Survivorship in potted Populus deltoides x Populus nigra hybrids in response to gradual soil water depletion

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Survivorship in potted Populus deltoides x Populus nigra hybrids in response to gradual soil water depletion

Tete Severien Barigah, Marie Douris, Marc Bonhomme, Eric Badel, Régis Fichot, Franck Brignolas, Hervé Cochard

To cite this version:

Tete Severien Barigah, Marie Douris, Marc Bonhomme, Eric Badel, Régis Fichot, et al.. Survivorship in potted Populus deltoides x Populus nigra hybrids in response to gradual soil water depletion. Colloque du groupe Xylème, Institut National de la Recherche Agronomique (INRA). FRA., Apr 2011, Nancy, France. 18 diapos. �hal-00964541�

1

Survivorship in potted Populus deltoides

x Populus nigra hybrids in response to

gradual soil water depletion

Barigah TS

, Douris M

, Bonhomme M

, Badel

, Fichot R

,

Brignolas F

, Cochard H

1_{INRA, UMR547 PIAF, F-63100 Clermont-Ferrand, France}

2_{Université Blaise Pascal, UMR547 PIAF, F-63177 Aubière, France}

3_{Université d’Orléans, UFR-Faculté des Sciences, UPRES EA 1207 Laboratoire de}

Biologie des Ligneux et des Grandes Cultures (LBLGC), BP 6759, F-45067, France

4_{INRA, USC2030 Arbres et Réponses aux Contraintes Hydrique et}

Environnementales (ARCHE), BP 6759, F-45067, France

5_{INRA UR588 Amélioration, Génétique et Physiologie Forestières (AGPF), Centre}

Introduction

™ Recent climate projections Æ increase

in frequency and duration of intense

summer droughts

(Badeau et al., 2005;

Salinger et al., 2005; IPCC, 2007).

™ Studies on drought-induced plant

mortality and survivorship have

rekindled interest in foresters and

scientists’ communities.

™

Ψ

(a proxy of cavitation resistance)

was regarded as a bio-sensing device

(gauge) for drought sensitivity

detection in trees (Cochard et al. 2005)

™

Ψ

was reported to vary consistently

with the reputed habitat of tree species

(Hacke et al. 2000; Pockman and Sperry

2000; Brodribb and Cochard 2009)

™ But the effectiveness of Ψ

as a gauge

is not demonstrated yet!

Xylem pressure inducing 50% cavitation, MPa -8 -7 -6 -5 -4 -3 -2 -1 0 Buxus sempervirens Taxus baccata Crataegus monogyna Prunus spinosa Amelanchier ovalis Pinus Halepensis Quercus ilex Lonicera etrusca Quercus suber Pinus corsicana Cedrus atlantica Euonymus europaeus Carpinus betulus Pinus mugho Abies alba Pinus pinaster Picea abies Cytisus scoparius Pseudotsuga Quercus petraea Pinus cembra Pinus sylvestris Fagus sylvatica Populus nigra Fraxinus excelsior Quercus robur Pinus nigra Populus tremula Quercus rubra Betula pendula Salix caprea Juglans regia Salix caprea Alnus glutinosa Populus alba Populus trichocarpa Salix fragilis Populus euphratica Hygrophilous Mesophilous Xerophilous

The objectives of this study were to:

™ test and interpret the differences in response

to protracted summer drought of the chosen

unrelated poplar hybrids mainly

if the hybrid

with the lowest

Ψ

was the most resistant to

drought-induced cavitation,

™ link cutting’s leaf water potential and their

hydraulic features to volumetric soil moisture

content,

™ check for whether xylem dysfunction leads to

plant mortality.

Populus deltoides Bartr. ex Marsh x Populus nigra L.

unrelated hybrids

• Eco 28 (Ψ

=-2.41 MPa)

• I45-51 (Ψ

=-1.69 MPa)

• Robusta (Ψ

=-1.60 MPa)

(Fichot et al., 2009; Fichot et al., 2010)

• 3x50 current year cuttings of

Populus deltoïdes x Populus nigra hybrids fed in 20-liter pot each

• 3x8 harvested sprouts per week

• Relative radiation: 80% of full

sunlight

• Temperature: 15 - 30°C

• Relative humidity: 40-70%

• Daily drip irrigation for control

plants and water shortage for the others.

•

Time domain reflectometer

(Soil moisture content)

•

Pressure chamber

(Leaf and xylem water potential)

•

Xylem Embolism Meter

(Xylem native steady-state embolism)

•

Cavitron

(Water potential inducing 50 %

loss of conductance)

•

Microcalorimeter

(Bud respiration rates)

Microscope 0 r 0.51 Light Réservoir Amont Réservoir Aval Microscope Pression négative de sève P= -0.5 ρ ω2R2 Conductance du segment : K= (dr/dt) / 0.5 ρ ω2_[R2 _{– (R-r)}2_] Utilisation de la force centrifuge

(Cochard 2002, 2005)

• It comes as an evidence

that water shortage inhibits

growth in plant size

• Growth in height tended to

be the lowest in I45-51 but

the highest in diameter for

control plants while for

drought-treated plants,

Robusta tended to display

the highest growth in height

but did not differ in

diameter from others.

Growth in height for three Poplar hybrids versus dry-down span

Dry-down span, Week

0 1 2 3 4 5 6 7 H e ig h t, c m 25 50 75 100 125

Dry-down span, Week

0 1 2 3 4 5 6 7 D iam et er , m m 2 4.5 6.0 7.5 9.0 10.5

Droughted plants (Robusta) Control (Robusta) Droughted plants (Eco 28) Control (Eco 28) Droughted plants (I45-51) Control (I45-51)

Growth in diameter for three Poplar hybrids versus dry-down span

Droughted plants (Robusta) Control (Robusta) Droughted plants (Eco 28) Control (Eco 28) Droughted plants (I45-51) Control (I45-51)

•

Steep drop in volumetric soil

moisture content (VSMC): more

than 50% loss over 2 weeks

•

10% of maximum VSMC threshold

appeared to be critical.

Variation in predawn leaf water potential of Poplar hybrids versus dry-down span

Dry-down span, Week

0 1 2 3 4 5 6 7 P red aw n w a te r p o ten ti al, M P a -2.5 -2.0 -1.5 -1.0 -0.5 0.0 Eco28 y=-0.1957-0.1334*x-0.0293*x2; r2=0.9910, P=0.09 I45-51 y=-0.1941-0.1493*x-0.0111*x2; r2=0.9672, P=0.18 Robusta y=-0.2371-0.2201*x-0.0008*x2; r2=0.9999, P=0.01

Dynamic of volumetric soil moisture content within the pots of the Poplar hybrids versus dry-down span

Dry-down span, Week

0 2 4 6 8 Volume tric s o il m o is tur e c ont ent , % 0 10 20 30 40 Eco 28 I45-51 Robusta

•

Fast drop in

Ψp for Eco28

in comparison with the

other 2 hybrids.

Percent loss

conductivity (PLC)

increased sigmoid-like

along with decreasing in

volumetric soil moisture

content for all three

hybrids

Relationships between percent loss conductivity and volumetric soil moisture content versus dry-down span

0 20 40 60 80 100 120 0 10 20 30 40 PLC VSMC P e rc e nt l os s c onduc ti v it y , % 0 20 40 60 80 100 V o lu m e tri c s o il m o is tu re c o n te n t (V S M C ), % 0 10 20 30 PLC VSMC

Dry-down span, Week

0 2 4 6 8 0 20 40 60 80 100 0 10 20 30 PLC VSMC Eco 28 I45-51 Robusta

Over time, percent loss

conductivity increased

while predawn leaf water

potential dropped for all 3

hybrids.

Relationships between percent loss conductivity and predawn leaf water potential versus dry-down span

0 20 40 60 80 100 120 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 PLC Ψp P e rcent l o ss co nd uct iv it y , % 0 20 40 60 80 100 P redaw n l eaf w a te r p o te nt ia l, M P a -2.5 -2.0 -1.5 -1.0 -0.5 PLC Ψp

Dry-down span, Week

0 2 4 6 8 0 20 40 60 80 100 -2.5 -2.0 -1.5 -1.0 -0.5 PLC Ψp Eco 28 I45-51 Robusta

• Since drought inception

mortality occurred in 5

weeks in Eco 28 hybrids

but 2 weeks later in the 2

others

• None of Eco 28 individuals

survived 7 weeks after

drought inception roughly

when

Ψ

got below -1.0

MPa

•

Therefore, survivorship

was threatened the most in

Eco 28 hybrids

Relationships between percent loss conductivity and survivorship of Poplar hybrids versus dry-down span

0 20 40 60 80 100 0 20 40 60 80 100 -2.0 -1.5 -1.0 -0.5 0.0 S u rv iv o rs h ip , % 0 20 40 60 80 100 Per cent l o ss conduct iv it y , % 0 20 40 60 80 100 Pr edaw n l e af w a te r pot ent ial , M P a -2.0 -1.5 -1.0 -0.5 0.0

Dry-down span, Week

0 2 4 6 8 0 20 40 60 80 100 0 20 40 60 80 100 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 Survivorship PLC Ψp

• Bud respiration

rate for Eco28

control sprouts

was the highest in

comparison with

others

• The respiration

rate dropped to nil

only in Eco28

hybrids by week 8.

Bud respiration rate in 3 Poplar hybrids versus dry-down span

Dry-down span, weeks

0 2 4 6 8 10 Bud res p iration ra te , nmol g MS -1 s -1 0 5 10 15 20 25 30 Eco28 y=26.5431-2.1145*x-0.1462*x2; r2=0.9864, P=0.002 I45-51 y=21.8843-2.0644*x+0.0001*x2; r2=0.8495, P=0.06 Robusta y=17.1751-1.7685*x+0.0186*x2; r2=0.8277, P=0.07

14

Conclusion

Sprouts of Eco28 were the most

sensitive to the gradual soil

moisture depletion whatever the

morphological or the physiological

parameter we considered.

Therefore, we concluded that

using

Ψ

as a gauge to stand for

drought resistance in Poplar

hybrids does not hold.

However, we still believe that

Ψ

is relevant enough to sort out

samples of different species …

regarding the displayed picture.

Xylem pressure inducing 50% cavitation, MPa

-8 -7 -6 -5 -4 -3 -2 -1 0 Buxus sempervirens Taxus baccata Crataegus monogyna Prunus spinosa Amelanchier ovalis Pinus Halepensis Quercus ilex Lonicera etrusca Quercus suber Pinus corsicana Cedrus atlantica Euonymus europaeus Carpinus betulus Pinus mugho Abies alba Pinus pinaster Picea abies Cytisus scoparius Pseudotsuga Quercus petraea Pinus cembra Pinus sylvestris Fagus sylvatica Populus nigra Fraxinus excelsior Quercus robur Pinus nigra Populus tremula Quercus rubra Betula pendula Salix caprea Juglans regia Salix caprea Alnus glutinosa Populus alba Populus trichocarpa Salix fragilis Populus euphratica Hygrophilous Mesophilous Xerophilous

15

Perspectives

™ Check for the consistency of our

findings

™ Look into drought-induced

acclimation in hydraulic features of

newly produced shoots after the

release from water shortage.

Many thanks for

your attention

and

Our acknowledgements to

C. Bodet, C. Serre, P. Conchon,

P. Chaleil and A. Faure

Water potential at 50% loss conductance (Ψ₅₀) of newly released shoots (control) of the 3 poplar hybrids

Poplar hybrids

I45-51 Robusta Eco28

W at er p o te n tial at 50% lo ss co nduc ta n ce ( Ψ50 ), MPa -2.5 -2.0 -1.5 -1.0

Delayed-effects of drought spells

on newly released sprouts of Robusta plants

Week since drought inception

ROW0 ROW4 ROW6 ROW7

W a te r pot ent ial at 50% lo ss c o nd uc ta n ce ( Ψ50 ), M P a -2.4 -2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -1.8723 -1.7572 -1.8442 -2.0144

Delayed-effects of drought spells on newly released sprouts of I45-51 plants

Week since drought inception

IW0 IW4 IW6 IW7

Wa te r pot ent ial at 50% loss condu ct ance ( Ψ50 ), M Pa -2.4 -2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -1.7498 -1.4625 -1.6825 -1.4651

Delayed-effects of drought spells on newly released sprouts of Eco 28 plants

Week since drought inception

ECOW0 ECOW4 ECOW6

Wate r pote nt ial at 50% lo ss co nd ucta nce ( Ψ50 ), M Pa -2.4 -2.2 -2.0 -1.8 -1.6 -1.4 -1.2 -1.0 -1.6152 -1.6290 -2.1038 Eco 28 (Ψ₅₀=-2.41 MPa) I45-51 (Ψ₅₀=-1.69 MPa) Robusta (Ψ₅₀=-1.60 MPa) (Fichot et al., 2009 Fichot et al., 2010)

Relationships between bud respiration rate, gravimetric bud water content and survivorship in Poplar hybrids versus dry-down span

0 5 10 15 20 25 30 20 40 60 80 100 0.5 1.0 1.5 2.0 2.5 3.0 Respiration Survivorship GBWC Bud respiratio n rate, nm ol g MS -1 s -1 0 5 10 15 20 25 30 P e rcent indiv idu als aliv e, % 20 40 60 80 100 Grav imetric bud w a ter content (GBWC), g g -1 0.5 1.0 1.5 2.0 2.5 3.0 Respiration Survivorship GBWC

Dry-down span, weeks

0 2 4 6 8 10 0 5 10 15 20 25 30 0 20 40 60 80 100 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Respiration Survivorship GBWC Eco 28 I-45 51 Robusta

Relationships between bud respiration rate and gravimetric bud water content

Gravimetric bud water content, g g-1

0.0 0.5 1.0 1.5 2.0 2.5 3.0 Bud respiration rat e , nmol g MS -1 s -1 0 5 10 15 20 25 30 Eco 28 I45-51 Robusta y = -1.86 + 11.40 * x r2 = 0.9260, P < 0.0001

Gravimetric bud water content or

bud respiration rates can stand

for gauges of plant mortality.

Aquaporin

TIP1

expression and its regulation

in the growing root apex

under two levels of osmotic stress

Rémy Merret, Irène Hummel, Bruno Moulia, David Cohen et Marie-Béatrice Bogeat-Triboulot

UMR EEF INRA-UHP, Nancy UMR PIAF INRA-UBP, Clermont-Ferrand

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 2

Referential change

The root apex is a constant structure in which elements are continuously renewed

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 3 Distance from root tip (mm)

Growth = division + cell expansion

Growth = V_{cell production} x L_mature

Beemster et al, 2002

division -> no growth !

elongation

division + elongation cell

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 4

Biophysical model of cell expansion (Lockhart, 1965) - Motor : turgor pressure

- Controls : Cell wall extensibility & membrane hydraulic conductivity (Lp)

Control of cell expansion

cell wall extensibility

water Cell wall Tonoplast Plasma membrane Lp expansins, XET, … aquaporins solutes P

Aquaporin family : two main classes - PIP : Plasma membrane intrinsic protein - TIP : Tonoplast intrinsic protein

+ NIP, SIP, XIP … Zardoya (2005)

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 5

Context

ª Hydraulic limitation of cell expansion ?

ƒ Boyer versus Cosgrove

ƒ estimation from the magnitude of Growth Induced/Sustaining Water Potential Gradient ƒ about 3-4 bars in leaves and hypocotyls

ƒ never found in roots …

ZC ZM †: P Δ : Π {: REGR ª Involvement of Aquaporins … Wei et al, 2007 (Hukin et al, 2002) RE GR (h -1 ) Martre et al, 1999

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 6

Context and aims

ª Lp_tonoplast>> Lp_{plasma membrane} but Lp_tonoplast also important for rapid water balance between cytoplasm and vacuole

ª Aims of the study :

ƒ Which TIP1s are expressed in the Poplar root growth zone? ƒ How are the expression affected by osmotic stress?

ƒ On the basis of transcript accumulation and their changes, can we detect a link between cell expansion and the expression of some TIPs?

ƒ What can we learn from the regulation of TIPs expression? Gupta and Sankaramakrishnan, 2009

Pt : Populus trichocarpa

Os : Oryza sativa

At : Arabidopsis thaliana

Zm : Zea mays

8 TIP1s in Populus trichocarpa genome 4 paralog pairs (gene duplication)

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 7 Osmotic Stress (PEG)

Times (hour) Root growth rate (mm h -1 ) Control 100g/L PEG (90 mosmol/kg) 200g/L PEG (260 mosmol/kg)

• Cuttings of Populus deltoïdes x nigra cv Soligo (15 cm)

• Hydroponics : Hoagland ½ + phosphates

• Controled environment (21°C, 70 % relative humidity, 16h light regime)

Root growth under osmotic stress

3 contrasted steady growth rates

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 8

REGR in the root apex

Distance from root tip (mm)

REGR (h

-1 )

• similar REGR in [0.5-3.5 mm]

• high growth rate <=> long growth zone • low growth rate <=> short growth zone

Control Moderate Stress

High stress

Osmotic Stress (PEG)

Times (hour) Ro ot growth rate (m m h -1) Carbon particules labelling + Kineroot (Basu et al, 2007) 0h 5h 4h 3h 2h 1h

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 9

Transcript density quantification

1mm

250 ng/μL 10 ng/μL

Total RNA extraction

Reverse transcription on 100 ng RNA

Transcript linear density =

Transcript amount in 1 mm-long segment Quantitative PCR

Gene expression normalization (geNorm)

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 10

TIP1;3 and TIP1;8 very weakly accumulated 5 others : distinct accumulation patterns

PtTIP1;1 : reference gene (geNorm) Transc ript de nsity (a.u. m m -1) Transc ript de nsity (a.u. m m -1) Transc ript de nsity (a.u. m m -1) Transc ript de nsity (a.u. m m -1) Transc ript de nsity (a.u. m m -1) Transc ript de nsity (a.u. m m -1) Transc ript de nsity (a.u. m m -1) Mean ± s.e.m. (n=3)

TIP1s transcript density profiles

Distance (mm) Distance (mm)

Among 14 analysed genes,

TIP1;1 : the most stable gene across segments and treatments

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 11

Low impact of moderate stress on

TIP1s accumulation patterns (except PtTIP1;2)

… as on REGR profile

Mean ± s.e.m. (n=3)

Effect of moderate osmotic stress

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 12

Effect of high osmotic stress

Mean ± s.e.m. (n=3)

Control

High stress

High stress:

TIP1s accumulation patterns strongly and differently affected

Transcript

density (a.u.

mm

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 13 PtTIP1;4 Transcript density (a.u mm -1 )

Distance from root tip (mm)

REGR (h

-1

)

Changes of REGR profile are accompanied by similar changes in TIP1;4 accumulation patterns

PtPIP2;6

Transcript

density

(a.u mm

-1 )

Distance from root tip (mm)

Transcript density (a.u mm -1 ) REGR (h -1 )

Expression and REGR profiles

Moderate Stress

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 14

Transcript is not a proxy of functional protein … but a change of expression is costly : -> change of transcript density has a sense in terms of

maintenance/increase in protein level

ª for the 3 growth states, REGR and TIP1;4 patterns overlap

-> clue for implication of TIP1;4 in cell expansion

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 15

Mature leaf

t

t

Treatment

Regulation of gene expression in a growing organ?

Mature organ : Gene regulation = temporal variation of transcript density

δρ/δt

In a growing organ, it is necessary to take into account

cells movement and their expansion.

Growing root

?

The continuity equation (issued from a fluid mechanics formalism) gives access to the material derivative of transcript density, i.e.,

Transcri p t a ccumul a ti on rate

Distance from root tip (mm)

Spatial pattern of

local transcript accumulation rate at a given time point

spline Continuity equation ( ) ( )x ( )x ( )x x x v ρ x ρ v t ρ D ∂ ∂ + ∂ ∂ + ∂ ∂ = 0h 5h 4h 3h 2h 1h

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 17

Growth trajectory : D = f (Time)

If steady state :

integration

Steady state and growth trajectory

spatial coordinates -> temporal coordinates

Spatial pattern of

transcript accumulation rate

in a ‘moving particule’ Transcri p t a ccumul a ti on rate

Distance from root tip (mm)

These eulerian patterns are valid for any particule for the “steady-state” time window

Time (min) Transcri p t a ccumul a ti on rate Mat e ri al der iva ti ve (a .u . m m -1 mi n -1 ) Time (min) A: Actin 11 Distance from root tip (mm) M at e ri al der iva ti ve (a .u . m m -1 mi n -1 ) Time (min) A: Actin 11 Distance from root tip (mm) Merret R. et al (2010) Spatio-temporal description

of regulation of gene expression in a ‘moving particule’

spline 0h 5h 4h 3h 2h 1h Continuity equation + steady state

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 19

Distance from root tip (mm)

Transcript

density

PtTIP1;4

PtPIP2;6

Distance from root tip (mm)

Transcript

density

Distance from root tip (mm)

Tran

script

accumulation rate

Distance from root tip (mm)

Tran script accumulation rate Control Moderate Stress High stress

Regulation of PtTIP1;4 and PIP2;6 expression

PtTIP1;4 under high stress : high expression without higher induction PtTIP2;6 under high stress : strong induction

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 20

Growth trajectory : D = f (Time)

If steady state : integration

Growth trajectories of the cells just finishing their expansion for the preceding 20h

Time (hours)

Distance from root tip (mm)

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 21

Contrasting spatial patterns but similar temporal patterns

The regulation of TIP1;4 expression seems to be temporally governed

Time (hours) Transcript accumulatio n rate Transcript accumulatio n rate

Distance from root tip (mm)

Control

100g/L PEG (90 mosmol/kg)

200g/L PEG (260 mosmol/kg)

Spatial and temporal patterns of regulation of TIP1;4 expression

Distance from root tip (mm)

Transcript

density

Distance from root tip (mm)

RE

G

Regulation of aquaporins expression in the root apex – Xylème 2011 – Avril 2011 - 22

Perpectives

ª analyse the regulation patterns of other TIP1s ª immunolocation of aquaporins

ª use the framework to analyse the molecular control of synchrony between cell division and cell elongation

Hydraulic safety

of Pinus pinaster needles

Katline Charra-Vaskou,

Régis Burlett, Sylvain Delzon,

Stefan Mayr

Institute of Botany, Innsbruck University, Austria

6th april 2011

Xylem colloquium, Nancy

UMR BIOGECO,

Bordeaux 1 university, France

Plant hydraulic resistance:

Aim:

Soil-plant-atmosphere continuum

Water potential in the plant

Water availability for tissues

Efficiency of the plant hydraulic system

Leonardo da Vinci Roots Axes Hydraulic architecture Leaves Introduction

Analysis of vulnerability to drought-induced loss of conductivity in needles of Pinus pinaster

Material:

Pinus pinaster

Twigs and needles

Aim

Methods: 1. Cavitron

2. Rehydration kinetics method

3. Ultrasonic acoustic emission analysis Introduction

Needle hydraulic conductivity

Xylem: conductivity is influenced by tracheid diameter, tracheid length and the number of pit connections xylem phloem transfusion tissue endodermis mesophyll epidermis resin channels xylem phloem

Cross section of Pinus pinaster needle

Extra-vascular pathway: transfusion tissue, mesophyll

Plant material

Pinus pinaster

Campus of Talence, Bordeaux mean length: 14 to 18 cm

needles

twigs

Material and method

Branches harvested the day before Cut under water

Rehydrated in refrigerator with plastic bag Needle preparation:

Resin channels blocked Resin does not block xylem

To avoid emptying of resin channels:

Needles immersed 1 hour in cold water ( 5 – 7°C)

many resin channels

Cavitron

Needles cut 2 times (15 minutes interval)

resin channel

xylem phloem

20 needles inserted in water reservoir

Problem: Measurements take long time (3 to 4h cavitron measurement per sample)

Preparation for cavitron measurements:

Cooled centrifuge and rotor (5 to 7°C)

Cavitron measurements:

Needle length (after cutting) : 14,5 cm

Cavitron

Temperature cavitron at 5 to 7°C during the measurement

Use of software “Cavisoft” (Regis Burlett, Biogeco) Procedure according to Cochard et al., 2005

Acoustic

2 well hydrated twigs 8 sensor

4 sensors per twig 2 Sensors on

STEMS 6 Sensors on _NEEDLES

Acoustic

While acoustic emission measurement were made, branches were slowly dehydrating (bench dehydration) and water potentials were measured every 6 to 12 hours (Scholander apparatus).

Needles used for UAE measurements stems used for UAE

measurements

Needles used for water potential measurements Material and method

Rehydration kinetics measurements

Material and method

6 well hydrated branches were slowly dehydrated (bench dehydration) from -0,3 to -3 MPa

2 needles: initial water potential (Scholander apparatus)

4 needles: rehydration measure and final water potential

Many times during dehydration of branches:

Measurements according to “Brodribb and Holbrook, 2003)

Before measurements, branches were bagged and put in the refrigerator to ensure homogeneous Ψ among needles

« Brodribb and Holdbrook , 2003 » Measurement of initial water potential (Ψ0) Measurement of final water potential (Ψf) Conductance K_N [mmol._m-_²._s-1._MPa-1_] Capacitance C_N [mol.m-2._MPa-1_] Time « t » in distilled water K_N = C_N * ln (Ψ0/ Ψf) / t Material and method

C_N = ΔRWC/ΔΨ * (DW/LA)*(WW/DW)/M Needle capacitance C_N:

DW: leaf dry weight (g); LA: leaf area (m2_{); WW: mass of leaf water at 100% RWC (g); M: molar mass of}

water (g.mol-1₎

Two-phase function fitted to pressure volume data for Pinus pinaster needles. (RWC threshold: 0,9)

y = 0,0744x + 1,0098 R2 _{= 0,908} y = 0,0843x + 1,0265 R2 _{= 0,8451} 0,0 0,2 0,4 0,6 0,8 1,0 1,2 -6 -5 -4 -3 -2 -1 0

water potential (MPa)

RW C (% ) pré TLP post TLP -1,5MPa Turgor loss point

K_N = C_N * ln (Ψ0/ Ψf) / t Needle conductance

Material and method

Results

Cavitron measurements

Vulnerability curves of Pinus pinaster needles and twigs

Ψ50 needles: -1,5 MPa Ψ50 twigs: -4,0 MPa -7 -6 -5 -4 -3 -2 -1 0 0 20 40 60 80 100 PL C ( % )

xylem pressure (P; MPa)

cavitron needles

cavitron axes (P50:-1,5MPa)

Ψ50 needles : -1,5 MPa Results

Acoustic measurements

Vulnerability curves of Pinus pinaster needles and twigs

Ψ50 twigs : -3,2 MPa -7 -6 -5 -4 -3 -2 -1 0 0 20 40 60 80 100 PL C ( % )

xylem pressure (P; MPa)

acoustic needles

acoustic axes (P50: -1,5)

Results

Rehydration kinetics measurements

Vulnerability curve of Pinus pinaster needles

Ψ50 needles: -0,5 MPa

-7 -6 -5 -4 -3 -2 -1 0

xylem pressure (P; MPa)

0 20 40 60 80 100 PL C ( % ) rehydration (P50: -0,5MPa)

Discussion

Pinus pinaster needles vulnerability

-0,5MPa (rehydration)

-1,5 MPa (cavitron)

-1,5 MPa ( acoustic)

Ψ50 needles

Quite similar results between cavitron and acoustic High Ψ50 with rehydration kinetics method

-7 -6 -5 -4 -3 -2 -1 0

xylem pressure (P; MPa)

0 20 40 60 80 100 PL C ( % ) rehydration cavitron needles acoustic needles cavitron axes acoustic axes (P50: -0,5MPa) (P50:-1,5MPa) (P50: -4,0MPa) (P50: -1,5) (P50: -3,2MPa)

Discussion

Pinus pinaster vulnerability

Quite similar results between cavitron and acoustic High Ψ50 with rehydration method

Loss of conductivity in needles occurs before cavitation in branches

-0,5MPa (rehydration) -1,5 MPa (cavitron) -1,5 MPa ( acoustic) Ψ 50 needles Ψ 50 branches -4,0 MPa (cavitron) -3,2 MPa ( acoustic) -7 -6 -5 -4 -3 -2 -1 0

xylem pressure (P; MPa)

0 20 40 60 80 100 PL C ( % ) rehydration cavitron needles acoustic needles cavitron axes acoustic axes (P50: -0,5MPa) (P50:-1,5MPa) (P50: -4,0MPa) (P50: -1,5) (P50: -3,2MPa)

Discussion

Pinus pinaster vulnerability

Vulnerability of needles:

Is the needle capacitance related to Ψ50 in needles? Methodical problems? -0,5MPa -1,5 MPa -1,5 MPa needles acoustic cavitron rehydration Ψ50 twigs -3,2 MPa -4,0 MPa cavitation?

Collapse? (fast recovery) Something else?

Overall high Ψ50 in needles

-7 -6 -5 -4 -3 -2 -1 0

xylem pressure (P; MPa)

0 20 40 60 80 100 PL C ( % ) rehydration cavitron needles acoustic needles cavitron axes acoustic axes (P50: -0,5MPa) (P50:-1,5MPa) (P50: -4,0MPa) (P50: -1,5) (P50: -3,2MPa)

0 10 20 30 40 50 60 70 80 90 100 % cum UAE 600 550 500 450 400 350 300 250 M e an h it en er g y Cavitation ou collapse? Discussion

hit energy from twigs and needles during dehydration (acoustic measurements) 0 10 20 30 40 50 60 70 80 90 100 % cum UAE 0 20 40 60 80 100 120 140 160 180 200 M ean h it en er g y

Most energy between 20 to 40

% of cum UAE Less energy than in twigs

“cavitation pattern” in twigs Complex pattern

Discussion

Cavitation ou collapse?

Epifluorescence technique on frozen samples

On fresh to dried needles: No observed collapse

Ψ max: -3,91MPa (80% PLC) Ψ measure: -0,43MPa 0 10 20 30 40 50 60 70 80 Time (minutes) 0 20 40 60 80 100 120 140 C o n d u c tiv it y [ m o l.s -1 .M P a -1 ] PLC (%) -273% -72% 10% 54% 75% 77% Capacitance

High capacitance in Pinus

pinaster needles

Time to recovery the loss of conductivity reached at -3,9MPa at -0,4MPa with cavitron

-3 -2,5 -2,0 -1,5 -1,0 -0,5 0 Psi 50 (Pressure,MPa, at 50% PLC) 0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 C a p a c ita n c e (m o l.m -2 .M P a -1 )

Needles capacitance VS needles vulnerability (P50)

acoustic

cavitron

rehydration

Pinus pinaster

Pinus mugo alpin Pinus nigra (Johnson) Pinus ponderosa (Johnson)

Picea abies alpin

Discussion

Capacitance

Pinus nigra and Pinus ponderosa of „Johnson, 2009,PCE“

Linear relation between Ψ50 and capacitance

More vulnerable needles have a bigger capacitance

Acknowledgments:

Acknowledgments:

Fonds zur Förderung der Wissenschaftlichen Forschung

University of Innsbruck and Bordeaux Institute of Botany and UMR Biogeco

Thanks

1

Miraculous xylem refilling in

plants

Hervé Cochard

UMR PIAF

2

% embolism

Water potential, MPa

S

to

m

ata

l

co

n

d

u

ct

an

ce

Previous paradigms

Plants operate near the point of xylem dysfunction

Stomatal closure prevents xylem embolism

3 Jour de l'année 150 200 250 300 350 % Em bolie 0 20 40 60 80 100

Hydratés

Déshydratés

H20

H20

Previous paradigms

4

How plants were able to recover from xylem embolism?

Stem diam eter, µm -250 0 250 500 750 PLC 0 20 40 60 80 100 Pressure, kPa 0 5 10 15 20 25 Leaf flush F M A M J

Fagus sylvatica

2- Xylem recovery

2

Positive

xylem

pressures

Cambial

growth

1- Xylem refilling

1

5

Air bubble at

atmospheric

pressure (P

)

2τ/r

P

P

Physics of xylem refilling

(Yang and Tyree 1992)

Xylem sap

saturated with air

at P

pressure

For the bubble to collapse:

P

xyl

> P

gaz

- 2

τ/r

If r = 30 µm P

> -5kPa

No transpiration + root pressure to

compensate gravitational forces

6

Can xylem vessels refill if P

xyl

< - 2τ/r ?

Many reports in the literature of refilling

when P

< - 2τ/r and transpiration is high

Cryo-SEM : virtually all the studies

Acoustic technique : many reports

7

-0.7 MPa

-0.7 MPa

1

0

µ

m

Evidence from the Cryo-SEM technique

8 Solar Time 0 6 12 18 24 30 36 42 48 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Percent em bolised vessels

Percent loss conductance

Water LN2

Cryo-SEM

+ Px = 0

Cryo-SEM

Hydraulic

Diurnal trends of embolism in walnut petiole

(Cochard et al 2000)

LN2

P

<<0

P

=0

9

Vitis (Salleo et al 1989, Brodersen et al 2010, Zufferey et al 2011)

Laurus (Salleo et al 1996, Tyree et al 1999, etc…)

Pinus (Edwards et al 1994

Zea (McCully et al 1998)

Fraxinus (Zwieniecki et al 1998)

Acer (Zwieniecki et al 1998)

Picea (Zwieniecki et al 1998)

Helianthus (Nardini et al 2008)

Orysa (Stillet et al 2005)

Populus (Secchi et al 2010)

…

Evidence for xylem refilling while P

xyl

< - 2τ/r ?

What is this novel mechanism?

“Do we need a new paradigm?”

10

P

xyl

> P

gaz

- 2

τ/r

Hydraulic isolation ?

11 Air Re filling ve sse l Ψ _PX > -2T/r Functiona l ve sse l Ψ _PX < < -2T/r Con ta ct ce lls W a ter Solute tra ns port La rge s olute s W a ter W a ter Solute tra ns port Solute tra ns port La rge s olute s La rge s olute s

Hypotheses for a “novel” refilling mechanism

The “large organic solute” hypothesis

(Hacke & Sperry 2003)

Large organic solute flow into the vessel causing a water flow and the refilling

Pit membrane acts as an osmotic membrane

12

Hypotheses for a “novel” refilling mechanism

The “pit valve” hypothesis

(Holbrook & Zwieniecki 1999)

Embolised vessels have to be

hydraulically isolated Solute flow into the vessel causing a water flow and the refilling

13

More insights into this “novel” refilling mechanism

Phloeme is important (girdling exp) Salleo et al 2003, Bucchi et al

2003

Starch and sucrose is implicated in the mechanism : Salleo et al 2006

Requires energy (Proton pump) Salleo et al 2004

Aquaporins are implicated Sakr et al 2003, Secchi & Zwieniecki 2010

Sensing and triggering the refilling

-Wall vibration Salleo et al 2008

-Chemical sensing (Zwieniecki and Holbrook 2009)

-Simulations Vesela et al 2003 Gas convection not diffusion

-Water condensation instead of liquid water flow (Zwieniecki and

Holbrook 2009)

15

a

b

c

d

16

Xylem pressure, MPa

-5 -4 -3 -2 -1 0 PLC 0 20 40 60 80 100 Exponential Sigmoidal

Conclusions

‘Miraculous’ refilling seems to exist

Less and less miraculous

More explicitely included in our experiments

New paradigms

Stomatal

conductance

How generic?

Mechanisms ?

Functional benefit ?

What’s new at Bordeaux?

Taxonomic diversity of conifers and species measured for cavitation resistance

resistance

Genera species Sampled

species Remaining sp. sampled genera Araucariaceae 3 41 3 38 1 Cephalotaxaceae 1 11 3 8 1 Cupressaceae 30 133 32 101 13 Ph ll l d 1 4 1 3 1 Phyllocladaceae 1 4 1 3 1 Pinaceae 11 228 34 194 7 Podocarpaceae 18 186 10 176 0 S i d it 1 1 1 0 1 Sciadopityaceae 1 1 1 0 1 Taxaceae 5 23 6 17 2 Total 70 627 90 537 26

Abies kawakamii Athrotaxis selaginoides Pinus sylvestris Pinus albicaulis Athrotaxis cupressoides Araucaria araucana Pinus cembra Metasequoia glyptostroboides Dacrycarpus dacrydioidesPinus wallichiana Taxodium distichum Agathis australis

Cavitation resistance of 100

conifer species

Chamaecyparis obtusaPseudotsuga menziesii Picea abies Abies grandis Abies lasiocarpa Pinus nigra Cunninghamia lanceolata Abies f orrestii Chamaecyparis pisifera Pinus hartwegii Abies f abriSaxegothaea conspicua Taiwania cryptomerioidesAbies kawakamii

conifer species

Cryptomeria japonicaPinus edulis Abies alba

Sciadopitys verticillata Pinus contorta Pinus ponderosa Podocarpus nubigenusAbies cilicica Tsuga canadensis Sequoiadendron giganteumPinus mugo Pinus pinaster

Pinus f lexilis Chamaecyparis obtusa

Seq oia semper irens Pinus radiata Pinus pinea Larix decidua Tsuga chinensis Podocarpus salignusLarix occidentalis Thuja plicata Picea engelmannii Pinus uncinata Pseudolarix amabilis Abies pinsapoAraucaria hunsteinii Chamaecyparis lawsonianayp j p Chamaecyparis nootkatensis Cedrus atlantica Fitzroya cupressoides Podocarpus totara Taxus cuspidata Lagarostrobos franklinii Torreya grandis Pinus halepensisAustrocedrus chilensis Ginkgo biloba Podocarpus acutifolius Pilgerodendron uviferumPodocarpus nivalis Sequoia sempervirens

High variability of P50 Cephalotaxus harringtoniaPhyllocladus trichomanoides alpinus Taxus baccata

Podocarpus henkelii Podocarpus latifolius Taxus brevif olia Torreya calif ornica Juniperus communis Podocarpus elongatus Torreya nucif era Thujopsis dolabrata Halocarpus bidwillii Prumnopitys andinaChamaecyparis nootkatensis

g y

from 2.1 (Agathis)

to 16 MPa (Callitris collu)

C l b Callitris oblonga Actinostrobus pyramidalis Cupressus sempervirens Cupressus dupreziana Juniperus scopulorum Callitris rhomboideaPlatycladus orientalis Juniperus osteospermaCupressus torulosa Cephalotaxus wilsoniana Af rocarpus falcatus Cephalotaxus f ortunei Cedrus deodara Cephalotaxus harringtonia -16 -14 -12 -10 -8 -6 -4 -2 0

Callitris columellarisCallitris preissii Callitris gracilis Cupressus glabrag

Different levels of variation in cavitation resistance

Conifers Pinus Pinus pinaster

Coca a Pinus flexilis Pinus hartwegii Pinus sylvestris Oria a Pinus mugo Pinus pinaster Pinus flexilis San-Cipriano a Pinus contorta Pinus ponderosa Mimizan a Pinus pinea Pinus uncinata Pinus edulis Tamrabta a Pinus halepensis Pinus radiata -4.3 -3.8 -3.3 P₅₀(MPa) -5 -4 -3 -2 -1 0 P₅₀(MPa)

Lamy et al., submitted, PLoS ONE Delzon et al., PCE, 2010

Objectives

Objectives

1 Extend the existing 1. Extend the existing database for P50 and other traits (wood density, anatomy)

anatomy)

2. Construct a phylogeny of conifers using online of conifers, using online sequence databases (NCBI)

3. Test for evolutionary convergence versus

evolutionary conservatism evolutionary conservatism Correlate P50 with other mesured traits.

Height-related effects on cavitation resistance

Height related effects on cavitation resistance

in maritime pine trees

Sylvain Delzon, Mélanie Lucas, Régis Burlett, Hervé Cochard

Eucalyptus regnans measured at 132 6 m in 1872 Eucalyptus regnans measured at 132.6 m in 1872 near Watts river, Victoria, Australia

It h

l

b

b li

d th t

It has long been believed that senescence

is an inevitable consequence of ageing in all

plants and animals.

old animal old trees

How to test the hydraulic limitation hypothesis ?

How to test the hydraulic limitation hypothesis ?

F = K

_L

* 

F=0 7

F=0 35

• small tree

• tall tree

VPD = 3kPa VPD = 3kPa Transpiration decrease

F=0.7

F=0.35

F=0.7

cavitation

homeostasis

g

=100

g

=50



= - 4



= - 4



= - 7.5

g

100



4

K

=0.1



4



7.5

 = - 3.5

K

=0.2

S il

 = - 3.5

 = - 7.0



= - 0.5

_

_{= - 0.5}

Soil

Leaf

Leaf--specific hydraulic conductance

specific hydraulic conductance

K

Leaf-specific hydraulic conductance (K_L) versus tree height

Substantial decrease in hydraulic conductance with

y

increasing tree height

Which hydraulic compensation mechanisms occur?

Which hydraulic compensation mechanisms occur?

Which hydraulic compensation mechanisms occur?

Which hydraulic compensation mechanisms occur?

 hydraulic adjustment: decrease in leaf to sapwood ratio (A

:A

)

increase in soil to leaf water potential gradient (decrease in minimum leaf

water potential (

))

 production of xylem tracheids with increased permeability (higher

d

ifi h d

li

d

ti it (k ))

sapwood-specific hydraulic conductivity (k

))

 increased water storage in the stem

g

Compensation mechanisms

Compensation mechanisms

Leaf / sapwood area ratio versus tree 0

0 10 20 30

Tree height (m)

Leaf-specific hydraulic conductance

0 10 20 30 40

A L / AS (x10 2

m2 m-2)

A

/ A

Leaf / sapwood area ratio versus tree

height Leaf specific hydraulic conductance versus leaf / sapwood area ratio

Hydraulic compensation

(decrease in A

: A

)

Compensation is

incomplete

Consequence for stomatal conductance

Consequence for stomatal conductance

Stomatal conductance versus air vapor pressure deficit (VPD)( )

 Stomatal closure allows to

maintain miminum water potential

maintain miminum water potential

above the critical threshold in tall

trees

 Decrease in stomatal conductance

induces both transpiration and

i il ti

d li

assimilation declines

Summing up

Summing up

cst

?

K

gs



1

1



?

cst

cst

?

K

VPD

A

A

h

gs



/

to

lation ximum

s in

re

he ma

x

gs th

Height (m)

H

Homeostasis in maritime pine tree ?

Homeostasis in maritime pine tree ?

Time

Heure 0

0:00 4:00 8:00 12:00 16:00 20:00 0:00

Heure

Wet soil Dry soil

Heure

Time

91 yrs (sol sec)

32 yrs (sol sec)

-0.5 M pa) 0 0:00 4:00 8:00 12:00 16:00 20:00 0:00 ) Time Time -1.5 -1

-0.5 32 yrs (sol sec)

54 yrs (sol sec)

54 yrs (sol très sec)

32 yrs (sol très sec)

-1  L ( M 10 yrs -1 -0.5  L (M p a ) -2.5 -2 1.5 -2 -1.5 _{32 yrs}y 54 yrs 91 yrs _Threshold -2 -1.5 10 yrs_{32 yrs} 54 yrs 91 yrs

Needle water potential measurements carried out across the chronosequence

Relationship between cavitation

Relationship between cavitation

i

d

i i

i l

i

d

i i

i l

resistance and minimum water potential

resistance and minimum water potential

Do cavitation resistance (safety) and specific

Do cavitation resistance (safety) and specific

hydraulic conductivity (efficiency) remain

constant with increasing tree height?

Maritime pine chronosequence

Maritime pine chronosequence

12 even

12 even--aged stands

aged stands

28m

B f

l

(3

12 even

12 even aged stands

aged stands

63

Before canopy closure (3

stands)

63

2m

24m

2 old stands

6

15 m

8 m

12

19

33

Stand age: 5; 6; 7; 12;

12

19

5 mature stands

14; 19; 22; 33; 45; 61 et

63 years

Vulnerability curves

Vulnerability curves

Pressure (MPa) Pressure (MPa) Pressure (MPa)

P

Evolution of cavitation resistance

Evolution of cavitation resistance

0 10 20 30 40 50 60 70

Tree age (yrs)_{Tree age (yr)}

3 2

0 10 20 30

Tree height (m)Tree height (m)

2 -3 6 -3.4 -3.2 Pa) _R2 _{0 8573} -3.6 R2= 0 8639 -3.4 -3.2 M Pa) R2 _{= 0.8639} R2 = 0.8573 -4 -3.8 3.6 P 50 (M R 2 _{= 0.8573} R 0.8639 -4 -3.8 P 50 ( M -4.2 -4.2

Significant linear trend according to tree height

ll

i

i

i

b

Evolution of xylem

Evolution of xylem--specific hydraulic

specific hydraulic

conductivity

conductivity

s

)

M

Pa

s

K

(m

M

K

k reaches an optimum at 15 m height and then

k

reaches an optimum at 15 m height and then

decreases as trees grow taller

Hydraulic compensation mechanisms

Hydraulic compensation mechanisms

 SAFETY: YES

As cavitation resistance increased with increasing tree height, tall trees

could reach lower minimum water potential, thus increasing soil to leaf

t

t

ti l

di

t

water potential gradient

 EFFICIENCY: NO

 EFFICIENCY: NO

Tall trees did not produce more efficient xylem and had even lower xylem

specific hydraulic conductivity.

specific hydraulic conductivity.

 Hydraulic adjustments that enhanced the ability to cope with vertical

Hydraulic adjustments that enhanced the ability to cope with vertical

gradients of increasing xylem tension were attained at the expense of

reduced water transport capacity and efficiency

Height

Height--related effect within tree crown

related effect within tree crown

Height

Height related effect within tree crown

related effect within tree crown

Safety

Efficiency

Safety

Burgess et al. 2006 PCE

No trade-off in Sequoia sempervirens

Perspectives: xylem anatomy

Perspectives: xylem anatomy

Pit membrane properties (margo

Pit membrane properties (margo

flexibility, torus overlap and valve

effect) highly correlated with P50

Delzon et al. 2010 PCE

Domec et al. 2008 PNAS Domec et al. 2008 PNAS

Torus overlap increased in

Douglas-fir trees along a height

di

t f 85

Dulhoste

Dulhoste

Rapha

Rapha

ë

ë

l

l

Rada 

General 

General 

Hypothesis

_Hypothesis

Our case

Tropical Mountains

Tropical Mountains

Daily water deficit

High VPD

Tropical Mountains

Seasonal deficit

•

Decreased rainfall 

HYPOTHESIS

HYPOTHESIS

•

Species  adapted  to  higher  altitude  in  the 

ecotone present mechanisms to improve their 

water  status  under  conditions  of  greater 

deficit.

OBJETIVOS

OBJETIVOS

•

Determine  the  minimum  water  potential  of 

three species of cloud forest‐páramo ecotone

in adult individuals in the field.

•

_{Identify  their  various  components  of  water} 

potential.

•

Characterize  the  behavior  of  the  response  of 

stomata  to  leaf  water  potential  in  these 

species, adult leaves.

Material & methods

Material & methods

SPECIES

SPECIES

•

Diplostephium venezuelense

SPECIES

SPECIES

•

Libanothamnus neriifolius

SPECIES

SPECIES

•

Miconia jahnii

Minimun (Ψ

_lmin

) and predawn(Ψ

_lpd

)

leaf water potential .