a newly characterized regulatory mechanism modulates the ... · nic2 erfs activate orca promoter...
TRANSCRIPT
A Newly Characterized Regulatory Mechanism Modulates the NIC2 Locus
that Controls Nicotine Biosynthesis
Ling YuanSitakanta Pattanaik
Priyanka PaulSanjay Singh
Barunava Patra
The Kentucky Tobacco Research and Development CenterUniversity of Kentucky
USA
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Nicotine Biosynthesis
Nicotine
Nornicotine
PNAS 114:6133-6138, 2017
E4/5/10
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ERFbHLHUnknown
Gene Regulation of Nicotine Biosynthetic Pathway
Pyridine ring Pyrrolidine ring
Modified from: MGG 290: 11-21, 2015
• ERFs: Ethylene Response Factors, APETALA2/ERF TFs.
• ERFs regulate molecular response to biotic/abiotic signals by binding to sequences containing AGCCGCC motifs (the GCC box), a cis-acting element.
• Some ERFs form clusters
Transcription factors (TFs)
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Two Types Gene Clusters• Metabolic enzyme gene clusters – non-homologous• TF gene clusters - homologous
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• The ERF clusters in Catharanthus, tomato, potato, and tobacco contain 5-12 TFs
• ERF cluster (ORCA) in Catharanthus controls production of terpenoid indole alkaloids
• NIC2 cluster in tobacco is critical for nicotine biosynthesis
• NIC2 and ORCA clusters are an excellent system for studying gene regulation
ORCA5 ORCA3 ORCA4 ORCA6 ORCA2
GAME9-L1 GAME9-L2 GAME9-L3 GAME9 GAME9-L4
GAME9-L1 GAME9-L2 GAME9-L3 GAME9-L4 GAME9 GAME9-L5 GAME9-L6 GAME9-L7
ERF163 ERF104C ERF221 ERF115 ERF168 ERF17 ERF179 ERF17L1ERF91L1 ERF189 ERF17L2 ERF17L1
ERF29 ERF16 ERF130 ERF210 ERF17L3 ERF199
Periwinkle
(C. roseus)
(70 kb)
Tomato
(S. lycopersicon)
(104 kb)
Potato
(S. tuberosum)
(230 kb)
Tobacco
(N. tabacum)
NIC2N. tomentosiformis (660 kb)
N. sylvestris (320 kb)
The ERF Clusters
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Do ERFs within a cluster regulate each other?
Jasmonic acid
ERF Clusters Are Controlled by Hormonal Signal Pathways and Post-translational Regulation
Phosphorylation(Activation and suppression)
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ERFs in Cluster Regulate Each Other
Promoter activation assay: ORCA 3/5 activate 4But not when
promoter is mutatedORCA 5 self-activating
Yeast one hybrid:ORCA 3/5 bind to ORCA 4 promoter
EMSA:ORCA 3/5 bind to ORCA 4 promoter
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C. roseus (periwinkle)
ORCA4
ORCA5
N. tabacum (tobacco)
ERF168
ERF115
GAME9-like 1
GAME9-like 2
S. lycopersicum (tomato)
GAME9-like 2
GAME9-like 3
S. tuberosum (potato)
ATG
ATG
ATG
ATG
ATG
ATG
ATG GAME9-like 4
ATG GAME9-like 7
ERF179ATG
ATG
GC-rich motifs
TCCCGGC, CGCTCCTC
AGCCCGCCC
TGGCACCT, GGCCAAGC
GGGTGGCA
TGCCTCCC
GATGGGCT
GCCCCACC
GCCCCACC, CACCAGCC
AGCCCGCT, AGCACCCC, AGCCCGCT
GCCTCAC, AGCCCGCT
GCCCGGC, AGCCCGACC
1kb
1kb
1kb
1kb
1kb
1kb
1kb
1kb
1kb
1kb
1kb
ATG
ERF Binding Sites Present in the ERF Promoters
Can ERF clusters mutually regulate (are they interchangeable)?
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Mutual Regulation of NIC2 and ORCA ERFs
Inter-species promoter activation by ERFs
NIC2 ERFs activate ORCA promoter NIC2 and ORCA ERFs activate NIC2 promoters
Dominant NIC2 ERF repressor does not activate NIC2 promoters
NIC2 ERF115 promoter contains 2 ERF binding sites,both are critical for ERF189 activation
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0
0.2
0.4
0.6
0.8
1
EV-1 EV-2 OE-1 OE-2
*
*
Nic
oti
ne
(%D
W)
Rel
ati
ve
exp
ress
ion
*
*
**
(b) (c)
EV 189-OE-1 189-OE-2
Rel
ati
ve
exp
ress
ion
**
(d) (e)
(a)
0
1
2
3
4
5PMT-Promoter
0
2
4
6
8
10
CN ORCA3 ERF189 ERF221
STR-Promoter QPT-Promoter
CN ERF221 ORCA3 ORCA4 ORCA5
Fold
A
ctiv
ity
CN ERF221 ORCA3 ORCA4 ORCA5
a
b
c
d
a
b
c
d
a
a a
b
c c
Reciprocal ERF Overexpression Increased Alkaloid Accumulation
ORCA/NIC2 ERFs activate pathway enzyme gene promoters
Overexpression of CatharanthusORCA5 increased nicotine accumulation in tobacco
Overexpression of NIC2 ERF189increased TIA accumulation in Catharanthus
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OR3OR5 OR4
C. roseus ORCA cluster N. tabacum NIC2 cluster
221 189168 17 179 17L1 J5L1104C
17L2
17L2
Inter-species
Intra-cluster
ORCAs
STR
GC-rich motif GC-rich motif
PMT/QPT
ERF189/221
OR3OR5 OR4
(f)
115
Intra-cluster and Inter-species ERF Regulation
• ERF clusters, e.g. NIC2, are critical in biosynthesis of alkaloids.
• We uncovered an intra-cluster regulation that is conserved in different species.
• The previously uncharacterized mechanism underscores the complexity of gene regulation of alkaloid biosynthesis.
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Yuan Lab (people who do the work)Dr. Barunava Patra
Dr. Craig Schluttenhofer
Kathy Shen
Dr. Sanjay Singh
Dr. Ji-Min Shin
Dr. Priyanka Paul
Dr. Jayadri Ghosh
Patrick Perry
Dr. Yongliang Liu
Dr. Yongqing Li
Dr. Hongxia Wang
Xiaoyu Liu
Liyuan Zhang
Dr. Ruth McNees
CollaboratorsDr. Sitakanta Pattanaik
Dr. Jingxue Wang
Dr. Ying Wang
Dr. Wei Ma
Dr. Xueyi Sui
Dr. Bingwu Wang
Dr. Xiaoyun Jia
Dr. X. Joe Zhou (UK)
Dr. Kurt Wagschal (USDA)
Dr. Erich Grotewold (Michigan St U)
KTRDCCorporate
sponsors
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