Flooding tolerance: escape or quiescence

Rens Voesenek Department of Plant Ecophysiology Utrecht University, The Netherlands http://www.bio.uu.nl/ecophysiology/

Flooding occurs world-wide and the numbers increase

www.dartmouth.edu/~floods/

Flooding is a major environmental stress for most plants

Consequences: Slow diffusion of gases Reduced levels of O2 and CO2

Problems: Energy crisis Carbohydrate starvation Reactive oxygen species Toxicity Water status

Contents
Sensing of low oxygen levels in plants Two Rumex species: an ecological model in flooding research Regulation of fast petiole growth to escape from submergence

An unrooted phylogenetic tree of Arabidopsis ERF proteins

Nakano, T., et al. Plant Physiol. 2006;140:411-432

Copyright 2006 American Society of Plant Biologists

Overexpression of RAP2.12 results in increased submergence survival and enhanced expression of hypoxia core genes

Air

Sub De-sub

Removal of 13 amino acids at the N terminal side of RAP2.12 affects expression of hypoxia marker genes and submergence survival

Air

Sub

De-sub

Localisation of RAP2.12 is affected by hypoxia and N terminal manipulation

Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilisation O2 %

Normoxia
MCRAP2.12

Anoxia

Methionine aminopeptidase

O2
C*RAP2.12

C-

RAP2.12

Hypoxia responsive genes

Arginine transferases Arg-C*RAP2.12

PROTEOLYSIS6 Ubq Arg-C*RAP2.12

26S proteasome

Oxygen sensing in plants mediated by an N-end rule pathway for protein destabilisation

Contents
Sensing of low oxygen levels in plants Two Rumex species: an ecological model in flooding research Regulation of fast petiole growth to escape from submergence

Rumex species are exposed to different flooding intensities and show contrasting elongation responses upon submergence

# floods/duration floods (d)

Duration Floods (d)

# floods Ra Rp Ra Rp

Rumex species exploit two different survival strategies upon flooding

Ordination diagram of vegetations in flood-prone river areas

Deep floods

Transient-deep floods

Rarely flooded

Long-shallow floods
Shallow floods

Short floods

Long floods
Voesenek et al., 2004. Ecology 85: 16-27

Ethylene accumulates rapidly in submerged Rumex

Submerged petioles of R. palustris and R. acetosa do not have low oxygen concentrations
R. palustris
25

Oxygen concentration in kPa

20

15

Light

10

Dark
0 200 400 600 800 1000

Inside distance from adaxial petiole side in m

R. acetosa
22

Oxygen concentration (kPa)

20 18 16 14 12 10 8 6 4 2 0 200 400 600 800 1000 1200

Light

Dark

inside distance from adaxial petiole side

Shoot emergence is not functional in R.acetosa due to low porosity

Contents
Sensing of low oxygen levels in plants Two Rumex species: an ecological model in flooding research Regulation of fast petiole growth to escape from submergence

Rumex palustris

Ethylene down-regulates ABA in R. palustris but not in R. acetosa

Rumex acetosa

Benschop et al. (2005) Plant Journal 44: 756-768

Auxin stimulates petiole elongation upon submergence

[IAA] pg/mg FW

Adaxial

Abaxial Time after submergence (h)

R. palustris
Petiole elongation (mm/48 h)
50 40 30

GA is required for submergence-induced petiole elongation

20 10

Control
25

c s

S S + GA Paclobutrazol

GA1 concentration (ng/gDW)

R. palustris
20

GA1 (ng/g DW) after 24 h

15

Submerged

Air C2H4 C2H4 Air

10

Control

0 0 5 10 15 20 25 30 35 40 45 50

Time after submergence (h)

Expansin transcription and protein abundance increases upon submergence

R. palustris

R. acetosa

Summary
Submergence

Auxin

Ethylene

NCED

Cell wall acidification ABA RpEXPA1 RpGA3ox1

GA Expansin proteins

Petiole elongation

Transcriptome analysis using RNA seq technology

air & submerged petioles R. palustris R. acetosa petioles: 4h air, 4h submerged petioles: 10h air, 10h submerged

normalized cDNA library

non-normalized cDNA library

454 sequencing (450 bp) & de novo assembly

Illumina/solexa sequencing (75bp)

Reference transcriptome
(blast against Arabidopsis for annotation)

air map solexa reads

submerged

air

submerged

R. acetosa

R. palustris

Identified gene families in both species (469 are differentially expressed)

3,116

11,036

2,992

P-value = 0.01, |FC|>1

R. acetosa R. acetosa

39

33

42

10 116
R. palustris 97

11

68 R. palustris

48

R. acetosa 39 75 10 73 R. palustris 48

R. acetosa 16

158
R. palustris

97

Log2(CPM)/kb (counts per million per kb)

Log2(Fold Change)

R. acetosa
Air Sub

R. palustris
Air Sub
R. acetosa R.palustris

Fuzzy K clustering of transcript abundance of 469 DEGs

Log fold change
R.ace
I

R.pal
2.49 2.85
3.78 4.90

XTH 33 ATEXLA3
KIDARI Peroxidase 32

0.54 0.98
-0.55 0.80

II

II

III IV

III

Ammonium Tr.2

-0.36

-2.16

IV V

AP2 trans.factor Lipoxygenase1 RAP2.12 XTH 7 EIN4 HB1 NCED4

NA NA 0.69 1.42 1.61 -0.33 -0.86

2.41 1.92 1.75 3.71 1.94 2.48 -2.13

V
VI

VI VII VIII IX X
VII

VIII

Alpha-amylase1

3.73

NA

IX X

GA2OX ATEXPB1

-2.29 NA

-2.78 -2.03

Gene families with a significant species X treatment interaction (P<0.01)

P. trichocarpa M. truncatula B. bistachyon
** * ** *

R. communis

A. thaliana

R. acetosa R. palustris

name basic helix-loop-helix (bHLH) dehydration responsive protein (RD22) peroxidase no hit MAPKK HSP/Chaperone SAM-dependent methyltransferase transposon protein bHLH/kidari peroxidase GTP binding (RHD) protein kinase AUX/IAA protein kinase (WAG1) lipid transfer protein/protease inhibitor zinc finger (COP1) germin no hit NAD+ ADP-ribosyltransferase (SRO5) non symbiotic haemoglobin chitinase 2OG-Fe(II) oxygenase subtilase SnRK1 beta subunit endopeptidase inhibitor unknown dehydrogenase/reductase unknown protein MYB (59) lipoxygenase glutathion S-transferase dehydration responvive related glutathion S-transferase haloacid dehalogenase hydrolase glycoside hydrolase/chitinase WD40 beta propeller sulfotransferase cytochrome P450 aspartyl protease unknown protein no hit UDP-glycosyltransferase pectin methylesterase inhibitor WRKY (54) annexin basic helix-loop-helix (bHLH)

*** *** ** * *** *** **

*** *** *** * ** *** * *** ** *

*** ** *** *** * ** *** *** ** *** *** *** *** *** *** ** *** *

** ** ** * *** *** *** *** *** *** *** *** ** ** ** *** ** ** ** *** ** *** ** ** *** *** *** *** *** * * ** ** ** ** ** *** *** ***

** ** * * *** *** *** *** * * ** *** * *** *** *** *** ** ** * ** ** ** ** ** ** ** * * * ** * * * *** *** *** *** *** *** *** *** *** ** ** ** * ** ** *** * ** ** ** * *** *** *** *** *** *** *** * *** *** *** *** *** *** *** *** * ** *** *** ** ** ** ** ** ** * ** *** ** *** *** ** * ** * ** ** ** ** * *** * * * *

** *** *** *** * ** *** ***

O. sativa

P. trichocarpa

M. truncatula

B. bistachyon

R. communis

A. thaliana

R. acetosa R. palustris

Per64 Per32 Hb1 COP1

name basic helix-loop-helix (bHLH) dehydration responsive protein (RD22) peroxidase no hit MAPKK HSP/Chaperone SAM-dependent methyltransferase transposon protein bHLH/kidari peroxidase GTP binding (RHD) protein kinase AUX/IAA protein kinase (WAG1) lipid transfer protein/protease inhibitor zinc finger (COP1) germin no hit NAD+ ADP-ribosyltransferase (SRO5) non symbiotic haemoglobin chitinase 2OG-Fe(II) oxygenase subtilase SnRK1 beta subunit endopeptidase inhibitor unknown dehydrogenase/reductase unknown protein MYB (59) lipoxygenase glutathion S-transferase dehydration responvive related glutathion S-transferase haloacid dehalogenase hydrolase glycoside hydrolase/chitinase WD40 beta propeller sulfotransferase cytochrome P450 aspartyl protease unknown protein WAG1 no KDR SHY2 hit UDP-glycosyltransferase pectin methylesterase inhibitor WRKY (54)

*** *** ** * *** *** **

*** *** *** * ** *** * *** ** ** *

*** ** *** *** * ** *** *** ** *** *** *** *** *** *** ** *** *

** ** ** * *** *** *** *** *** *** *** *** ** ** ** *** ** ** ** *** ** *** ** ** *** *** *** *** *** * * ** ** ** ** ** *** *** *** *

** ** * * *** *** *** *** * * ** *** * *** *** *** *** ** ** * ** ** ** ** ** ** ** * * * ** * * * *** *** *** *** *** *** *** *** *** ** ** ** * ** ** *** * ** ** ** * *** *** *** *** *** *** *** * *** *** *** *** *** *** *** *** * ** *** *** ** ** ** ** ** ** * ** *** ** *** *** ** * ** * ** ** * *** * ** ** * * * *

O. sativa

KIDARI and COP1: regulators of underwater shoot elongation?

Light signals (R, B)

Degradation

COP1

HFR1 HY5 LAF1

KIDARI

Light phenotype (non-elongated)

Major findings:
1. Rumex palustris and R. acetosa have different field locations characterized by contrasting flooding regimes.
2. R. palustris is characterized by ethylene-mediated petiole elongation upon submergence, R. acetosa by ethylene-driven inhibition of elongation. 3. Regulation of endogenous ABA plays an important role in the contrasting elongation responses of the two studied Rumex species. 4. RNA seq transcriptome analysis revealed approx. 14,000 gene families in both species. 5. In R. acetosa 140 families are differentially expressed upon submergence in light, 376 in R. palustris. 6. RNA seq results confirm important roles of ABA, auxin and cell walls in submergenceinduced petiole elongation. 7. Important novel genes are discovered that demonstrate very strong expression differences between the two species.

Acknowledgements
Francesco Licausi Pierdomenico Perata Joost van Dongen Julia Bailey-Serres Eric Visser Rashmi Sasidharan Ronald Pierik Dick Vreugdenhil Ole Pedersen Hans van Veen Divya Vashist Robert Vreeburg Joris Benschop Martijn van Zanten Ankie Ammerlaan Rob Welschen

Conference ISPA 7-11 October 2013 IRRI, Los Baos

Expression of Rumex orthologues of RAP2.12 correlates with flooding tolerance

Licausi et al., 2011, Nature

The Methionine-Cysteine amino acid sequence at the N-terminal part of RAP2.12 qualifies it as a candidate substrate of the N-end rule pathway

Methionine aminopeptidase

Arginine transferase

Proteolysis 6

 Ethylene stimulates petiole elongation in R. palustris and inhibits it in R. acetosa Petiole elongation is caused by cell elongation and is equally distributed over the petiole

Petiole length (% of air control)

R.palustris R.acetosa

 isotigs with < 50 reads were discarded and isotigs with < 30 reads per kb were discarded significant regulation P < 0.001