Agave Genomics in Support cambiodesign.org of CAM Engineering · CAM vs. non-CAM (i.e. C3, C4, NVP)...

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


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


Research Goals

Inform genetic improvement of Agave for

biomass production

Provide genomics information for creating

synthetic CAM machinery in C3 plants


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


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


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


Transcriptome Sequencing of A. americana

strand-specific library


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


Gene Co-expression Modules

Mature Leaf

Co

-ex

pre

ss

ion

mo

du

le ID

Software:WGCNA


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


Functional Enrichment in Co-expression

Modules: Gene Ontology (GO)

MMred MMsalmon


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


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)


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


Possible Stomata Control Gene

Coursol et al. 2003.

Nature 423:651-654

Sphingosine-1-phosphate phosphatase


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


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


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


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:


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


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


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


Evolution of CAM-specific clock


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


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.


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

Agave Genomics in Support cambiodesign.org of CAM Engineering · CAM vs. non-CAM (i.e. C3, C4, NVP)...

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