50!
75!
50!
11!3! 11!3!
Supplemental Figure S3. Overexpression of OsCPK4 in transgenic rice. A, Northern blot analysis of wild-type (WT) and transgenic OsCPK4 rice plants (T0 generation). Total RNAs were isolated from leaves of 15 day-old transgenic rice plants, subjected to formaldehyde-containing agarose gel electrophoresis and hybridized with a 32P-labeled cDNA probe encoding the N-terminal domain of OsCPK4 (variable region among rice CPKs). Longer exposure times allowed detection of the endogenous OsCPK4 transcripts (results not shown). Lower panel show ethidium bromide staining of RNA samples. Boxes denote the lines analysed in this work. B, 2D-PAGE of protein extracts from transgenic and vector control plants. Protein extracts (20 µg each) were probed with antiserum prepared against the N-terminal domain of OsCPK4. No serological reaction occurred when the preimmune serum was used to probe protein extracts from roots of either control or OsCPK4-rice plants (results not shown).
OsDREB2B!Os05g27930!Os09g35030!
OsDREB1A!1.2!
0.9!
0.6!
0.3!
0.0!3! 11! 1! 14!pC! OsCPK4!
1.2!
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0.0! 3! 11! 1! 14!pC! OsCPK4!
0.0000!
0.3000!
0.6000!
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1!
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4! 0.0000!
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0.6000!
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1.2000!
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Os09g35030 ! Os05g27930 !
0.0000!
0.3000!
0.6000!
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ONAC045!Os11g03370!1.2!
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0.0!3! 11! 1! 14!pC! OsCPK4!
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OsNAC6!Os07g48450 !1.2!
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0.0! 3! 11! 1! 14!pC! OsCPK4!
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B!
0.0000!
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Os03g45280!OsLEA24!
1.2!
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0.0!3! 11! 1! 14!pC! OsCPK4!
0.0000!
0.3000!
0.6000!
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1.2000!
1!
2!
3!
4!
Os11g26760!OsLEA27!
1.2!
0.9!
0.6!
0.3!
0.0!3! 11! 1! 14!pC! OsCPK4!
0.0000!
0.3000!
0.6000!
0.9000!
1.2000!
1!
2!
3!
4!
OsLEA19a!Os05g46480!1.2!
0.9!
0.6!
0.3!
0.0!3! 11! 1! 14!pC! OsCPK4!
0.0000!
0.3000!
0.6000!
0.9000!
1.2000!
1!
2!
3!
4!
Os02g44870!OsLEA23!
1.2!
0.9!
0.6!
0.3!
0.0!3! 11! 1! 14!pC! OsCPK4!
A!
Rel
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l
0.0000!
0.3000!
0.6000!
0.9000!
1.2000!
1!
2!
3!
4!
Os05g28210!OsLEA21!
1.2!
0.9!
0.6!
0.3!
0.0!3! 11! 1! 14!pC! OsCPK4!
Supplemental Figure S4!
Supplemental Figure S4. Expression of salt-associated genes in roots of rice plants overexpressing OsCPK4. Transcript levels were determined by qRT-PCR using total RNA from roots. Specific primers for the indicated genes were used (Supplemental Table SIV). Five independently generated homozygous lines and three independent vector control lines were examined. Each RNA was prepared from a pool of roots of 10 plants. Representative results are shown for two OsCPK4 lines (lines 1 and 14) and two vector control lines (lines 3 and 11). A, Expression of LEA genes: OsLEA19a and OsLEA21; and Dehydrin genes, OsLEA23, OsLEA24 and OsLEA27. LEA genes are named according to Wang et al., (2007). B, Expression of transcription factor genes: OsDREB1A, OsDREB2B, OsNAC6, and OsONAC45. No significant differences were observed between the OsCPK4 and empty vector (pC) plants for any of the genes analysed.
Supplemental Table SI. cis-‐related motifs identified in the 2000bp upstream region of OsCPK4. The PLACE database (http://www.dna.affrc.go.jp/PLACE/) was used to perform the analysis. Dehydratation and ABA-responsive motifs are listed by alphabetical order
Motif name Number motifs Sequence Description
ABRELATERD1 4 ACGTG ABRE-like sequence required for etiolation-induced expression of erd1 (early responsive to dehydration) in Arabidopsis
ABRERATCAL 2 MACGYGB "ABRE-related sequence" or "Repeated sequence motifs" identified in the upstream regions of 162 Ca2+responsive upregulated genes
ACGTATERD1 10 ACGT ACGT sequence required for etiolation-inducedexpression of erd1 (early responsive to dehydration) in Arabidopsis;
BP5OSWX 1 CAACGTG OsBP-5 (a MYC protein) binding site in Wx promoter (induced by salt, drought and ABA)
DPBFCOREDCDC3 3 ACACNNG
bZIP transcription factors, DPBF-1 and 2 (Dc3 promoter-binding factor-1 and 2) binding core sequence; Found in the carrot Dc3 gene promoter; Dc3 expression is normally embryo-specific, and also can be induced by ABA.
GT1GMSCAM4 8 GAAAAA GT-1 motif found in the promoter of soybean (Glycine max) CaM isoform, SCaM-4; Plays a role in salt-induced SCaM-4 gene expression.
LTRECOREATCOR15 1 CCGAC
Core of low temperature responsive element (LTRE) of cor15a gene in Arabidopsis; ABA responsiveness; Light signaling mediated by phytochrome is necessary for cold- or drought- induced gene expression through the C/DRE in Arabidopsis.
MYB1AT 5 WAACCA MYB recognition site found in the promoters of the dehydration-responsive gene rd22 and many other genes in Arabidopsis
MYBATRD22 1 CTAACCA Binding site for MYB (ATMYB2) in dehydration-responsive gene, rd22; ABA-induction;
MYBCORE 3 CNGTTR
Binding site for all animal MYB and at least two plant MYB proteins ATMYB1 and ATMYB2, both isolated from Arabidopsis; ATMYB2 is involved in regulation of genes that are responsive to water stress in Arabidopsis
MYCATERD1 2 CATGTG MYC recognition sequence necessary for expression of erd1 (early responsive to dehydration) in dehydrated Arabidopsis; NAC protein bound specifically to the CATGTG motif
MYCATRD22 2 CACATG Binding site for MYC (rd22 BP1) in Arabidopsis dehydration-resposive gene,; ABA-induction
MYCCONSENSUSAT 14 CANNTG MYC recognition site found in the promoters of the dehydration-responsive gene rd22 and many other genes in Arabidopsis
Biological functionGene ID Description Fold change
LOC_Os01g51890 Endonuclease/exonuclease/phosphatase domain containing protein -2.08LOC_Os02g33390 pectinesterase inhibitor domain containing protein -3.22, -2.19LOC_Os02g44230 Trehalose-6-phosphate phosphatase (OsTPP) -2.20LOC_Os10g40640 Glycosyl transferase 8 domain containing protein -2.35
LOC_Os01g51920 Similar to GmCK1p (Choline kinase) -3.26, -2.04LOC_Os01g62430 C2 ((Calcium/lipid-binding domain, CaLB) domain containing protein -2.20LOC_Os02g11070 Similar to Fatty acid elongase 1 -3.11LOC_Os03g47940 GDSL-like lipase/acylhydrolase -2.32, -2.08LOC_Os05g34700 GDSL-like lipase/acylhydrolase -2.27
LOC_Os01g67980 Cysteine endopeptidase -2.24LOC_Os02g39570 Amino acid-binding ACT domain containing protein 13.26LOC_Os12g39360 aspartic proteinase nepenthesin precursor 2.10
LOC_Os03g63870 Nucleic acid-binding, OB-fold domain containing protein 2.12LOC_Os04g58810 CAF1 family ribonuclease containing protein -2.40, -2.21LOC_Os05g36280 Histone H3 -2.13LOC_Os06g06510 Histone H3 -2.45, -2.17LOC_Os07g43400 SWIM zinc finger family protein -3.67, -2.95none RNA-directed DNA polymerase (reverse transcriptase) domain containing protein (AK121448) -3.62LOC_Os11g05730 Histone H3 -2.05
OthersLOC_Os02g47470 Cytochrome P450 -3.63LOC_Os03g18030 Leucoanthocyanidin dioxygenase -2.09LOC_Os04g41960 NADP-dependent oxidoreductaseLOC_Os05g12040 Cytochrome P450 51 -2.51LOC_Os05g28210 OsLEA21 (EMP1) -2.20LOC_Os06g10210 Chalcone isomerase domain containing protein -2.12LOC_Os07g42280 von Willebrand factor type A domain containing protein -2.37, -2.61LOC_Os07g46670 early response to dehydration 15-like protein -2.39LOC_Os08g40850 Mitochondrial carrier protein -2.71LOC_Os11g29290 Cytochrome P450 -2.30, -2.07LOC_Os12g39310 Cytochrome P450 -2.22
2. Cellular processesLOC_Os02g13660 meiosis 5 -2.61LOC_Os02g56540 Kinesin motor domain containing protein -2.34, -2.04LOC_Os07g41310 COBRA-like protein -4.45
3. Oxidative stressLOC_Os01g73220 Peroxidase -2.11LOC_Os07g48040 Peroxidase -2.18LOC_Os08g37660 Plastocyanin-like domain containing protein -2.44miR osa-miR528 2.42
4. Transcription and RNA bindingLOC_Os01g01840 helix-loop-helix DNA-binding domain 2.08, 2.38LOC_Os01g64310 No apical meristem (NAM) protein domain containing protein -2.93LOC_Os02g08440 WRKY71 -2.46LOC_Os02g15810 High mobility group, HMG1/HMG2 domain containing protein -2.76LOC_Os02g35329 RING-H2 finger protein ATL3F -2.03LOC_Os02g39580 ZOS2-12 - C2H2 zinc finger protein -2.29, -2.00LOC_Os02g45450 CRT/DRE binding factor 1 -4.11LOC_Os02g51350 OsFBK10 - F-box domain and kelch repeat containing protein -6.46, -2.03LOC_Os02g52210 zinc finger, C3HC4 type domain containing protein -5.52, -2.58LOC_Os03g08330 ZIM domain containing protein -2.25LOC_Os03g26870 Similar to Salt-responsive WD40 protein 5 -2.24, -2.03LOC_Os03g28940 ZIM domain containing protein -2.04LOC_Os03g32230 ZOS3-12 - C2H2 zinc finger protein -2.07
DNA/ RNA modification
Supplemental Table SII. Genes misregulated in leaves of plants overexpressing OsCPK4 relative to vector control plants, identified by microarray analysis. Up-regulated genes are shown in bold letters
1. Metabolism Carbohydrates
Lipids
Amino acids and Proteins
LOC_Os04g23550 Basic helix-loop-helix family protein -3.35, -2.18LOC_Os04g32480 Zinc-finger protein -2.20LOC_Os09g25060 WRKY transcription factor 76 2.30LOC_Os09g35010 Dehydration-responsive element-binding protein 1B -2.82LOC_Os09g35030 DRE-binding protein 1A -2.48LOC_Os10g25230 ZIM domain containing protein -2.57, -2.51LOC_Os11g45740 MYB family transcription factor -2.27, -2.01miR osa-miR159c 2.00
5. Signal transduction and Intracellular traffickingLOC_Os01g41420 Amino acid transporter, transmembrane domain containing protein -5.04LOC_Os01g50420 Serine/threonine protein kinase domain containing protein -2.41LOC_Os01g65110 POT family protein (Oligopeptide transporter) -2.02LOC_Os02g43410 Oligopeptide transporter OPT superfamily protein -2.40LOC_Os02g54600 Similar to MAP kinase kinase -3.09LOC_Os03g07170 lactose permease 2.78, 2.19LOC_Os04g49690 FERONIA receptor-like kinase -2.05
6. Ion transportLOC_Os03g19420 OsNAS2_Nicotianamine synthase 2 -6.21LOC_Os03g19427 OsNAS1_Nicotianamine synthase 1 -6.79
7. UnknownLOC_Os01g09220 Similar to transposon protein CACTA, En/Spm sub-class -2.40LOC_Os01g29330 Protein of unknown function DUF679 family protein -2.16LOC_Os01g49750 Expressed protein 2.53LOC_Os01g58350 Conserved hypothetical protein -2.40LOC_Os01g61340 Expressed protein 4.06LOC_Os01g72360 Conserved hypothetical protein -3.59, -3.54LOC_Os03g02470 Expressed protein 3.22LOC_Os03g43100 Conserved hypothetical protein 2.54LOC_Os03g50670 Retrotransposon protein 3.20LOC_Os03g51390 Expressed protein -2.05LOC_Os04g02490 expressed protein 7.62LOC_Os04g41900 Expressed protein -2.06LOC_Os05g03960 Expressed protein -2.77LOC_Os05g08900 Expressed protein -2.45, -2.17LOC_Os05g37520 Tetratricopeptide-like helical domain containing protein -2.77LOC_Os05g46840 Proline-rich protein -2.27, 2.04LOC_Os06g16040 Transposase, Ptta/En/Spm -6.01LOC_Os07g03040 Expressed protein -3.61LOC_Os07g26100 Conserved hypothetical protein 6.94LOC_Os07g33280 Conserved hypothetical protein 4.02LOC_Os08g31950 Expressed protein -2.00LOC_Os10g3124 Conserved hypothetical protein 2.03, 3.14LOC_Os10g36610 UP-9A 2.72LOC_Os11g11694 Retrotransposon protein 2.60none Hypothetical gene 2.38none Expressed protein 2.37none Non-protein coding transcript 3.64
Supplemental Table SIII. List of primers used for OsCPK4 cloning purposes and mutant analysis. Restriction site sequences are underlined. PCR target Oligo ID Sequence Usage
NOS NOS-BHI-SmaI-F 5'- CCGGATCCCGGGGATCGTTCAAACATTTGGCAA -3' Overexpression (pC::pUbi) NOS-KpnI-HIII-R 5'- CCGGTACCAAGCTTGTTTGACAGCTT -3'
OsCPK4 (cds)
OsCPK4-BHI-F 5'- CCCGGATCCACCATGGGCGCGTGCTTCTCATC -3' Overexpression (pC::pUbi::nos) OsCPK4-SmaI-R 5'- CGGCCCGGGTCACAGGGGTTGTGGATTTGGA -3'
OsCPK4 (cds)
OsCPK4-BHI-F 5'- CCCGGATCCACCATGGGCGCGTGCTTCTCATC -3' Subcellular localization (pC::pUbi::GFP::nos) OsCPK4-SacI-R 5'- CGGGAGCTCCAGGGGTTGTGGATTTGGAGGT -3'
OsCPK4 (N-terminal domain)
N-ter.OsCPK4-BHI-F 5’- CCGGATCCATGGGCGCGTGCTTCTCATCCCA -3' Antibody production (pGEX-4T-3) N-ter.OsCPK4-SmaI-R 5'- GGCCCGGGCTACCTCCCCTCGAAGTCCCTCT -3'
2D-00040 mutant p1 5'- CAACCAAGGAGGACTCACCT -3'
cpk4 mutant analysis p2 5'- GAAGGCAAGGGGATGCAGAAGA -3' p3 5’- GCTAGAGTCGAGAATTCAGT -3’
1D-03351 mutant p1 5'- GGTGCCAGCTGTCTAGAACTAA -3'
cpk4 mutant analysis p2 5'- CTAGGGCCATAAGGCCATTACT -3' p3 5'- CACGTGGTGAATGGCATCGTT -3'
Supplemental Table SIV. List of primers used for expression analysis of rice genes by qRT-PCR.
Oligo ID Accession number Sequence
OsCPK4 Os02g03410 Forward: 5′- CGTGTGCAGCATGCAGATAA -3′ Reverse: 5′- TGCGATGAATACGTGCAATCA-3′
OsCPK4 (transgen) Os02g03410 Forward: 5′- TCCAAGAGGACCTCCAAATCC -3′ Reverse: 5′- AAATGTTTGAACGATCCCCG-3′
OsLEA19a (LEA3) Os05g46480 Forward: 5'- ACCGGCAGCGTCCTCCAACA-3'
Reverse: 5'- CACACCCGTCAGAAATCCTC-3' OsLEA21 (EMP1) Os05g28210 Forward: 5'- CAAACACAAGCCACCCTTCC-3'
Reverse: 5'- GCTAGCCTGACGTACGCTGC-3' OsLEA23 (Dehidrin, DIP1) Os02g44870 Forward: 5'- GTGAGACCAGGCCATGGTTG -3'
Reverse: 5'- TGCAGTGCAGAAAAAGCACC -3' OsLEA24 (Dehydrin) Os03g45280 Forward: 5'- GGCATGGTTTGCCTTTGTG -3'
Reverse: 5'- TTACAAGGCACCGTGCAGC -3' OsLEA26 (Dehydrin, RAB16B) Os11g26750 Forward: 5'- GATGGGAGGAAGGAGGAAGAA -3'
Reverse: 5'- TGCTGGTTGTTGCCCTTGT -3' OsLEA27 (Dehydrin, RAB16C) Os11g26760 Forward: 5'- CTTCTGCCTTGGGACGGAT -3'
Reverse: 5'- CAAGGGTAAAACCGACACGG-3'
OsDREB1A Os09g35030 Forward: 5’- CCACACTCGAGCAGAGCAAAT -3’ Reverse: 5’- GCTTGATCCCGCACATCTTC -3’
OsDREB1B Os09g35010 Forward: 5’- TCTCCGGCGGAGACCTTC -3' Reverse: 5’- CCGGCAACACGTCCTTGT -3'
OsDREB2B Os05g27930 Forward: 5’- CAGCCCGGAAGAAAAAGCG-3’ Reverse: 5’- GCTCCTGCTGATTGTTGAGC-3’
OsNAC6 Os07g48450 Forward: 5’- TGGAAATCGATCAAACGCAG -3’ Reverse: 5’- ATGCCAGTACGTAGCATCCGT -3’
ONAC45 Os11g03370 Forward: 5’- AACCTCCACAACGACAACCC-3’ Reverse: 5’- TGCACACAACCCACTCATCCT-3’
WRKY71 Os02g08440 Forward: 5’- GCCTGCCCTGTCAAGAAGAA -3' Reverse: 5’- TCGCCACGAGGATCGTGT -3'
WRKY76 Os09g25060 Forward: 5’- AGGAGCAGGGAGAGCATGG -3' Reverse: 5’- CCTGATGCCTGTTGCTGTTG -3'
Thioredoxin Os02g42700 Forward: 5’- AAGCTTGTTGCTATGGCTCCA-3’ Reverse: 5’- GCATGCAGCTAGCATTCGC -3’
Laccase-5 Os10g20610 Forward: 5'- ATACATCTCTCCCCCCACCAC -3' Reverse: 5'- TTCATGGCCTTCCGTCCTAG -3'
Aspartic proteinase Os12g39360 Forward: 5’- CAGAGCGTGATCGGCAACTA -3' Reverse: 5’- CATTGGCGAGGTCGTAGAGC -3'
OsCYP Os02g02890 Forward: 5’- GTGGTGTTAGTCTTTTTATGAGTTCGT -3’ Reverse: 5’- ACCAAACCATGGGCGATCT -3’
OsUbi1 Os06g46770 Forward: 5’- TTCCCCAATGGAGCTATGGTTT -3’ Reverse: 5’- AAACGGGACACGACCAAGG -3’
SUPPLEMENTAL METHODS
Rice transformation
For overexpression of the OsCPK4 gene, a pCAMBIA1300-derived vector
containing the maize ubiquitin 1 (ubi) promoter and the nopaline synthase (nos)
terminator was obtained. For this, the ubi promoter was obtained by digestion with
HindIII and BamHI of the pAHC17 plasmid DNA (Christensen and Quail, 1996) and
cloned into the HindIII and BamHI- digested pCAMBIA1300 vector (pC::pUbi
plasmid). Next, the nos terminator fragment was obtained by PCR amplification using
primers NOS-BHI-SmaI-F and NOS-KpnI-HIII-R. The amplified fragment was digested
with BamHI and KpnI and then inserted in the pC::pUbi plasmid, resulting in the
pC::pUbi::nos plasmid (BamHI and SmaI as unique cloning sites). Finally, the OsCPK4
DNA fragment was PCR-amplified from the full-length OsCPK4 cDNA sequence
(clone 001-032-C08; KOME (Knowledge-based Oryza Molecular biological
Encyclopedia)), using the primers OsCPK4-BHI-F and OsCPK4-SmaI-R, containing the
BamHI and SmaI restriction sites at the 5’ and 3’ end, respectively, of the OsCPK4
coding sequence. The amplified OsCPK4 fragment was then cloned into the BamHI and
SmaI sites of the pC::pUbi::nos vector, resulting in the pC::pUbi::OsCPK4::nos vector
(plasmid used for OsCPK4 overexpression in rice). Nucleotide sequences of the PCR
primers used for preparation of the plant expression vector are listed in Supplemental
Table SIII. Transgenic rice (O. sativa cv. Nipponbare) plants were produced by
Agrobacterium-mediated transformation (A.tumefaciens EHA105 strain) as previously
described (Sallaud et al., 2003).
For Northern blot analysis of transgenic rice plants, 10 µg of total RNAs were
subjected to 1.2% formaldehyde-containing agarose gel electrophoresis, transferred to
nylon membranes (Hybond-N, Amersham) and fixed by ultraviolet cross-linking. The
nucleotide sequence encoding the N-terminal domain of the OsCPK4 gene (Met1-Arg58)
was PCR amplified from the full-length OsCPK4 coding sequence (clone 001-032-
C08), from the KOME database using the primers N-ter.OsCPK4-BHI-F and N-
ter.OsCPK4-SmaI-R (Supplemental Table SIII), labelled with [α-32P]dCTP and used as
probe. Hybridization was performed in phosphate buffer [125 mM Na2HPO4, pH 7.2,
7% (w/v) SDS, 1 mM EDTA] at 65°C overnight and washed three times for 20 min in
20 mM Na2HPO4 (pH 7.2), 1% (w/v) SDS, 1 mM EDTA at 65°C. The hybridized blot
was exposed overnight to a phosphor screen, and visualized with a Bio-Rad Personal
FX phosphorimager.
Analysis of oscpk4 mutants
Two T-DNA insertional (KO) lines were identified in the Postech Rice T-DNA
Insertion Sequence Database, the 2D-00040 (D00062) and the 1D-03351 (B08233)
mutants (Dongjin and Hwayoung background, respectively). Genotyping of the cpk4
mutants was performed with a PCR-based approach (see primer sequences in
Supplemental Table SIII). DNA was extracted from leaves of rice plants according to
the method of Murray and Thompson (1980) but using MATAB (0.1 M TrisHCl, pH
8.0, 1.4 M NaCl, 20 mM EDTA, 2% MATAB, 1% PEG 6000, and 0.5% sodium
sulphite) as the extraction buffer. Genomic DNA was isolated and used for PCR
genotyping of the cpk4 mutants.
Microarray construction and analysis.
Oligonucleotides for the rice transcriptomic array were designed using the Tethys
software (Oryzon Genomics, Barcelona, Spain). Tethys is an oligonucleotide design
algorithm that performs in silico thermodynamic simulation of the hybridization
procedure previously used in the design of gene expression or genome hybridization
arrays. The final array design based on the release 6.1 of the MSU Rice Genome
Annotation Project contained 3 x 29450 probes (3 technical replicates per probe)
representing approximately 20750 genes. Microarray slides, each containing two
microarrays, were synthesized by Agilent using ink-jet printing and in situ
oligonucleotide synthesis.
Total RNA (0.5 µg) amplification and labelling with Cy3 or Cy5 was carried out
using the MessageAmp™ RNA amplification kit from Ambion (Applied Biosystems).
Cy3- and Cy5-labelled cRNAs were combined and hybridized to the microarray for 17h
at 60°C using Agilent's gaskets G2534-60002, G2534A hybridization chambers and
DNA Hybridization Oven G2545A, according to the manufacturer's instructions. Raw
data were obtained using Agilent's DNA Microarray Scanner G2505B and Feature
Extraction software (v10.1). The raw fluorescence intensity data were processed using
the Polyphemus™ software, developed at Oryzon Genomics.
Subcellular localization of OsCPK4
The OsCPK4 DNA fragment was PCR-amplified from the full-length OsCPK4
cDNA sequence (clone 001-032-C08) from the KOME collection using the primers
OsCPK4-BHI-F and OsCPK4-SacI-R, which generated the BamHI and SacI restriction
sites at the 5’ and 3’ end of the OsCPK4 coding sequence (Supplemental Table SIII).
The PCR product was digested with BamHI and SacI and inserted upstream of and in
frame with the green fluorescent gene (GFP), in a pCAMBIA1300-derived vector
harbouring the maize ubiquitin 1 promoter. The procedure for bombarding onion
(Allium cepa) epidermal cells was conducted as described (Murillo et al., 2003) using a
Biolistic PDS-1000/He gene gun system (BIO-RAD). Onion cells were also
transformed using the plasmid for expression of the GFP gene alone. The expression of
the OsGFP4-GFP fusion gene was observed by confocal microscopy (Olympus
FV1000 spectral inverted confocal microscope) at 5 h after bombardment. To confirm
plasma membrane localization, cells were plasmolysed with 0.75 M mannitol for
15min.
Bacterial Expression, Purification of the OsCPK4 Protein, and Preparation of the
Antiserum
The sequence encoding the N-terminal domain of the OsCPK4 gene (Met1-Arg58;
Supplemental Fig. SII) was cloned into the BamHI and SmaI restriction sites of the
expression vector pGEX-4T-3 (GE Healthcare) for production of a recombinant C-
terminal Glutathione-S-transferase (GST)-tagged protein in E. coli BL21 (DE3) strain.
Primers used for cloning are listed in Supplementary Table SIII. For purification of the
fusion protein, cells from the IPTG (isopropyl-D-thiogalactopyranoside)-induced
culture were lysed by incubation with lysozyme (1 mg mL-1, 2 h on ice). After
centrifugation of the bacterial lysate, the recombinant protein was recovered in the
supernatant. Purification of the recombinant protein was carried out by affinity
chromatography on glutathione-Sepharose 4B resin (Amersham Pharmacia Biotech)
and cleaved with thrombin (Sigma). Production of the OsCPK4 protein in E. coli was
monitored by SDS-PAGE analysis of bacterial extracts. Polyclonal antibodies were
raised in rabbits by multiple subcutaneous injections of the purified N-terminal domain
of OsCPK4 protein. Four weekly injections (100 µg each injection) were made and the
rabbits were bled 8 days after the third and four injection. Blood samples were collected
from the marginal ear vein. Preimmune serum was collected from the rabbits 1 week
prior to immunization. The antibody was then used to examine the accumulation of
OsCPK4 in protein extracts obtained from roots of transgenic rice plants.
Preparation of Protein Extracts, 2D-PAGE and immunoblotting
Protein extracts were prepared from root tissues as previously described (Romeis et
al., 2000). All protein concentrations were determined with the Bio-Rad dye reagent and
BSA as a standard. SDS-PAGE was performed according to the method of Laemmli
(1970). For 2D-PAGE, protein extracts in 7 M urea, 2 M thiourea, 2% CHAPS, 1%
immobilized pH gradient (IPG) buffer and 2% DeStreak reagent were separated on IPG
strips (7.5 cm, pH gradient 3–11) (http://www.gehealthcare.com) in the first dimension.
Second dimension was performed by SDS-PAGE (12.5% acrylamide in separation gel)
on a miniprotean apparatus for 90 min at 120V. Blots were blocked in PBS-T buffer
(1XPBS = 0.05 M Na phosphate, pH 7.5, 0.15 M NaCl containing 0.1% Tween) and
10% nonfat dry milk for 1h at room temperature and incubated overnight at 4ºC in the
same buffer containing the anti-OsCPK4 antiserum at a dilution of 1:1000, washed four
times in PBS-T, and then incubated with protein A–peroxidase (Sigma) at a dilution of
1:10000 for 1h at room temperature. Following four washes in PBS-T, peroxidase
activity was made visible by incubating the blot with ECL Western Blotting Substrate
(Pierce) for 5min on an LAS-4000 Image analyzer (Fujifilm). (Wang et al., 2007)
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