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Agave Genomics in Support
of CAM Engineering
Xiaohan Yang
Biosciences Division
Oak Ridge National
Laboratory
C4-CAM Conference
August 09, 2013
cambiodesign.org
2 Managed by UT-Battelle for the U.S. Department of Energy Presentation_name
Acknowledgements
Oak Ridge National Laboratory
Biosciences Division
Hengfu Yin
David J. Weston
Anne Borland
Timothy J. Tschaplinski
Sara Jawdy
Henrique Cestari De Paoli
Priya Ranjan
Gerald A. Tuskan
Environmental Sciences Division
Stan D. Wullschleger
CINVESTAV, Mexico
June Simpson
María Jazmín Abraham-Juarez
DOE Joint Genome Institute
Stephen M. Gross
Jeffrey A. Martin
Zhong Wang
Axel Visel
University of Tennessee
Jianzhuang Yao
Hao-Bo Guo
Hong Guo
University of Nevada-Reno
John Cushman
University of Liverpool
James Hartwell
This research was funded by ORNL LDRD and the DOE Office of Science, Genomic Science Program
3 Managed by UT-Battelle for the U.S. Department of Energy Presentation_name
High land-use efficiency
Low water-use footprint
Minimal detrimental
effects on the environment
Minimal interference
with food production
Requirements for an Ideal
Bioenergy Crop
4 Managed by UT-Battelle for the U.S. Department of Energy Presentation_name
Research Goals
Inform genetic improvement of Agave for
biomass production
Provide genomics information for creating
synthetic CAM machinery in C3 plants
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Candidate Gene Strategy
Comparative
genomics
Gene expression
pattern
Protein-protein
interaction
CAM-specific
gene/family
expansion
PPI module
Co-expression
module/GRN
Transcriptome sequencing, 3 Agave species
6 Managed by UT-Battelle for the U.S. Department of Energy Presentation_name
Plant Materials for RNA-Seq Analysis
Agave americana 'marginata’
Mature leaf (8 time points)
Young leaf (3 time points)
Shoot tip
(meristem)
Stem
Rhizome
Root
7 Managed by UT-Battelle for the U.S. Department of Energy Presentation_name
Developmental Progression of CAM in Agave
americana is linked to Leaf Succulence
-2
-1
0
1
2
3
4
Time
Net
CO
2 u
pta
ke (
μm
ol C
O2 m
-2 s
-1)
Leaf 1
Leaf 2
Leaf 3
Leaf 4
R² = 0.9993
0
40
80
120
160
200
1 1.4 1.8 2.2 2.6 3 3.4 3.8
Succulence (kg m-2) N
et d
ark
CO
2 u
pta
ke
(m
mo
lm-2
)
Anne Borland
1:00 pm 9:00
pm 5:00 am 1:00 pm
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Transcriptome Sequencing of A. americana
strand-specific library
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Transcriptome Assembly
HiSeq reads Contig set1
454 reads
Trinity
Contig set2
CAP3
Unigene set (Strand calibrated: 5’ -> 3’)
(expressed in at least 2 tissues; >5 rpkm)
Mapping with HiSeq
reads
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Gene Co-expression Modules
Mature Leaf
Co
-ex
pre
ss
ion
mo
du
le ID
Software:WGCNA
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Distribution of Genes among the
Co-expression Modules
MMblack, 710
MMblue, 6134
MMbrown, 1890
MMcyan, 688
MMgreen, 2710
MMgreenyellow, 314
MMgrey, 7537
MMlightcyan, 2283
MMmagenta, 1801
MMmidnightblue, 540
MMpink, 1854
MMpurple, 580
MMred, 1235
MMsalmon, 590
MMtan, 2156
MMturquoise, 9716
MMyellow, 2700
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Functional Enrichment in Co-expression
Modules: Gene Ontology (GO)
MMred MMsalmon
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NVP
Comparative Genomics: Clusters of
Orthologous Gene Groups
Arabidopsis thaliana
Populus trichocarpa
Solanum tuberosum
Musa acuminata
Brachypodium distachyon
Oryza sativa
Sorghum bicolor
Zea mays
Setaria italica
Agave americana
Agave deserti
Agave tequilana
Selaginella moellendorffii
Physcomitrella patens
Chlamydomonas reinhardtii
CAM/Agave
NVP (Non-
vascular
plants)
C4
C3_moncot
C3_dicot
14130 14759
14626
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Co-expression Modules in the Agave-
specific Ortholog Groups
*
*
*
*
* **
**
0
5
10
15
20
25
% o
f th
e g
en
e s
et
Co-expression module
All
Agave-specific
*Significant (Adjusted P-value < 0.01)
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Functional Enrichment in Gene
Families in CAM/Agave Lineages
Gene families expanded in the Agave/CAM-lineage
(CAM>C3dicot, CAM>C3monocot, CAM>C4, and CAM>NVP)
CAM
C3_dicot C3_monocot
C4 NVP
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Possible Stomata Control Gene
Coursol et al. 2003.
Nature 423:651-654
Sphingosine-1-phosphate phosphatase
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Sphingosine-1-phosphate phosphatase
Agam Aam347390 Agam Aam053622 Agde Locus38946v1rpkm2.14 7 Agte Locus34930v1rpkm4.06 9 Agam Aam053623 Agde Locus27139v1rpkm6.09 9 Agte Locus40480v1rpkm2.68 5 Agam Aam222744 Muac GSMUA Achr5P24700 001 Muac GSMUA Achr2P12440 001 Muac GSMUA Achr4P28620 001 Seit Si035879m Seit Si005314m Zema GRMZM2G115612 T01 Zema GRMZM2G417009 T01 Zema GRMZM2G062377 T01 Sobi Sb01g004520.1 Brdi Bradi1g04840.1 Orsa Os03g59070.1 Agam Aam092848 Agte Locus80774v1rpkm0.81 6 Agde Locus40684v1rpkm1.85 6 Agde Locus26967v1rpkm6.19 6 Agte Locus33848v1rpkm4.41 7 Agam Aam080231 Potr Potri.006G196200.1 Potr Potri.016G062100.1 Arth AT3G58490.1 Sotu PGSC0003DMP400002048 Semo 405511 Phpa Pp1s11 360V6.1 Phpa Pp1s22 422V6.1
68
99
75
89
95
64
89
94
9366
97
97
96
100
83
88
93
100
71
99
97
92
10099
99
Agam = Agave americana
Agte = A. tequilana
Agde = A. deserti
Arth = Arabidopsis thaliana
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Protein-protein Interaction (PPI):
A Machine Learning Approach
Random Forests Classification
Co- Expression
Gene- Ontology
Phylogenetic- Profiling
Domain- Similarity
Protein Protein
Interaction Score
SR-MCL
PPI networks
CAM-Gene Enrichment Analysis
CAM-related PPI networks
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PPI Prediction in Agave
PDB ID: 3UJG Aam048341:Aam083646
Homology Modeling by MOE2012
~4,000 most variable genes
(Expression among 15 samples)
PPI network
~700 clusters
49 cluster enriched with
CAM-associated genes
(~600 genes)
Random Forest
MCL Clustering
Functional
enrichment
Co-expression module: MMred
PPCK1 and its interaction partner
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Gene Regulatory Network
Computational prediction: Transcription factor (TF) - target gene
pair
Case study: Potential TF candidates for the phosphoenolpyruvate
carboxylase kinase 1 (PPCK1) gene
Three different methods:
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Case I: Diurnal Expression Patterns of
Photosynthetic Electron Transport Genes
The control of PET chain reaction plays pivotal roles in the regulation
of photosynthesis via coordination of energy state and metabolism.
Several PET genes displayed higher levels of expression in the night
compared to Arabidopsis
Agave
Arabidopsis
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Agave
Arabidopsis
Case II: Diurnal Expression Patterns of CAM
Modules Genes
(A) (B) (C)
Row z-score
Lower
expression
Higher
expression
Carboxylation Decarboxylation Stomatal movement
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Blue light Red/far red light Cry1
Cry1 Phot2
FKF2
ELF3
PIF3
CCA1
PhyB
TOC1
GI
CHE
APRR7
LUX
ZTL APRR5
CCR2
AMY3
DPE1
RVE1
PORA CDF2
GWD1
Agave
Arabidopsis
Case III: Circadian Clock
Row z-score
Lower
expression
Higher
expression
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Evolution of CAM-specific clock
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Summary
Identified co-expression modules associated with CAM features
and circadian clock
CAM vs. non-CAM (i.e. C3, C4, NVP) comparison revealed CAM-
specific genes/family expansion, providing new insights into the
evolutionary origin of CAM
PPI and GRN networks are under active development;
Experimental data are needed to confirm the computational
prediction
Future study: Co-expression + Promoter analysis (from Agave
genome sequence)
Note: The Agave genome (~4GB) is being sequenced at ORNL
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Working Hypotheses
Hypothesis I: The diversification of clock gene
family (i.e. CAM-specific clock gene(s)) drives
the CAM evolution.
Hypothesis II: The dynamics of photosynthetic
electron transport (PET) system in CAM is
different than that in C3.
Hypothesis III: Sphingolipid signaling plays an
important role in stomatal closure in CAM.
27 Managed by UT-Battelle for the U.S. Department of Energy Presentation_name
34th New Phytologist Symposium
“Systems Biology and Ecology of CAM Plants”
Organizing Committee
Xiaohan Yang (ORNL)
Anne Borland (Newcastle University and ORNL)
John Cushman (Un. of Nevada-Reno; local organizer)
Stan Wullschleger (ORNL),
Joseph Holtum (James Cook University)
James Hartwell (University of Liverpool)
Outcome
A Virtual Special Issue for the journal New Phytologist
A CAM roadmap focusing on hypotheses and questions to stimulate research in both basic (e.g. CAM photosynthetic system, evolution) and applied areas (e.g. biofuel, CAM engineering)
http://www.newphytologist.org/symposiums/view/5
Dream
Mission
Lake Tahoe, CA, USA; July 15th to 18th, 2014
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