Engineering The Timing Of Leaf Starch Accumulation - Arabidopsis "proof Of Concept"
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&OHJOFFSJOHUIF5JNJOHPG-FBG4UBSDI"DDVNVMBUJPO 4FBO&8FJTF ;BDIBSZ++BSPV 5IPNBT%4IBSLFZ .JDIJHBO4UBUF6OJWFSTJUZ %FQBSUNFOUPG#JPDIFNJTUSZBOE.PMFDVMBS#JPMPHZ 4UBSDI"DDVNVMBUJPOJO5SBOTHFOJD1MBOUT
3BUJPOBMF 5IJTTVCQSPKFDUGPDVTFTPOJODSFBTJOHUIFZJFMEPGFBTJMZEFHSBEFEQPMZNFST TVDIBTTUBSDI JOMFBWFT5IFHPBMJTUPNBLFMFBWFTBCFUUFSGFFETUPDLGPSB DFMMVMPTJD FUIBOPM GFSNFOUPS5IF NBKPS BEWBOUBHFT PG UIF BQQSPBDI JO UIJT TVCQSPKFDUJT QPUFOUJBMGPSIJHIFSZJFMEBOE FBTFBOEDPNQMFUFOFTTPG GFSNFOUBUJPO 5IF NBKPS CPUUMFOFDL JT SFEVDFE ZJFME JO NPTU TUBSDI BDDVNVMBUJOHNVUBOUT
0WFSDPNJOHUIF:JFME1FOBMUZ
b.
35S
alcR
alcA
GWD 400bp
GWD 400bp
Intron
Intron
ALCR ALCR Ethanol added
E
GWD 400bp GWD 400bp
E
E
Target GWD DNA or mRNA
Dicer GWD
400
bp
GWD
400
bp
160
GWD KO
120 80 40
WD -G c l A
Ai RN
WT
0 0
2
4
6
Day
8
10
12
14
RISC
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
'JSTU"SBCJEPQTJTUIFO$PSO
$PODVSSFOU XJUI UIF "SBCJEPQTJT XPSL 3/"J DPOTUSVDUT UBSHFUFE UP UIF DPSO IPNPMPHVFPG(8%BOE4FYIBWFCFFONBEF5IFTFXJMMCFQMBDFECFIJOEUIF BMDPIPMJOEVDJCMFQSPNPUFS BOECFIJOEUIFDPOTUJUVUJWFQSPNPUFSPGVCJRVJUJO 5IFUSBOTGPSNBUJPOTBSFCFJOHDBSSJFEPVUCZ%S)FJEJ,BFQQMFSBUUIF6OJWFSTJUZ PG8JTDPOTJO.BEJTPO
120 80
Ai RN e s ata ph os h -P Alc
Phosphatase KO Alc Spray
Sag12p
Starch (µmol Glu g-1FW)
a.
Starch (µmol Glu g-1FW)
5PEPUIJTXFVTFE3/"JOUFSGFSFODF 3/"J BHBJOTUUIFHMVDBOXBUFSEJLJOBTF FO[ZNF (8% BOE UIF QIPTQIPHMVDBO QIPTQIBUBTF #PUI FO[ZNFT BSF OFDFTTBSZ GPS TUBSDI EFHSBEBUJPO JO MFBWFT 5IFTF 3/"J DPOTUSVDUT XJMM CF DPVQMFEUPBTFOFTDFODFJOEVDFEQSPNPUFS 4"( PSBOBMDPIPMJOEVDJCMF QSPNPUFS 'JH $POTUSVDUTGPSUIFWBSJPVTQSPNPUFSTBOEJOWFSUFESFQFBUT XFSFNBEFCZ#JP#BTJDJO.BSLIBN0OUBSJP$BOBEB XXXCJPCBTJDDPN
'JHVSF4FOFTDFODF*OEVDFE3/"J4UBSDI"DDVNVMBUJOHQMBOUT 1IPUPHSBQIT XFSF UBLFO BOE MFBWFT XFSF TUBJOFE XJUI *,* GPS TUBSDI JO UIF NPSOJOHXIFOTUBSDIMFWFMTBSFUIFMPXFTU5IF(8%LOPDLPVUQMBOUIBTIJHI TUBSDIMFWFMTCVUBTJHOJmDBOUHSPXUIQFOBMUZ5IFQMBOUXJUIUIFTFOFTDFODF JOEVDFE 3/"J HFOF BHBJOTU (8% EPFT OPU FYIJCJU B EFDSFBTF JO HSPXUI BOE BGUFSNPOUITDPOUBJOTIJHIMFBGTUBSDIMFWFMTDPNQBSBCMFUPUIF(8%,0MJOF
Alc Spray
5PBUUFNQUUPPWFSDPNFUIFZJFMEQFOBMUZXFCMPDLFETUBSDIEFHSBEBUJPOBUB TQFDJmDEFWFMPQNFOUBMUJNFQPJOUMBUFJOUIFMJGFDZDMFPGUIFQMBOU&BSMZJO QMBOU HSPXUI DBSCPO GSPN TUBSDI SFJOWFTUFE JO MFBWFT DBVTFT FYQPOFOUJBM HSPXUI 0ODF TVöDJFOU QMBOU NBTT JT QSFTFOU UP DPOUBJO UIF DBSCPO NFUBCPMJTNDBOCFTXJUDIFEUPDBVTFBDDVNVMBUJPOPGTUBSDI
40
WT
0 0
2
4
6
Day
8
10
12
14
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
3BUJPOBMF 5IJTTVCQSPKFDUGPDVTFTPOJODSFBTJOHUIFZJFMEPGFBTJMZEFHSBEFEQPMZNFST TVDIBTTUBSDI JOMFBWFT5IFHPBMJTUPNBLFMFBWFTBCFUUFSGFFETUPDLGPSB DFMMVMPTJD FUIBOPM GFSNFOUPS5IF NBKPS BEWBOUBHFT PG UIF BQQSPBDI JO UIJT TVCQSPKFDUJT QPUFOUJBMGPSIJHIFSZJFMEBOE FBTFBOEDPNQMFUFOFTTPG GFSNFOUBUJPO 5IF NBKPS CPUUMFOFDL JT SFEVDFE ZJFME JO NPTU TUBSDI BDDVNVMBUJOHNVUBOUT
0WFSDPNJOHUIF:JFME1FOBMUZ
b.
35S
alcR
alcA
GWD 400bp
GWD 400bp
Intron
Intron
ALCR ALCR Ethanol added
E
GWD 400bp GWD 400bp
E
E
Target GWD DNA or mRNA
Dicer GWD
400
bp
GWD
400
bp
160
GWD KO
120 80 40
WD -G c l A
Ai RN
WT
0 0
2
4
6
Day
8
10
12
14
RISC
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
'JSTU"SBCJEPQTJTUIFO$PSO
$PODVSSFOU XJUI UIF "SBCJEPQTJT XPSL 3/"J DPOTUSVDUT UBSHFUFE UP UIF DPSO IPNPMPHVFPG(8%BOE4FYIBWFCFFONBEF5IFTFXJMMCFQMBDFECFIJOEUIF BMDPIPMJOEVDJCMFQSPNPUFS BOECFIJOEUIFDPOTUJUVUJWFQSPNPUFSPGVCJRVJUJO 5IFUSBOTGPSNBUJPOTBSFCFJOHDBSSJFEPVUCZ%S)FJEJ,BFQQMFSBUUIF6OJWFSTJUZ PG8JTDPOTJO.BEJTPO
120 80
Ai RN e s ata ph os h -P Alc
Phosphatase KO Alc Spray
Sag12p
Starch (µmol Glu g-1FW)
a.
Starch (µmol Glu g-1FW)
5PEPUIJTXFVTFE3/"JOUFSGFSFODF 3/"J BHBJOTUUIFHMVDBOXBUFSEJLJOBTF FO[ZNF (8% BOE UIF QIPTQIPHMVDBO QIPTQIBUBTF #PUI FO[ZNFT BSF OFDFTTBSZ GPS TUBSDI EFHSBEBUJPO JO MFBWFT 5IFTF 3/"J DPOTUSVDUT XJMM CF DPVQMFEUPBTFOFTDFODFJOEVDFEQSPNPUFS 4"( PSBOBMDPIPMJOEVDJCMF QSPNPUFS 'JH $POTUSVDUTGPSUIFWBSJPVTQSPNPUFSTBOEJOWFSUFESFQFBUT XFSFNBEFCZ#JP#BTJDJO.BSLIBN0OUBSJP$BOBEB XXXCJPCBTJDDPN
'JHVSF4FOFTDFODF*OEVDFE3/"J4UBSDI"DDVNVMBUJOHQMBOUT 1IPUPHSBQIT XFSF UBLFO BOE MFBWFT XFSF TUBJOFE XJUI *,* GPS TUBSDI JO UIF NPSOJOHXIFOTUBSDIMFWFMTBSFUIFMPXFTU5IF(8%LOPDLPVUQMBOUIBTIJHI TUBSDIMFWFMTCVUBTJHOJmDBOUHSPXUIQFOBMUZ5IFQMBOUXJUIUIFTFOFTDFODF JOEVDFE 3/"J HFOF BHBJOTU (8% EPFT OPU FYIJCJU B EFDSFBTF JO HSPXUI BOE BGUFSNPOUITDPOUBJOTIJHIMFBGTUBSDIMFWFMTDPNQBSBCMFUPUIF(8%,0MJOF
Alc Spray
5PBUUFNQUUPPWFSDPNFUIFZJFMEQFOBMUZXFCMPDLFETUBSDIEFHSBEBUJPOBUB TQFDJmDEFWFMPQNFOUBMUJNFQPJOUMBUFJOUIFMJGFDZDMFPGUIFQMBOU&BSMZJO QMBOU HSPXUI DBSCPO GSPN TUBSDI SFJOWFTUFE JO MFBWFT DBVTFT FYQPOFOUJBM HSPXUI 0ODF TVöDJFOU QMBOU NBTT JT QSFTFOU UP DPOUBJO UIF DBSCPO NFUBCPMJTNDBOCFTXJUDIFEUPDBVTFBDDVNVMBUJPOPGTUBSDI
40
WT
0 0
2
4
6
Day
8
10
12
14
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