STRESS ON PLANTS UNIVERSITÉ DE LUXEMBOURG26 SEPTEMBRE 2013

Raquel Folgado Casado

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ENVIRONMENTAL STRESSThis is any factor extern to the plant, which has a negative influence on the developmentFor thousands of years of evolution, plants have developed defence mechanisms

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Response: Structural and functional changes produced in the plant against stress

1. Heritable changes that were included in the genome2. Transitional modifications, reversible. Transient phenotypic

expression

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Introduction

PLANT RESPONSE TO STRESS

Alarme Phase

• They decrease or stop their basic physiological functions. They reduce their vigor.

Resistance Phase

• Accommodation of cellular metabolism to the new conditions, activation of repair processes and appropriate expression of morphological adaptations.

• It reaches a new optimal physiological state for the new conditions

Depletion Phase

• If the stressful situation is maintained for long and the plant stops its functions

Regeneration Phase

• The stress disappears and the plant reaches an optimum physiological state again

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Stress/ response cycles are routine events in the life of a plant

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Introduction

THE STRESS FACTORSThe biotic factors

• large and small animals, • other plants, • insects, • bacteria, fungi, viruses • nematodes.

The abiotic factors

• Drought: hydric stress• Salt stress: excess of salts in the soil• Thermic stress: heat, cold and freezing temperatures• Anaerobic stress: ponding and flooding• Stress by environmental pollutants: SO2, herbicides, metals, ozone• Nutritional stress: deficiency in minerals• Mechanical stress: wind, soil compaction• Stress by injuries or wounds

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Introduction

THE ROUTE OF STRESS SIGNAL

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Perception of plant stressor. The external

stimulus must be transformed into an internal

signal

Signal processing.

Amplification, integration into

the transmission

routes

Regulation of gene

expression

Protein products that enable stress

tolerance

Source : Hirayama & Shinozaki (2010)

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Introduction

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Source : Spinelli et al(2011)

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Introduction

BIOTIC STRESS

The entrance of the pathogen to the host plant is linked to the secretion of enzymes: cutinases, cellulases, pectinases and proteases.

The plant actives enzymes which strengthen cell wall, synthesizes phytoalexins (protease inhibitors) and activates the selective cell death of some cells.

Biotic Stress

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Biotic factors: large and small animals, other plants, insects, bacteria, fungi, viruses and nematodes.

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ABIOTIC STRESS

Water stress (drought)Salt stress: excess of salts in the soilThermic stress: heat, cold and freezing temperaturesAnaerobic stress: ponding and floodingStress by environmental pollutants: SO2, herbicides, metals, ozoneNutritional stress: deficiency in mineralsMechanical stress: wind, soil compactionStress by injuries or wounds

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Abiotic Stress

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WATER STRESS

The plant and its water environment.

The absorbed water from the soil by the roots is carried to all parts of the plant. Part is removed to the air through transpiration.

Abiotic Stress: Hydric Stress, Osmotic Stress

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To the right are rated values of water potential (Megapascals, MPa) of different partners

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WHEN IS THE PLANT IN WATER DEFICIT?Water deficit: the amount of transpired water is greater than the amount of absorbed waterThe reactions of plants to drought are dependent on:

• Speed of water evaporation• Duration of water deficit• Species (also variety, genotype)

At cell level, reactions are function of • Organ • Cell type• Developmental state of the plant

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Abiotic Stress: Hydric Stress, Osmotic Stress

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WATER IS NECESSARY FOR:

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• The photosynthesis: primary donor of

electrons

• The growth

• The turgor

• The movements

• The refreshment by evapotranspiration

• The absorption and the transport of solutes

Abiotic Stress: Hydric Stress, Osmotic Stress

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WATER STRESS, OSMOTIC STRESS

Drought stressSalt stress

Cold-freezing stress

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Common responses Specific responses

Abiotic Stress: Hydric Stress, Osmotic Stress

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SALINITY6 % of the earth surface is affected by sality (about 20% of watered cultures)Natural salinity

• Sea water:• Na+ : 10 g/kg 470 mM• Cl- : 20 g/kg 550 mM

Salinity induced by the agriculture

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Abiotic Stress: Hydric Stress, Osmotic Stress

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SALINITYPlants generally do not use neither the Na+ nor the Cl-Salinity-> hyper-osmolarity and ionic toxicity

GlycophytesHalophytes :

• need more electrolytes for optimal growth ([NaCl] soil: 20 to 500 mM), used as osmoticum

• extrusion of Na + (specialized cells)• vacuolar compartmentalization• transport to young aerial parts (limitation of Na + to the

root level)

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Abiotic Stress: Hydric Stress, Osmotic Stress

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COLD

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Membrane protection is essential

Abiotic Stress: Hydric Stress, Osmotic Stress

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THE WATER PASSES THROUGH THE ROOT BY THREE ROUTES:

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Abiotic Stress: Hydric Stress, Osmotic Stress

(a) Via transcellular = through the cell membrane (b) Via symplaste = From cell to cell through plasmodesms(c)Via apoplaste = Between the cells or through death cells

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RESPONSES TO WATER STRESS: NON ADAPTED PLANTS

Cell expansionProtein synthesisNitrate reductase

activityIncrease in ABA

Decreased cytokininStomatal closure

Decrease in photosynthesis

Decrease in respirationWilting

Senescence

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Diminution of water potential into the soil

Abiotic Stress: Hydric Stress, Osmotic Stress

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THE STOMATA

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Headquarters of gas exchange (O2, CO2) and place of transpiration(evaporation of water in the form of vapour).Transpiration: open stomata, CO2 fixation from atmosphere (dissolved form, for photosynthesis)

Closure of the stomata pendant hydric stress

Abiotic Stress: Hydric Stress, Osmotic Stress

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RESPONSES TO WATER STRESS: ADAPTED PLANTS (XEROPHYTES)

Anatomic and morphologic adaptations

• Root system at the soil surface• Deep root system• Water accumulation• Reduction of leave surface

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Abiotic Stress: Hydric Stress, Osmotic Stress

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Abiotic Stress: Hydric Stress, Osmotic Stress

Metabolic adaptations• Photosynthesis (crassulacean acid metabolism; CAM)• Biosynthesis of protective compounds (osmotic, structural)• Establishment of detoxification systems (reactive oxygen

species, ROS)• Repair systems

RESPONSES TO WATER STRESS: ADAPTED PLANTS (XEROPHYTES)

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Abiotic Stress: Hydric Stress, Osmotic Stress

CAM PLANTS:

Modification of C4-metabolism: CO2 capture and photosynthesis are separated in time and spaceNight:• Open stomata

• CO2 absorption

• CO2 fixation to a 3C-compound (pyruvate) to form another 4C-compound (oxalacetate and then malate)

• Malate is accumulate into the vacuoles during the night

Day:• The stomata close (to limit the loss of water)

• Malate is converted into a 3C-compound (pyruvate) and CO2 (Calvin C.)

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THERMIC STRESSDegree of saturation of the membrane lipids

• Plants that are adapted to heat increase the % of saturated fat acids in the glycerolipids

• Plants that are adapted to cold increase the % of insaturated fat acids

Accumulation of anti-freeze proteins (AFP)

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Abiotic Stress: Thermic Stress

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ANAEROBIC STRESSHypoxia-anoxia: roots into inundated soils

• Krebs cycle and respiration are blocked• ATP production stops• Active transport of protons into the vacuole is inhibited• cytoplasm is acidified• Roots metabolism stops

The aerenchyma is extended from the leaves to the roots in plants adapted to the absence of oxygen (ethylene)

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Abiotic Stress: Anaerobic Stress

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THE POLLUTANTS Xenobiotics: industrial or agricultural activities

• Acid rain Les pluies acides• Increase of ozone concentration in the tropasphere • Metals: Mn, Fe, Zn, Cu, Al, Cd, Hg, Ni, Pb (they affect the

root growth and formation). Cd and Pb can also enter into the alimentary chain

The phytochelatines (PC) join to metals and they are stored into the vacuoles

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Abiotic Stress: Les pollutants

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GENERAL RESPONSES TO THE STRESSIncreased levels of phytohormones• The induction of the synthesis may be due to changes in the volume

and cell turgor

Reduction of leaf growth and stomatal closure.• The ABA (Abscisic acid) is transported through the xylem of the aerial

part of the plant

Ethylene biosynthesis• Senescence

Synthesis of antioxidants

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GENERAL RESPONSES TO THE STRESS

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STRESS RESPONSES: PROTEINS

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ProteinsProteomicsWhat makes it

happen?

MetabolitesMetabolomics

What happen?

GenesGenomics

What is possible?

mRNATranscriptomics

What seems to happen?

OMICs

Gene expressionmRNA

DNA sequencingMutations

PolymorphismEpigenomics

Small moleculesMetabolites

IdentificationExpression

LC-MS driven

Phenotype/Cellular Response

Enzyme modulation

INTRODUCTION

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Study of abiotic stress

29Dep. Environment and Agro-biotechnologies (EVA), CRP- GL, Luxembourg.Laboratory of Tropical Crop Improvement, KU Leuven, Belgium.

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ABIOTIC STRESS

AND ITS RELEVANCE

IN CRYOPRESERVATION:

POTATO, A CASE OF STUDY

Cortesia Paolo_EFE

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THE POTATO

-Rich in carbohydrates- Source of minerals and

vitamins

- Enormous genetic diversity: breeding potential

• Solanaceae Family• Solanum spp.

-Herbaceous annual -Originated in Andean Region-One of the most important crops

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Variability in temperature and precipitationMicroclimatic regions

THE ORIGIN OF POTATO DIVERSITY

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PLANT MATERIAL

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Solanum commersonii Solanum tuberosum cv Désirée

EXPERIMENTAL SETUP

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Cryoprocedure: Droplet-Vitrification

DEHYDRATION

CRYOPROTECTION

FREEZING-STRESS

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Differentiate Survival of Tip, Tip Growth and Plant RecoveryS. commersonii S. tuberosum cv

Désirée

Survival

Tip growth

Recovery

Metabolite analysis (e.g. carbohydrates,)

Proteomic analyses for different

stressors

Different approaches have been used during this experiment to provide a wider picture of what happened to the plant

Phenotyping plants

Control Stress

Study of abiotic stress in potatoINTRODUCTION

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HPAEC-PAD (High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection)

Advantages:1. It allows the direct quantification of carbohydrates at pmol levels2. It provides highly selective separations

CHs targeted in this study:

METABOLOMIC APPROACH

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 33.0-20

0

20

40

60

80

100

120

140 120116STRESSPOT #25 [modif ied by User, 4 peaks manually assigned] S-3 14.1 1/10 IntAmp_1nC

min

1 -

Gal

acto

se -

6.5

17

2 -

Glu

cose

- 7

.142

3 -

Suc

rose

- 7

.684

4 -

Xyl

ose

- 8.

175

5 -

Fruc

tose

- 9

.550

6 -

Mel

ibio

se -

11.

792

7 -

Raf

finos

e -

14.2

92

8 -

16.9

679

- S

tach

yose

- 1

7.39

2

Concentration: 9.50 mM

100.00

9.50

GalactoseGlucose

Sucrose

Xylose

Fructose Raffinose

Stachyose Cellobiose

MaltoseRhamnoseArabinose

Melibiose

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PROTEOMIC APPROACHDIGE experiment

Proteins

Proteins are involved in almost all cellular processes and fulfil many functions

PROTEOMICS

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The study of proteins

“the study of the proteome” may be defined as a large-scale analysis of the properties of all the proteins expressed by the genome of cells or tissues, at one time point (dynamics).

TissuesOrgans

Organism

What is proteomics? PROTEOMICS

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Subcellular / cellular level

High through-put2D Gel

nano-LC

The whole proteome

Define the proteome of a cell or tissue

Control modified

Selected proteins

Provide means of comparing proteomes (e.g. stressed vs normal states)

defining co-stimulated and co-regulated proteins

Multiprotein complexes affinity purification

Relevant proteins

global or.. functional proteomicsPROTEOMICS

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First dimension

Second dimension

PROTEOMICSSeparation proteins

Gel electrophoresis: 1 or 2 dimensions for the separation; on 2D gels, one can see isoforms from the same protein family

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Protein extract 1Label with fluor 1

Protein extract 3Label with fluor 3

Mix labeled extracts

Excitation wavelength 1

Image analysis: Overlay images

Excitation wavelength 2

Excitation wavelength 3

Separate by 2D PAGE

Image for each fluor

Protein extract 2Label with fluor 2

DiGE technology PROTEOMICS

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Advantages:1. Multiplexing2. Use of an internal standard for

all proteins which can be run on all gels in a set of experiments.

Sucrose

Glucose Fructose

Raffinose

Stachyose

Oxidative homeostasis-related prots

77% recovery

35%recovery

8%recovery

S. commersoniiSucrose

+

+

+

Sucrose

Glucose Fructose

Raffinose

Stachyose

Oxidative homeostasis-related prots

29% recovery

DésiréeSucrose

+

+

Sucrose

Glucose Fructose

Raffinose

Stachyose

Oxidative homeostasis-related prots

72% recovery

S. commersoniiCold

+

-

+Sucrose

Glucose Fructose

Raffinose

Stachyose

Oxidative homeostasis-related prots

13% recovery

DésiréeCold

- -

+

DésiréeControl

S. commersoniiControl

+