Evolution of Plant Stress Responsiveness:Evolution of Plant Stress Responsiveness:Genome-wide and Gene Family Level AnalysisGenome-wide and Gene Family Level Analysis

Shin-Han ShiuDepartment of Plant Biology

KBS, 1/18, 2008

OutlineOutline

Major interests and why

Gene families and stress responsiveness: The interplay between gene family expansion, duplication

mechanism, and the elusive selection pressure

The Receptor Kinase family as an example One of the biggest plant gene families and their involvement

in plant biotic interactions

If there is enough time, the short story on plant pseudogenes When can you can a gene pseudogene?

Major interestsMajor interests

Molecular evolutionary

patterns

Genetic basisof adaptation

Source of selection pressure: abiotic and biotic stress conditions

Target of selection: duplicate genes

Where does all these duplicates come fromWhere does all these duplicates come from

+

Whole genome duplication

Tandem duplication

Segmental duplication

Replicative transposition

Plasticity of plant gene contentsPlasticity of plant gene contents

3 whole genome duplications in the Arabidopsis thaliana lineage over the past ~150 million years

More “recent” retentions in plants

*: Number of orthologous groups in shared families between Arabidopsis and rice.**: Number of genes in shared families.

120,000

Arabidopsisgene content:

21,000**

60,000

30,000

15,000*

Expected Observed

Shiu et al. (2006) PNAS

Plant Gene Family Evolution: Major questionsPlant Gene Family Evolution: Major questions

What is the rate of gene gains in plants?

Do certain types of genes have higher gain rate?

What is the influence of duplication mechanisms?

Finally, how does genes that are responsive to stresses behave?

AtGenExpress microarray dataset22 stress conditions

Measuring Lineage-specific GainMeasuring Lineage-specific Gain

Orthologous group and lineage-specific gain Reconcile species and gene trees

Retention rate along the Retention rate along the A. thaliana A. thaliana lineage lineage

Diminishing rate of retention over time

M R P A

5,774.5±72.8

Moss Rice PoplarArabidopsis

6,508.9±109.7

8,029.4±133.8

6,995

8,474

10,050

Tree-based

Similarity-basedA-M

A-R

A-P

Number of gene gains

700 MYA

200 MYA

100-120MYA

11.3 - 14.4

47.5 - 70.0

32.0 - 42.4

Gain rate(gain/MY)

1

2

3

1

2

3

1521-1576 3.0- 3.2

734-1479 9.2-18.5

5774-6995 48.1-58.3

Retained (R) Rate (R/My)

Expansion at the gene family level Expansion at the gene family level

Lineage-specific gains per family in one plant lineage are moderately correlated with gains in the other lineage.

y = 0.32xr 2 = 0.33

0

30

60

90

120

150

0 50 100 150 200 250 300

A. thaliana lineage-specific gains

cificeps-e

gaenil

sneta

p.P

ga

ins

LRR

Protein kinase

PPR

NB-ARCC1

PPR

P450

Protein kinase

UDPGT

LRR

Kinesin

AP2

Moss

Moss

Moss

1

1

4

3

4

1

At 9

Moss 5

Rensing et al. (2008) Science

E.g. a family with 3 OGs

Expansion at the orthologous group level Expansion at the orthologous group level

Enrichment:

Exp

Obs

N

Nlog

−0.7 0.7log2(Obs/Exp)

0

Sp.Branch

size

Moss

A. t.

2

3

Orthologousgroup

Moss

A. t.

log(OG size)

log

(fre

q)

Two major patterns in OG expansion Two major patterns in OG expansion

Convergent expansion Single lineage expansion

5

4

2

3

1

0

0 1 2 3 4 5 > 6

> 6

0 1 2 3 4 5

5

4

2

3

1

0

> 6

> 6

5

4

2

3

1

0

0 1 2 3 4 5

> 6

> 6

Arabidopsis thaliana Arabidopsis thalianaArabidopsis thaliana

sso

M

eciR

ral

p

−0.7 0.7log2(Obs/Exp)

0Enrichment:

Exp

Obs

N

Nlog

Expansion patterns and duplication mechanisms Expansion patterns and duplication mechanisms

Comparison of ratios between tandem and non-tandem genes e.g. for A-M orthology

Expansion patternOG type Method for defining OG

Convergent 1 Single-lineage2P values

3

Similarity 0.17 (756/4500) 0.30 (848/2918) 2.2×10-23

A-MTree 0.16 (831/5297) 0.40 (1443/3566) 9.4×10-88

Similarity 0.31 (959/3115) 0.47 (644/1375) 3.1×10-12

A-RTree 0.27 (844/3073) 0.50 (1631/3294) 2.3×10-33

Similarity 0.29 (1141/3944) 0.60 (741/1234) 7.2×10-38

A-PTree 0.26 (1014/3930) 0.64 (1578/2452) 1.0×10-83

Convergent Single-lineage

Tandem 756 848

Non-tandem 4500 2918

Ratio 0.17 0.30

Summary I Summary I

Duplicate gene turn over But even though some of them are retained for millions of

years, the majority of them will be lost over hundreds MY time scale.

The degree of lineage-specific expansion is similar at the family level but with substantial variation

Expansion patterns fall into two major categories Convergent expansion Single lineage expansion

Orthologous group with single lineage expansion Tend to be enriched in tandemly repeated genes

What's so special about tandem genesWhat's so special about tandem genes

Duplication rate (event per unit time):

Whole genome duplication: 1 event / ~50 million years

Tandem duplication: multiple events / generation

Rate of recombination

Recombination rate: Pathogen attack > control Lucht et al., 2002. Nature.

Recombination rate: Tandem > non-tandem Zhang & Gaut, 2003. Genome Res.

Gene family expansion and functional bias Gene family expansion and functional bias

Question: What types of genes tend to experience expansion? What is the influence of duplication mechanism?

Classification of genes: In OG without expansion In OG with expansion

Gene Ontology, controlled vocabulary describing Gene functions:

e.g. protein kinase, involved in attaching phosphates onto self or other proteins, serving as a molecular switch.

Biological processes involved e.g. serine/threonine phosphorylation, the process of attaching

phosphate onto amino acid ser or thr. Location within the cell

e.g. plasma membrane

Functional bias of gene retention Functional bias of gene retention

None

Moss Rice Poplar Arabidopsis

Plasma membraneLipase activityCopper ion-bindingResposes to ABAHypersensitive responseCell wall organization

Translation

Ribosome, Apoplast,Membrane,Cell wall, Cytosol,UDP-glycosyl transferase,Receptor activity,Ca2+ homeostasis,Enzyme inhibitor,Electon transport,Phosphorylation,Stress response

A-M

A-R

A-P

Stress response categories over-represented in the vascular plant lineage

Cellular component categories: T vs. NT Cellular component categories: T vs. NT

Tandem: Extracellular region, cell surface, endomembrane Non-tandem: cytosol, cytoskeleton, nucleus

Biological process categories: T vs. NT Biological process categories: T vs. NT

Tandem: kinases, glycosinolate transferase, toxin responses

Non-tandem: regulation & hormone metabolism

response tostimulus

cellularprocess

physiologicalprocess

defenseresponse

response tochemicalstimulus

response tobiotic stimulus

responseendogenousstimulus

response toabioticstimulus

responsestress

cellularphysiological

process

cellularmetabolism

transport

generationprecursor metand energy

toxinmetabolism

phosphorusmetabolism

metabolism

localization

secondarymetabolism

glycosinolatemetabolism

responsetoxin

response drug

response toother

organismresponse tobacterium

peptidetransport

drug transport

lipidtransport

establishmentlocalization

response toosmotic stress

regulationbiologicalprocess

regulationcellularprocess

cellcommunication

regulationcell physiolprocess

regulationcellular

metabolism

hormonemetabolism

regulationphysiological

process

regulationmetabolism

responsehormonestimulus

responseABA

stimulus

signal

Biological process categories: T vs. NT (contd.)Biological process categories: T vs. NT (contd.)

response tostimulus

cellularprocess

physiologicalprocess

defenseresponse

response tochemicalstimulus

response tobiotic stimulus

responseendogenousstimulus

response toabioticstimulus

responsestress

cellularphysiological

process

cellularmetabolism

transport

generationprecursor metand energy

toxinmetabolism

phosphorusmetabolism

metabolism

localization

secondarymetabolism

glycosinolatemetabolism

responsetoxin

response drug

response toother

organismresponse tobacterium

peptidetransport

drug transport

lipidtransport

establishmentlocalization

response toosmotic stress

regulationbiologicalprocess

regulationcellularprocess

cellcommunication

regulationcell physiolprocess

regulationcellular

metabolism

hormonemetabolism

regulationphysiological

process

regulationmetabolism

responsehormonestimulus

responseABA

stimulus

signal

Tandem: response to stimuli, various transport functions Non-tandem: cell-cell communication and hormone

response

Stress responsivenessStress responsiveness

Expression data set: Arabidopsis thaliana Under 22 abiotic and biotic stress conditions

Definition: stress responsiveness For a given gene

ET: Expression level under stress condition Ec: Expression level under mock treatment control

If ET >> Ec: Significant UP regulation

If ET << Ec: Significant DOWN regulation

Question: do stress responsive genes tend to be those that are gained throughout plant evolution?

Expansion of responsive genes and conditionsExpansion of responsive genes and conditions

Response Up regulation Down regulation

OG type A-M A-R A-P A-M A-R A-P

Statistical test Exp1 T/N2 Exp T/N Exp T/N Exp T/N Exp T/N Exp T/N

Abiotic stress conditions3

UV-B + T + T + T + N

Wounding + T + + + +

Cold4C + N + N + N

Heat + N + N

Drought + + T + + +

Salt + + +

Osmotic + + +

Biotic stress conditions3

AvrRpm1 + + +

DC3000 + + +

Flg22 + T + T + + +

GST-NPP1 + T + T + T

HrcC- + T + T + T

HrpZ + + +

P. infestans + T + T + T

Psph + T + T + T

Genes in expanded OGs tends be enriched in stress responsive genes

+: significant at the 5% level

Stress responsiveness and duplication Stress responsiveness and duplication mechanismsmechanisms

Response Up regulation Down regulation

OG type A-M A-R A-P A-M A-R A-P

Statistical test Exp1 T/N2 Exp T/N Exp T/N Exp T/N Exp T/N Exp T/N

Abiotic stress conditions3

UV-B + T + T + T + N

Wounding + T + + + +

Cold4C + N + N + N

Heat + N + N

Drought + + T + + +

Salt + + +

Osmotic + + +

Biotic stress conditions3

AvrRpm1 + + +

DC3000 + + +

Flg22 + T + T + + +

GST-NPP1 + T + T + T

HrcC- + T + T + T

HrpZ + + +

P. infestans + T + T + T

Psph + T + T + T

Enrichment of tandemly over non-tandemly expanded genes under biotic conditions

Significant at the 5% level

T: tandem >> non-tandemN: non-tandem >> tandem

Tandem genes tend to be “bioticly” responsive Tandem genes tend to be “bioticly” responsive

This does not mean biotic responsive genes tend to be tandem Among GO molecular function categories that are enriched in

genes respond to biotic stresses: Tandem >> non-tandem

Non-tandem >> tandem

DNA binding

transferaseglycosy

1MPRrva

UDP-glycosy

nucleic actranscription factortranscription regulator

kinase_activity

bindingion bindingmetal ion bindingtransition metal ion bindingcarbohydrate bindingoxidoreductase

0003CD

1PPN-TSG22glF

ZprH

snatsefni-Phps

Summary II Summary II

Over the course of plant evolution, retention rate: Stress response genes >> genome average

True for genes up-regulated in both biotic and abiotic stress conditions

Influence of duplication mechanism, particularly for biotic stress conditions, retention rate: Tandem >> non-tandem

However, genes responsive to biotic stimuli are not necessarily tandem Depend on their location in the signaling network e.g. Plant receptor kinase: biotic -> tandem e.g. Transcription factors -> non-tandem, presumably WGD

Receptor KinaseReceptor Kinase

Shiu & Bleecker (2001) Science’s STKE

ArabidopsisTransmembrane Kinase 1

Functional bias: the Receptor-Like Kinase familyFunctional bias: the Receptor-Like Kinase family

Shiu & Bleecker (2001) PNAS

The Kinase superfamilyThe Kinase superfamily

Family size differences imply differential expansion Kinase: >1000 in A. thaliana, >1600 in Oryza sativa RLK/Pelle: ~600 in At, ~1200 in Os

Animal homolog: Drsophila: Pelle Mammalian: IRAKs

Shiu et al. (2004) Plant Cell

Receptor kinase configurationReceptor kinase configuration

Chlamydomonasreinhardtii

Ostreococcustauri

Physcomitrellapatens (M)

Oryza sativa (O)

Arabidopsisthaliana (A)

Populustrichocarpa (P)

356

73

256 356

73

256

388148

462

376

159

911

453

187

1003

388148

462

388148

462

376

159

911

376

159

911

453

187

1003

453

187

1003

424

2

93

424

2

424

2

9393

RLKRLCKOther Kinases

RLKRLCKOther Kinases

ECD Kinase

Kinase

InnovationInnovation

LysM

GDPD

CHASE

LRR

LRR

LRR

Thaumatin

ThaumatinThaumatin

GH18

GH18

DUF26

DUF26DUF26

Functional bias: motivated by RLK studiesFunctional bias: motivated by RLK studies

Shiu et al., 2004 Plant Cell

Stress responsiveness of RLKs Stress responsiveness of RLKs

RLKs are more responsive to stress than genome average

Response Up regulation Down regulation

Statistical test RLK T/N RLK T/N

Abiotic stress conditionsUV-B O T O NWounding O O NDrought U naCold4C O NHeat USaltOsmotic O O N

Biotic stess conditionsFlg22 O T NGST-NPP1 O THrcC- O T OP.infestans O T OPsph O T O NHrpZ O T NAvrRpm1 O O NDC3000 O

Stress responsiveness of RLKs Stress responsiveness of RLKs

Tandem RLKs are more responsive to biotic stress than non-tandem RLKs

Response Up regulation Down regulation

Statistical test RLK T/N RLK T/N

Abiotic stress conditionsUV-B O T O NWounding O O NDrought U naCold4C O NHeat USaltOsmotic O O N

Biotic stess conditionsFlg22 O T NGST-NPP1 O THrcC- O T OP.infestans O T OPsph O T O NHrpZ O T NAvrRpm1 O O NDC3000 O

Stress responsiveness and tandem RLKsStress responsiveness and tandem RLKs

Responsiveness (R) of an RLK subfamily For subfamilies with ≥ 10 genes i: subfamily j: condition UP: # of up-regulated genes DN: # of down-regulated

j i

ji

j i

ji N

DNR

N

UPR or

The “RLK swarm” modelThe “RLK swarm” model

In the context of biotic stress signaling In the context of biotic stress signaling networksnetworks

T > NT

NT > T

NT > T

T > NT

Summary IIISummary III

Innovation in the RLK/Pelle family Most RK configuration established > 700 million ago. Plenty evidence of domain shuffling, but the rate is not high. Shuffled domains suggest involvement in biotic stress

perception.

History of expansion 4 major turnover patterns Substantially more recent gains in poplar and rice Mostly involved subfamilies with lots of tandem repeats

Stress responsiveness RLK > genome average Tandem > non-tandem Biotic > abiotic Stress responsive genes are not necessarily tandem

Plant pseudogenesPlant pseudogenes

Pseudogenes are: Genomic DNA sequences similar to normal genes but non-

functional

For protein coding genes, non-functional to many means: They have frameshift mutation or premature stop codons They are not transcribed into mRNA They exhibit signatures of neutral selection

Pseudogene numbers and family size Pseudogene numbers and family size

Gene family size is generally correlated with the number of pseudogenes in the family in question.

0 200 400 600 800

00

20

40

60

80

01

02

1

Domain family size

sene

go

dues

pf

ore

bm

uN

F-box

LRR_1

Pkinase_tyrPkinase

LRRNT-2

RRM1

Myb_DNA_binding

P450

PPRNB-ARC

Family size (S)1 Slope2 Spearman's rank (ρ) p-value

Overall 0.1247 0.5484 <2.2e-16

S < 10 0.0967 0.3000 <2.2e-16

10 ≤ S < 25 0.1259 0.2291 3.94e-5

25 ≤ S < 50 0.1950 0.2307 0.0209

50 ≤ S < 100 0.1650 0.3317 0.0152

S > 100 0.1177 0.6042 3.20e-4

Selection pressure on pseudogenes Selection pressure on pseudogenes

Pseudogenes still show signatures of purifying selection

Determine pseudogene expression Determine pseudogene expression

Tiling microarray Cover the whole genome, regardless of the annotation Can distinguish sense and antisense transcripts

Exon

UTR

Intron

Cis-regulatory elements

Novel genes

MAR (Matrix attachment regions)

Transcript array

Tiling array

Selection pressure on pseudogenes Selection pressure on pseudogenes

Pseudogenes still show signatures of purifying selection

A. Arabidopsis

B. Rice

Summary IVSummary IV

Relationships between gene family sizes and the numbers of pseudogenes Positively correlated Larger gene families tend to loss more frequently than

smaller families

Pseudogene still shows signature of purifying selection Mostly may due to the fact that pseudogenization event

occurred relatively recently

Pseudogenes are still expressed Significantly higher than intron antisense expression In rice, pseudogene expression is even as high as that

among presumably functional genes

AcknowledgementAcknowledgement

Lab members Melissa Lehti-Shiu Gaurav Moghe Cheng Zou

Past member Kosuke Hanada, RIKEN

Collaborators Jeff Conner Gregg Howe, PRL Rong Jin, CSE Doug Schemske Mike Thomashow, PRL

Funding:

Takk!Takk!

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