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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