Stomatal behaviour of irrigated Vitis vinifera cv Syrahfollowing partial root removal

V ZuffereyAC and D R SmartB

AStation de recherche Agroscope Changins-Waumldenswil ACW CP 1012 CH-1260 Nyon (Switzerland)BDepartment of Viticulture and Enology University of California One Shields Avenue Davis CA 95616 USACCorresponding author Email vivianzuffereyacwadminch

Abstract We examined stomatal behaviour of a grapevine cultivar (Vitis vinifera L cv Syrah) following partial rootremoval under field conditions during progressively developing water deficits Partial root removal led to an increase inhydraulic resistances along the soil-to-leaf pathway and leaf wilting symptoms appeared in the root-pruned plantsimmediately following root removal Leaves recovered from wilting shortly thereafter but hydraulic resistances weresustained In comparisonwith the non-root pruned vines leaves of root-pruned vines showed an immediate decrease in bothpre-dawn (yPD) andmidday (yleaf) leaf water potential The decline inyPD was unexpected in as much as soil moisture wasnot altered and it has been shown that axial water transport readily occurs in woody perennials Only ~30 of the functionalroot systemwas removed thus leaving the systemmainly intact forwater redistribution Stemwater potential (yStem) and leafgas exchanges of CO2 (A) andH2O (E) also declined immediately following root pruning The lowering ofyPDyleafyStemA andEwas sustainedduring the entire growing seasonandwas not dependent on irrigationduring that timeThis and a closerelationship between stomatal conductance (gs) and leaf-specific hydraulic conductance (Kplant) indicated that the stomatalresponsewas linked to plant hydraulics Stomatal closure was observed only in the root-restricted plants and at times of veryhigh evaporative demand (VPD) In accordance with the Ball-Berry stomatal control model proposed by Ball et al (1987)the stomatal sensitivity factor was also lower in the root-restricted plants than in intact plants as soil water availabilitydecreasedAlthoughyPDyStem andyLeaf changedmodestly and gradually following root removal gs changed dramaticallyand abruptly following removal These results suggest the involvement of stomatal restricting signals being propagatedfollowing removal of roots

Additional keywords gas exchanges grapevine leaf specific hydraulic conductance leaf water potential root pruningstomatal conductance

Received 20 March 2012 accepted 7 September 2012 published online 8 October 2012

Introduction

The control of stomata and therefore gas exchanges of CO2

and H2O is a highly complex phenomenon where the exactcoordination of many apparent mechanistic signals remainsuncertain The complexity of stomatal behaviour resides in theexistence of feedback controls that interact with a wide range ofenvironmental conditions (Jones 1998) A general distinction ismade between CO2 feedbacks which probably operate either byinternal CO2 concentration (Ci) (Morison 1987Mott 1988) or byrate of assimilation (Wong et al 1985) Plant hydraulic controlsare apparently dependent on specific aspects of stomatal or plantwater status (Jarvis and Davies 1998) hydraulic continuityand xylem pressure gradients in the soilndashplantndashatmospherecontinuum (Sperry et al 2002)

Of the factors that stomata respond to the difference in watervapour pressure between ambient air and the saturation vapourpressure at the same temperature (VPD) and photosyntheticphoton flux density (PPFD) are of primary importance Severalinvestigations suggest the response to VPD is in fact sensed

through transpiration demand (Mott and Parkhurst 1991) andmediated by negative feedback via the water status of cellsassociated with the stomatal apparatus (Monteith 1995) Theobserved relationship between stomatal response evaporativedemand and plant water loss suggests this effect is linked to planthydraulics and plays a role in yleaf regulation (Oren et al 2001)Under water stress conditions the progressive closure of stomataoccurs as VPD rises and as the hydraulic conductance along thesoil-to-leaf pathway (Kplant) decreases so that yleaf remainsabove a critical threshold value preventing xylem cavitationand hydraulic failure (Tyree and Sperry 1988) A positivecorrelation between hydraulic and stomatal conductances hasbeen described in several studies (Meinzer and Grantz 1990Saliendra et al 1995 Comstock 2000 Zufferey et al 2011) butat a relatively constant yleaf These observations suggest afeedback link between gs and some form of hydraulic signal(Fuchs and Livingston 1996 Nardini et al 2001) Indeed whenxylem hydraulic conductance is reduced experimentally usingvarious techniques such as stem notching (Sperry et al 1993)

CSIRO PUBLISHING

Functional Plant Biology 2012 39 1019ndash1027httpdxdoiorg101071FP12091

Journal compilation CSIRO 2012 wwwpublishcsiroaujournalsfpb

root and leaf removal (Teskey et al 1983 Meinzer and Grantz1990) defoliations (Hubbard et al1999) or induced air embolism(Hubbard et al 2001) stomatal conductance decreases Theseobservations indicated that hydraulic conductance (K) throughthe plantndashsoil system to supply water for transpiration forms anintegral and dynamic part of the stomatal control mechanism(Sperry et al 2002 Franks 2004) The relationship observedbetween the leaf specific hydraulic conductance Kplant and gstherefore may not represent a direct effect ofKplant on gs butmayinstead occur indirectly through the effect of Kplant on leaf waterstatus followed by the effect of leaf water status on gs (Saliendraet al 1995 Brodribb andHolbrook 2003Addington et al 2004)

Many studies have suggested that stomatal opening maybe controlled by leaf water potential (yleaf) although someevidence suggests that stomata respond to root or soil waterstatus independent of any change in yleaf (Davies and Zhang1991 Tardieu et al 1996 Jones 1998) For some time it has beenknown that different plant species known as lsquodrought-avoidingplantsrsquo (Smart and Combe 1983) or lsquoisohydric plantsrsquo have atendency to adjust their stomata so as to maintain a relativelystable leaf water status as environmental conditions change(Chaves et al 1987 Tardieu and Simonneau 1998 Schultz2003) In contrast in lsquoanisohydric plantsrsquo yleaf decreasessharply as the evaporative demand rises during the day and ismore negative in plants suffering from drought than inwell-watered plants Both the leaf water potential and leafconductance decline with increasing soil water potential in thiscase The spectrum in stomatal lsquostrategyrsquo between isohydric andanisohydric plants is determined by the degree of influence ofleaf water status on stomatal control for a given concentration ofABA in the xylem the predominant message arriving from rootsin contact with drying soil (Davies et al 1994) Various studiescarried out on grapevines have indeed demonstrated that ABAwas able to influence leaf stomatal conductance (Loveys 1991Stoll et al 2000 Lovisolo et al 2002) However Correia et al(1995)noted in their experimentsonvines that evengswas closelyrelated to ABA from roots This was true in the morning when gswas at a maximum but there was a poor correlation over awhole day In addition observations made by Schultz (2003)in Vitis showed a strong correlation between gs and Kplant thusarguing for a more exclusive stomatal control mechanism basedon hydraulic conductance in the soil-plant system irrespective ofwhether stomatal regulation resembled isohydric or anisohydricbehaviour

Partial root removal may influence the hydraulic conductancealong the soil-to-leaf pathway since it may change root shootratio so that demand exceeds supply or because ABA signallingfrom the root systemmaybe altered Forwoodyperennials plantsprevious investigations reported root removal diminished gs andyleaf when gt50 of roots were removed from Abies amabilisDouglas ex J Forbes but notwhenlt50were removed (Teskeyet al 1983) For sugarcane removal of 20 of roots resultedin a 10 decline in gs with no significant alteration in yleafInconsistencies in previous investigations and a general lackof comprehensive information makes it unclear whether therestriction of gs was related to leaf water supply (hydrauliccontinuity) or changes to water status In order to help resolvethese issues we examined stomatal response in relation to planthydraulics and environmental co-factors (VPD air humidity) that

would limit gs We conducted the investigation under conditionsof progressively developing water deficit and removed rootsfrom well watered versus non-irrigated soil quadrants ofindividual grapevines We hypothesised that irrigation wouldrelieve negative feedback to gs by restoring water continuity(Smart et al 2005) and hydratedwater status of leaves (yPDyleafystem)

Materials and methodsPlant material and treatments

The investigation was conducted in a 04 ha vineyard at theUniversity of California Davis USA (38320N 121460W)during the summer of 2004 and 2005 Rows of 30 5-year-oldSyrah grapevines grafted onto Vitis ripariaVitis rupestrisCouderc cv 3309C rootstock were divided into three sets(plots) of six vines with two guard vines between plots andfour guard vines on the East and West ends of the rows Eachexperimental row (block) had two guard rows on either side Thisyielded a randomised complete block design with the experimentreplicated three times The site was planted on a Yolo sandy clayloam soil and had a planting density of 1900 vines handash1 Duringthe period this investigation was conducted daily maximumtemperatures were predominantly above 30C and minimumtemperatures were above 10C Water relations and 2-yeargrowth data were detailed in a previous report (Smart et al2006) Approximately 3 weeks following anthesis (floweringon 31 May 2004) vines were carefully excavated at the baseand structural framework roots emerging from the trunk werepruned in an amount visually equal to ~25ndash35of the total cross-sectional area of framework roots emerging from the trunk Thispercentage varied somewhat because two roots were removed ofwhich there were generally 6ndash8 of relatively uniform size

We applied two root-pruning (RP) treatments For onetreatment (root pruning irrigated RPI) the two roots weremoved were taken from the East quadrant whichcorresponded to the side of the vine where a single dripirrigation emitter was allowed In the second treatment thetwo roots were cut from the West quadrant or non-irrigatedside of the vine (root pruning dry RPD) These two treatmentswere compared with a control without root pruning Soil wasexcavated from the control vines in the same way it was for theroot-pruning treatments but then the trunk and roots were re-buried without severing any roots In each plot the central fouradjacent vines were subjected to the root pruning and controltreatments and the population of leaves on those vines usedfor physiological measurements The vineyard was irrigated1 week before roots were pruned and then every 10ndash14 days at100 L vinendash1 after the end of June

Water relations measurements

Leaf water potential (yleaf) and stem water potential (ystem)measurements were obtained using a pressure chamber(Scholander et al 1965) according to the method by Turner(1988) Leaf water potential measurements were taken eitherduring the pre-dawn hours (0400ndash0500 hours pre-dawn waterpotential yPD) early afternoon (1400ndash1500 hours yleaf) orimmediately following leaf gas-exchange measurements yleaf

was measured on mature leaves that were well exposed to direct

1020 Functional Plant Biology V Zufferey and D R Smart

sunlightMidday stemwater potentials (ystem) weremeasured onnon-transpiring mature leaves by bagging them with a plasticsheet covered with aluminium foil for 10 minutes beforemeasuring water potential

Differences in stomatal behaviour and hydraulic conductivityof the root system have been related to hydraulic continuity usingthe leaf specific hydraulic conductance (Tyree and Sperry 1988)The leaf specific hydraulic conductance Kplant was estimatedfrom the relationshipbetween single leaf transpiration rate and thesoil-xylempotential differenceGenerally the soilwaterpotential(ysoil) has been assumed to be close to yPD (Breda et al 1995)with noted exceptions for desert shrubs (Donovan et al 2003) ornon-homogeneous soils (Ameacuteglio et al 1999) Thus we usedyPD as a reasonable proxy for soil water potential and estimatedKplant from the relationship between the single leaf transpirationrate (E) and the approximate soil-xylemwater potential difference(Sperry and Pockman 1993)

Kplant frac14 E=ethyPD yleaf THORN eth1THORNLeaf gas-exchange measurements and stomatal sensitivityLeaf gas-exchange measurements were conducted on maturewell exposed canopy leaves emerging from nodes above nodesthat carry fruit clusters the four treated vines within a treatmentreplicate We used an open-system gas-exchange module(LI-6400 Li- Cor Lincoln NE USA) and gas-exchangeparameters were calculated using the equations by vonCaemmerer and Farquhar (1981) Measurements were madebeginning before the phenological phase of pea size berries(10 days after root pruning) until harvest Photosynthetic rate(A) transpiration rate (E) and stomatal conductance (gs) weremeasured throughout the day between sunrise and sunset on6 separate days following root pruning From visualassessment of these datasets maximum photosynthetic rate(Amax) and maximum stomatal conductance (gmax) weresubsequently determined between 1000 and 1100 hours Theintrinsic water use efficiency (WUE) was calculated from theratio between the net assimilation rate (A) and the stomatalconductance (gs) which were recorded in the morning between1000 and 1100 hours

In order to examine stomatal response to environmentalfactors independent of root pruning treatments we evaluatedgs with respect to theBall-Berry stomatal controlmodel proposedby Ball et al (1987)

gs frac14 k ARH

Cathorn gs min eth2THORN

where A is net photosynthetic assimilation rate (mmolCO2m

ndash2 sndash1) RH is the relative humidity Ca is the CO2

concentration external to the leaf and k is the slope constantdescribing the sensitivity of stomata toRHACa and temperature(Ball et al 1987)

Statistical analyses

Statistical analyses were performed by analysis of variance(ANOVA) according to the main experimental design whichwas a randomised complete blocks design with one replication ofthe experiment per block (n= 3) Season-long data were analysedby ANOVA using a repeated-measures model (proc glm SAS

ver 82 Cary NC USA) Comparisons were conducted to testhypotheses related to root pruning treatments For meanseparation we conducted individual ANOVAs for each day(eg gmax and Amax) or hour (diurnal gs) All comparisonsdiscussed in the text and referred to as statisticallysignificantly different conform to the probability ofcommitting a Type I error at P 005 unless otherwise statedLinear multiple regressions (SAS ver 82) and non-linearregression (Systat Software Chicago IL USA SigmaPlotver 90) were used to identify relationships among vine waterpotential stomatal conductance photosynthesis and otherphysiological parameters derived from such measurements

Results

Water relations and gas exchange

Partial root removal led to a rapid decrease in viney (yPDyStem

andyleaf Fig 1andashc) This differencewas sustained over the entiregrowing season in the root-restricted plants (RPI and RPD)compared with intact plants Grapevines that had roots prunedfrom the irrigated area of the drip zone (RPI) showed the mostnegative levels of yPD yStem and yleaf The yPD measurements(Fig 1a) indicated that nocturnal water stress remained low inintact vines throughout the season (yPD lt ndash03MPa) whereas itwas moderate (yPD = ndash03 to ndash05MPa) in RPD and RPI vinesWater stress atmidday here defined asystem levels that fell belowroughly ndash10MPa and yleaf measurements below ndash12MPa atmidday (Fig 1b c) was greater in the root-pruned plants than inthe control plants In addition leaf wilting symptoms wereobserved in root-pruned vines only and expansive growth wasslowed (data not shown)

A large and immediate decrease in gmax and Amax wasobserved in root-restricted plants compared with the intactplants (Fig 1d e) gmax and Amax became more stable duringthe 50ndash60 days following root pruning after which both gmax

and Amax began to decline (Fig 1d e) No consistent statisticallysignificant difference in photosynthetic or stomatal conductancerate was observed between the two root restriction treatmentsnevertheless there was a trend for gmax and Amax to besystematically lower in PRI vines (root-restricted fromirrigation source) compared with PRD vines (root-restricted indry non-irrigated soil) There was an overall decreasing trendin the intrinsic WUE during the season The change in intrinsicWUE (Fig 1f) was higher in root-restricted vines than in controlsduring the first 50 days following root removal The RPI vinesshowed the highest levels of WUE of all studied treatments

A linear decay function was fitted to data relating gmax toyPD

(Fig 2a) The fitted curves were significantly different at thePlt 001 level Analysis of the relationship established betweengmax and yPD indicated gmax decreased more rapidly in root-restricted plants than in intact plants as water stress increased(Fig 2a) In other words the same level of available soil water asindicated byyPD gmax was lower in root-restricted plants than inintact plants Factors other than soil water availability appearedto play an important role in this relationship Nonetheless therelationship between gmax and yStem showed that the decrease ingmax with increasing water stress during the day was equivalent(same slope) for intact versus root-removed plants (Fig 2b)A curvilinear decay function was fitted to data relating Amax and

Stomatal behaviour and root removal Functional Plant Biology 1021

yPD (data not shown) At low levels of water stress(yPD gt ndash03MPa) Amax was greater for intact plants than root-restricted plants In contrast the adjusted levels of Amax for the

root-pruned vines remained not significantly different as waterstress increased during the first 25 days following root pruningthen decreased uniformly for all treatments as leaf senescenceensued at the end of the season (Fig 1e)

Stomatal sensitivity and response to VPD

Significant linear relationshipswere obtainedbetween the [A(RHCa)] and gs for both root pruned (pooled) and non-root prunedcontrols (Fig 3) The proposed stomatal sensitivity factor k washigher in vines without root removal than for root-pruned vines

The relationships between VPD andA gs andE are presentedin Fig 4 and are representative for diurnal observations made on25 June 9 July and 16 August Observations made over these3 days with light-saturating conditions indicated that A and gsdecreased as VPD increased (Fig 4a b) independent of rootpruning treatment The decrease in gs in relation to VPD wasbest characterised by a decreasing linear function in the root-restricted plants as well as in intact plants (Fig 4a) gs varied withVPD between the treatments for the same VPD where the root-restricted plants systematically showed lowergs values than intactplantsApositive correlationwasobservedbetweenVPDand leaf

00

ndash01

ndash02

ndash03

ndash04

ndash05

ndash06

ndash03

ndash06

ndash09

ndash12

ndash15

ndash03

ndash06

ndash09

ndash12

ndash15

ndash18

300

250

200

150

100

50

20

15

10

5

0

80

70

60

50

40160 180 200 220 240 260 160 180 200 220 240 260

ControlMidday Mid-morning

Mid-morning

Mid-morning

(c)

Midday(b)

Night(a) (d)

(e)

(f)

RPIRPD

F V H F

Day of year

ΨLe

af (

MP

a)Ψ

PD (

MP

a)Ψ

Ste

m (

MP

a)

g max

(mm

ol H

2O m

ndash2 s

ndash1)

Am

ax

(μm

ol C

O2

mndash2

sndash1

)in

trin

sic

WU

E (

Ag

s)(μ

mol

CO

2m

ol H

2O)

V H

Fig 1 Changes in (a) pre-dawnwater potential (yPD) (b) stemwater potential (yStem) (c) leafwater potential (yleaf) (d)maximumstomatal conductance (gmax) (e)maximumphotosyntheticrate (Amax) and (f) intrinsicwateruseefficiency (WUE)during the2004seasonwithdifferent rootpruning treatments 8 hours following the first pre-dawn leaf water potential measurement(JulianDay 152) 2ndash3 framework roots were removed from the quadrant of the vine trunkwherethe drip irrigation emitter was located (root-pruning irrigated RPI) or from the opposite sidequadrant (root-pruningdryRPD)ThefirstyStemmeasurementwas taken5daysbefore the root-pruning treatments (Julian day 147) and the first yleaf measurement 3 days following rootpruning (Julian day 155) F flowering V veraison H harvest Means se for nine leavesReprinted with permission of the American Journal of Enology and Viticulture

ΨPD (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14

g max

(m

mol

mndash2

sndash1

)

0

50

100

150

200

250

300

ΨStem midday (MPa)

(a) (b)

Control RPIRPD

R2=064

R2=077

R2=075

R2=087

Fig 2 Relationship between the maximal stomatal conductance (gmax) and(a) pre-dawn water potential (yPD) (b) and stem water potential (yStem) forcontrol vines (solid line) and root-pruning treatments together (dashed line)Data se for measurements on nine fully-exposed leaves per treatment

1022 Functional Plant Biology V Zufferey and D R Smart

transpiration E (Fig 4c) In comparison with the root-restrictedplants intact plants showed greater E values particularly at highlevels of VPD E values tended to increase more in intact plantswith increasing evaporative demand (greater slope) than in root-pruned plants

Leaf specific hydraulic conductance

Differences in stomatal behaviour were related to plant hydraulicproperties (Tyree and Sperry 1988) The leaf specific hydraulicconductanceKplant was estimated from the relationship of singleleaf transpiration rate to the soil-xylem potential difference sincethe soil water potential (ys) can be assumed to be very close to thepre-dawn leaf water potential (yPD) (Breda et al 1995) We noteexceptions from desert environments (Donovan et al 2003) andsoils with extreme heterogeneity in available water (Ameacuteglioet al 1999) Fig 5 shows the relationship between diurnal gs asa function of Kplant or yleaf for 3 different days Although norelationshipwas found to exist between gs andyleaf (Fig 5dndashf) gsand Kplant were found to be closely linked (Fig 5andashc) in all thestudy treatments analysed The lowest gs rates were observed onleaves of root-restrictedplants (RPI) andwere associatedwith thelowest values observed for Kplant

These data indicate that differences in stomatal behaviouramong the various treatments were linked to whole-plant

hydraulics Moreover this relationship existed despite of thefact that moderate differences were recorded in leaf areabetween the treatments at 113 22m2 vinendash1 (mean sen= 3) for the control 86 02m2 vinendash1 RPI and 9507m2 vinendash1 RPD (Smart et al 2006) Only the control andRPI treatments differed significantly and represented about a24 difference in leaf area It should be noted that treatmentswith the lowest stomatal sensitivity factor (root-restricted plants)also had the smallest leaf area per plant (Smart et al 2006)and consequently the lowest rate of soil water consumptionIn addition intact plants and root-removed plants showed acommon relationship between Kplant and the degree of waterstress as estimated either by yPD or by yStem (Fig 6)Nonetheless since E Kplant and gs do not represent completelyindependent variables some degree of auto-correlation may beimplicit in the relationships presented (see Schultz 2003)

Discussion

Partial root removal provided an experimental opportunity tomanipulate liquid as well as vapour phase resistances along

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300pre-veraison

Control RPIRPD

05 10 15 20 25 300

50

100

150

200

250

300post-veraison

A (hsCa)

k = 85

k = 62

k = 90

k = 55

Fig 3 Relationship between the [A (RHCa] and the stomatalconductance (gs) for adult leaves on Syrah vines at pre- and post-veraisonin 2004 Pre-veraison data is before Julian day 200 when fruit colouringwas first observed (Fig 1) Control vines (solid line) and root pruningtreatments together (dashed line) k is termed the stomatal sensitivityfactor (and represent the slope in the equation gs = g0 + k A (RHCa)see lsquoMaterials and methodsrsquo)

VPDa (kPa)

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300

A (

μmol

CO

2 m

ndash2 s

ndash1)

0

5

10

15

(a)

0 1 2 3 4

E (

mm

ol H

2O m

ndash2 s

ndash1)

0

2

4

6

8

(b)

(c)

Control RPIRPD

R2 = 079

R2 = 072

R2 = 050

R2 = 082

R2 = 068

R2 = 066

Fig 4 Relationship between (a) stomatal conductance (gs) (b) netphotosynthesis (A) and (c) transpiration (E) and the air vapour pressuredeficit (VPDa) during the course of 3 days (25 June 26 July and 16 August)yPDgt ndash03MPa by control plantsyPDgt ndash045MPa by root-restricted plantsLight-saturating conditions

Stomatal behaviour and root removal Functional Plant Biology 1023

the pathway of water movement in the soilndashplantndashatmospherecontinuum Removal of ~30 of the root system resulted in animmediate (24 h) and substantial decrease in gs and gas exchangeby photosynthesis and (Fig 1b d) Plant water status in contrastlagged slightly behind these changes (Fig 1a c e) These results

may concur with the hypothesis of a lag between an increase inroot water absorption to offset changes in diurnal transpirationdemand (Kramer and Boyer 1995) but suggests an inabilityof remaining roots to improve water uptake Axial waterredistribution can serve to rehydrate the entire vine (Smartet al 2005 Bauerle et al 2008) but in this case where rootswere left intact in irrigated soil and pruned from non-irrigatedsoil axial redistribution or an increase in water uptake couldonly apparently compensate for a portion of the water supplyLeaf wilting symptoms taken together with very negative valuesofyStem andyleaf were indicative of an increase in the hydraulicresistances between the soil and leaves during periods of highevaporative demand The accelerated decline in gmax and Amax atthe end of the season was consistent among treatments and waslikely a consequence of leaf aging (Fig 1b d)

Stomata respond to a variety of moisture related or watertransport factors under light-saturating conditions includinghumidity transpiration soil moisture and critical levels ofwater potential along the soilndashplantndashatmosphere continuum(Sperry et al 2002) Ball et al (1987) proposed the BWBindex as a means of evaluating the combined effects of theclimatic variables of air humidity and external Ca on gsA linear relationship was noted between the BWB index andgs (Fig 3) where apparent stomatal sensitivity was representedby the slope (k) of gs with ARHCa (Leuning 1990 Harleyand Baldocchi 1995) In all cases in the current investigationsensitivity of stomata was greater (higher k values) in theintact plants as compared with the root-restricted plants Thisindicated that internal signals in this case imposed by rootpruning treatments that caused immediate stomatal closure(Fig 1b) removed a portion of the stomatesrsquo ability torespond to external environmental factors of humidity and Ca

as empirically described by the BWB model Nonethelessstomates of both the intact and root pruned vines appearedequally sensitive to VPD throughout the season

The present results show that stomatal closure occurredfollowing an increase in VPD and was linked to a rise in thetranspiration rate (E) of the whole leaf in both intact and root-restricted plants Results of other studies have suggested thatthe closure of stomata with rising VPD appears to be more of afeedback response linked to some other aspect of transpiration(Shackel and Brinckmann 1985 Mott and Franks 2001) and leafwater loss rather than to a direct response to humidity at the leafsurface (Mott and Parkhurst 1991Monteith 1995) Neverthelessunder higher VPD leaf transpiration of root-pruned plants waslower than control plants An explanation for this phenomenonmay lie in part in the decrease ofyleaf in the root-restricted vinesand consequent fall in whole-plant water flux FurthermoreCi ofleaves of root-restricted plants was somewhat lower than intactplants (data not shown) and there was a and similar correlationbetween gs and A This confirmed in part that patchy stomatalclosure was of little significance in the present study and inconditions of progressive developing water deficit (Mott andBuckley 1998)

The observed relationship between stomatal behaviour VPD(Fig 4a) and plant water loss (Fig 4c) further suggests that theresponse to VPD was related to plant hydraulics and that itmay play a role in regulating leaf water potential (Oren et al2001)Underwater deficits progressive closure of stomata canbe

Kplant (mmol MPandash1 mndash2 sndash1)

g s (m

mol

mndash2

sndash1

)

50

100

150

200

250

30025 June

50

100

150

200

250

30026 July

0 2 4 6 8 10 120

50

100

150

200

250

30016 August

(a) (d)

(b) (e)

(c) (f)

Ψleaf (MPa)

ndash18ndash15ndash12ndash09ndash06ndash03

Control RPI

RPD

R2 = 087

R2 = 056

R2 = 071

Fig 5 (andashc) Stomatal conductance (gs) as a function of leaf specificconductance (Kplant) and (dndashf) of leaf water potential (yleaf) for differentroot pruning treatments during 3 days in 2004

ΨPD (MPa) ΨStem (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14 ndash16

Kpl

ant (

mm

ol M

Pandash1

mndash2

sndash1

)

0

2

4

6

8

10(a) (b)

Control RPIRPD

R2 = 075R2 = 077

Fig 6 Relationship between mid-morning leaf specific hydraulicconductance Kplant and (a) pre-dawn water potential (yPD) and (b)midday stem water potential (yStem) for different root pruning treatments

1024 Functional Plant Biology V Zufferey and D R Smart

observed when VPD increases and hydraulic conductance alongthe soil-to-leaf pathway (Kplant) decreases yleaf then remainsabove a critical threshold preventing xylem cavitation andhydraulic failure (Tyree and Sperry 1988) A positivecorrelation between hydraulic and stomatal conductances(Saliendra et al 1995 Comstock 2000 Addington et al2004) but a relatively constant yleaf suggests a feedback linkbetween gs and some form of hydraulic signal (Fuchs andLivingston 1996 Nardini et al 2001) Experimentallyreducing xylem-hydraulic conductance by various techniques(Sperry et al 1993 Hubbard et al 1999 2001) including rootpruning (Teskey et al 1983 Meinzer and Grantz 1990) bringsabout a decrease in gs thereby suggesting the hydraulicconductance for transpiration flux forms an integral anddynamic part of the stomatal control mechanism (Franks2004) Our observations from partial root removal in irrigatedversusnon-irrigated soils supports this contention in asmuchasgsand yleaf in RPI and RPD vines were shifted to different levelswith RPI having lower gs and yleaf

According to Meinzer (2002) true feed-forward control of gswith respect toyleaf would allow stomata to limit transpiration inanticipation of potential reductions in yleaf resulting fromrestricted available water in the soil environment or increasedsoil-to-leaf hydraulic resistance Experiments by Teskey et al(1983) on Abies amabilis and by Meinzer and Grantz (1990) insugarcane demonstrated that gs decreased in response to rootremoval with no change inyleaf This response can be interpretedas generating a hydraulic signal in the form of diminished leaf-specific hydraulic conductance following partial root pruning Ifstomata did not respond by restricting transpiration yleaf woulddecline In the present experiments gs of root-restricted plantsdecreased immediately asKplant fell during the day This lead to arapid drop in yleaf (and leaf wilting) in root-pruned plantscompared with intact plants This response differed from theapparent feed-forward stomatal behaviour in response to rootpruning where stomata appeared to overcompensate and causeyleaf to be noticeably less negative following root removal thanbefore (Meinzer and Grantz 1990)

Feed-forward behaviour of stomata with respect to regulationof yleaf has been attributed to the presence of chemical signalsthat modify stomatal opening and which are brought to theleaf in the transpiration flow (Davies et al 1994) In drought-avoiding plants (isohydric) the leaf water potential is unaffectedby soil water deficits and isohydric behaviour is linked to aninteraction between both chemical and hydraulic information(Tardieu and Simonneau 1998) In drought-tolerant species(anisohydric) a controlling influence of leaf water status on gsneed not be invoked Rather leaf water status is likely to vary as aconsequence of water flux through the plant which is controlledby stomatal conductance Schultz (2003) observed that thegrapevine cultivar lsquoSyrahrsquo behaves in an anisohydric mannerand that the differences in thewater-conducting capacity of stemsand especially petioles may be the origin of the isohydric andanisohydric signal regulating gs in grapevine Results from thepresent study showed that yleaf of lsquoSyrahrsquo fell sharply asevaporative demand rose During the daytime and when waterstress was accentuated as a result of partial root removal morenegative leaf water potentials were realised compared withwell-watered intact plants This provided some confirmation of

anisohydric behaviour but also suggested root and other signalsin the plant hydraulic path have a strong influence on stomatalbehaviour Other contradictory studies have been reported inthe literature (see review by Chaves et al 2010) showingthat the same cultivar could behave differently dependingon experimental conditions (eg intensity and duration ofwater deficits VPD and root system) and physiological oranatomical adjustments (eg cell osmotic adjustment wallelasticity vessel conductivity)

However no recovery of Kplant was observed during theseason in root-restricted plants in comparison with the intactcontrols An increase in the total root surface area for absorptionby the growth of new roots after partial root pruning couldimprove Kplant (Sperry et al 2002) and Alsina and co-workersverified this hypothesis for grape (Alsina et al 2011) It is notcertain that roots produced during a period of water stress(drought) will have sufficient water conductivity (Ewers et al2000)Atgt60 cmdepth inmoist soils root conductivity increasedbut gs did not increase substantially Either the failure to producenew roots or lower conductivity would help to explain whyKplant

in root-restricted vines did not regain hydraulic conductancelevels approaching those of the intact non-root pruned vinesduring the growing season Moreover it should be noted thatdiurnal measurements of Kplant conducted in the field may giverise to errors if they were conducted under non-steady-stateconditions (Hubbard et al 2001) Non-steady-state conditionsmight arise as a consequence of significant capacitance (Schultzand Matthews 1997 Smart et al 2005) or rapid changes inenvironmental conditions over time

Partial root removal may also have had an influence onembolism development as a consequence of rapid changes inwater potential and as a consequence may have contributed tostomatal closure by an embolism related hydraulic signal(Lovisolo and Schubert 1998) Salleo et al (2000) suggestedthat even cavitation could act as a hydraulicmodulator for stomataopening The impact of cavitation on total hydraulic conductanceand finally on gs would depend on the position of cavitation inthe root-leaf pathway and on the distribution of hydraulicresistances between roots stems petioles and leaves (Meinzer2002 Zufferey et al 2011) Root systems often constitute a largepart of the total plant hydraulic resistance (Tyree et al 1998) andthe relative contributions of stem and leaf resistance to theremaining shoot resistance can vary substantially (Choat et al2010) Leaf wilting symptoms together with the very low gsvalues observed in this study for root-restricted vines andparticularly in root-pruned plants in wet irrigated soils may beattributed in part to an increased vulnerability of xylem tocavitation coupled with a high evaporative demand Thesecavitation events may act in this way as a signal for stomatalclosure (Nardini et al 2001) or stomatal closure would occur toprevent cavitation (Cochard et al 2002)

Significant decreases in gs recorded in root-restrictedplants were closely related to a reduction in leaf-specifichydraulic conductance (Fig 5) However other factors suchas hormonal control or even a combination of chemical andhydraulic messages which various authors (Tardieu andDavies 1993 Tardieu and Simonneau 1998 reviewed byHolbrook et al 2002) have brought to light cannot be entirelyruled out and have not been directly evaluated in this

Stomatal behaviour and root removal Functional Plant Biology 1025

investigationTheobservation thatgmaxwasdifferent between theroot-restricted plants and intact plants at similaryPD representingdifferent soilwater status prospectedby the roots andpresumablytriggering ABA synthesis (or other hormonal factors such ascytokinins) suggests that either the signals were different innature or concentration or the receptor site sensitivity variedbetween the different root pruning treatments We also notethat yPD changed within 48 h following root pruning Thisindicated that root pruned vines were not able to refill xylemand recharge hydraulic status to the same extent as the intactcontrols despite of the fact that roots of RPD vines had rootsexposed to the same irrigation source as the controls This hasshown that axial redistribution and reverse flow of water occursto such an extent that rehydration of lateral roots occurs (Smartet al 2005 Burgess and Bleby 2006) This raises questionswith respect to root signalling If pruning of roughly 30 ofthe root system occurs then it might be a reasonable assumptionthat removal of 30 of fine roots and fine root tips occurs whichmay be the primary source of the root signals that influence gs(Bauerle et al 2008) Further this suggests that root signallingwas of lesser importance in the initial response of gs and A to rootpruning

References

AddingtonRNMitchell RJOrenRDonovanLA (2004)Stomatal sensitivityto vapor pressure deficit and its relationship to hydraulic conductance inPinus palustris Tree Physiology 24 561ndash569 doi101093treephys245561

AlsinaMMSmartDRBauerleTdeHerraldeFBielCStockertCMNegronCSaveR (2011)Seasonal changes ofwhole root systemconductanceby adrought tolerant grape root system Journal of Experimental Botany 6299ndash109 doi101093jxberq247

Ameacuteglio TArcher P CohenMValancogneCDaudet FDayauSCruiziat P(1999) Significance and limits in the use of predawn leaf water potentialfor tree irrigation Plant and Soil 207 155ndash167 doi101023A1026415302759

Ball JT Woodrow IE Berry JA (1987) A model predicting stomatalconductance and its contribution to the control of photosynthesis underdifferent environmental conditions In lsquoProgress in photosynthesisresearchrsquo (Ed J Biggins) pp 221ndash234 (Martinus Nijhoff DordrechtThe Netherlands)

Bauerle TL Richards JH Smart DR Eissenstat DM (2008) Importance ofinternal hydraulic redistribution for prolonging the lifespan of roots in drysoil Plant Cell amp Environment 31 177ndash186

Breda N Granier A Barataud F Moyne C (1995) Soil water dynamics in anoak stand I Soil moisture water potentials and water uptake by rootsPlant and Soil 172 17ndash27

Brodribb TJ Holbrook NM (2003) Stomatal closure during leafdehydratation correlation with other leaf physiological traits PlantPhysiology 132 2166ndash2173 doi101104pp103023879

Burgess SSO Bleby TM (2006) Redistribution of soil water to lateral rootsmediatedby stem tissuesJournalofExperimentalBotany57 3283ndash3291doi101093jxberl085

ChavesMMTenhunen JDHarley P LangeOL (1987)Gas exchange studiesin two Portuguese grapevine cultivars Physiologia Plantarum 70639ndash647 doi101111j1399-30541987tb04318x

Chaves MM Zarrouk O Fransisco R Costa JM Santos T Regalado APRodriguesMLLopesCM (2010)Grapevine under deficit irrigation hintsfrom physiological and molecular data Annals of Botany 105 661ndash676doi101093aobmcq030

Choat B DraytonWMBrodersen CRMatthewsMA Shackel KAWadaHMcElrone AJ (2010) Measurement of vulnerability to water stress-induced cavitation in grapevine a comparison of four techniquesapplied to long-vesseled species Plant Cell amp Environment 331502ndash1512

Cochard H Coll L Le Roux X Ameglio T (2002) Unraveling the effects ofplant hydraulics on stomatal closure during water stress in walnut PlantPhysiology 128 282ndash290 doi101104pp010400

Comstock JP (2000) Variation in hydraulic architecture and gas exchange intwo desert sub-shrubs Hymenoclea salsola (TampG) and Ambrosiadumosa Oecologia 125 1ndash10 doi101007PL00008879

Correia MJ Pereira JS Chaves MM Rodrigues ML Pacheco CA (1995)ABA xylem concentrations determine maximum daily leaf conductanceof field-grown Vitis vinifera L plants Plant Cell amp Environment 18511ndash521 doi101111j1365-30401995tb00551x

Davies WJ Zhang J (1991) Root signals and the regulation of growth anddevelopment of plants in drying soil Annual Review of Plant Physiologyand Plant Molecular Biology 42 55ndash76 doi101146annurevpp42060191000415

Davies WJ Tardieu F Trejo CL (1994) How do chemical signals work inplants that grow in drying soil Plant Physiology 104 309ndash314

Donovan LA Richards JH LintonMJ (2003)Magnitude and mechanisms ofpredawn disequilibrium between predawn and plant soil water potentialsin desert shrubsEcology84 463ndash470 doi1018900012-9658(2003)084[0463MAMODB]20CO2

Ewers BEOrenR Sperry JS (2000) Influence of nutrient versuswater supplyon hydraulic architecture and water balance in Pinus taeda Plant Cell ampEnvironment 23 1055ndash1066 doi101046j1365-3040200000625x

Franks PJ (2004) Stomatal control and hydraulic conductance with specialreference to tall trees Tree Physiology 24 865ndash878 doi101093treephys248865

FuchsEELivingstonNJ (1996)Hydraulic control of stomatal conductance inDouglasfir (Pseudotsugamensiesii (Mirb) Franco) and alder (Alnus rubra(Bong)) seedlings Plant Cell amp Environment 19 1091ndash1098doi101111j1365-30401996tb00216x

Harley PC Baldocchi DD (1995) Scaling carbon dioxide and water vapourexchange from leaf to canopy in a deciduous forest I Leaf modelparametrization Plant Cell amp Environment 18 1146ndash1156doi101111j1365-30401995tb00625x

Holbrook NM Shashidhar VR James RAMunns R (2002) Stomatal controlin tomato with ABA-deficient roots response of grafted plants to soildrying Journal of Experimental Botany 53 1503ndash1514 doi101093jexbot533731503

Hubbard RM Bond BJ Ryan MG (1999) Evidence that hydraulicconductance limits photosynthesis in old Pinus ponderosa trees TreePhysiology 19 165ndash172 doi101093treephys193165

Hubbard RM Ryan MG Stiller V Sperry JS (2001) Stomatal conductanceand photosynthesis vary lineary with plant hydraulic conductance inPonderosa pine Plant Cell amp Environment 24 113ndash121 doi101046j1365-3040200100660x

Jarvis AJ Davies WJ (1998) Modeling stomatal responses to soil andatmospheric drought Journal of Experimental Botany 49(SpecialIssue) 399ndash406 doi101093jxb49Special_Issue399

Jones HG (1998) Stomatal control of photosynthesis and transpirationJournal of Experimental Botany 49(Special Issue) 387ndash398

Kramer PJ Boyer JS (1995) lsquoWater relations of plants and soilsrsquo (AcademicPress San Diego CA)

Leuning R (1990) Modeling stomatal behaviour and photosynthesis ofEucalyptus grandis Australian Journal of Plant Physiology 17159ndash175 doi101071PP9900159

Loveys BR (1991) How useful is a knowledge of ABA physiology for cropimprovement In lsquoAbscisic acid physiology and biochemistryrsquo (Eds WJDavies HG Jones) pp 245ndash260 (Bios Scientific Publishers Oxford)

1026 Functional Plant Biology V Zufferey and D R Smart

sunlightMidday stemwater potentials (ystem) weremeasured onnon-transpiring mature leaves by bagging them with a plasticsheet covered with aluminium foil for 10 minutes beforemeasuring water potential

Differences in stomatal behaviour and hydraulic conductivityof the root system have been related to hydraulic continuity usingthe leaf specific hydraulic conductance (Tyree and Sperry 1988)The leaf specific hydraulic conductance Kplant was estimatedfrom the relationshipbetween single leaf transpiration rate and thesoil-xylempotential differenceGenerally the soilwaterpotential(ysoil) has been assumed to be close to yPD (Breda et al 1995)with noted exceptions for desert shrubs (Donovan et al 2003) ornon-homogeneous soils (Ameacuteglio et al 1999) Thus we usedyPD as a reasonable proxy for soil water potential and estimatedKplant from the relationship between the single leaf transpirationrate (E) and the approximate soil-xylemwater potential difference(Sperry and Pockman 1993)

Kplant frac14 E=ethyPD yleaf THORN eth1THORNLeaf gas-exchange measurements and stomatal sensitivityLeaf gas-exchange measurements were conducted on maturewell exposed canopy leaves emerging from nodes above nodesthat carry fruit clusters the four treated vines within a treatmentreplicate We used an open-system gas-exchange module(LI-6400 Li- Cor Lincoln NE USA) and gas-exchangeparameters were calculated using the equations by vonCaemmerer and Farquhar (1981) Measurements were madebeginning before the phenological phase of pea size berries(10 days after root pruning) until harvest Photosynthetic rate(A) transpiration rate (E) and stomatal conductance (gs) weremeasured throughout the day between sunrise and sunset on6 separate days following root pruning From visualassessment of these datasets maximum photosynthetic rate(Amax) and maximum stomatal conductance (gmax) weresubsequently determined between 1000 and 1100 hours Theintrinsic water use efficiency (WUE) was calculated from theratio between the net assimilation rate (A) and the stomatalconductance (gs) which were recorded in the morning between1000 and 1100 hours

In order to examine stomatal response to environmentalfactors independent of root pruning treatments we evaluatedgs with respect to theBall-Berry stomatal controlmodel proposedby Ball et al (1987)

gs frac14 k ARH

Cathorn gs min eth2THORN

where A is net photosynthetic assimilation rate (mmolCO2m

ndash2 sndash1) RH is the relative humidity Ca is the CO2

concentration external to the leaf and k is the slope constantdescribing the sensitivity of stomata toRHACa and temperature(Ball et al 1987)

Statistical analyses

Statistical analyses were performed by analysis of variance(ANOVA) according to the main experimental design whichwas a randomised complete blocks design with one replication ofthe experiment per block (n= 3) Season-long data were analysedby ANOVA using a repeated-measures model (proc glm SAS

ver 82 Cary NC USA) Comparisons were conducted to testhypotheses related to root pruning treatments For meanseparation we conducted individual ANOVAs for each day(eg gmax and Amax) or hour (diurnal gs) All comparisonsdiscussed in the text and referred to as statisticallysignificantly different conform to the probability ofcommitting a Type I error at P 005 unless otherwise statedLinear multiple regressions (SAS ver 82) and non-linearregression (Systat Software Chicago IL USA SigmaPlotver 90) were used to identify relationships among vine waterpotential stomatal conductance photosynthesis and otherphysiological parameters derived from such measurements

Results

Water relations and gas exchange

Partial root removal led to a rapid decrease in viney (yPDyStem

andyleaf Fig 1andashc) This differencewas sustained over the entiregrowing season in the root-restricted plants (RPI and RPD)compared with intact plants Grapevines that had roots prunedfrom the irrigated area of the drip zone (RPI) showed the mostnegative levels of yPD yStem and yleaf The yPD measurements(Fig 1a) indicated that nocturnal water stress remained low inintact vines throughout the season (yPD lt ndash03MPa) whereas itwas moderate (yPD = ndash03 to ndash05MPa) in RPD and RPI vinesWater stress atmidday here defined asystem levels that fell belowroughly ndash10MPa and yleaf measurements below ndash12MPa atmidday (Fig 1b c) was greater in the root-pruned plants than inthe control plants In addition leaf wilting symptoms wereobserved in root-pruned vines only and expansive growth wasslowed (data not shown)

A large and immediate decrease in gmax and Amax wasobserved in root-restricted plants compared with the intactplants (Fig 1d e) gmax and Amax became more stable duringthe 50ndash60 days following root pruning after which both gmax

and Amax began to decline (Fig 1d e) No consistent statisticallysignificant difference in photosynthetic or stomatal conductancerate was observed between the two root restriction treatmentsnevertheless there was a trend for gmax and Amax to besystematically lower in PRI vines (root-restricted fromirrigation source) compared with PRD vines (root-restricted indry non-irrigated soil) There was an overall decreasing trendin the intrinsic WUE during the season The change in intrinsicWUE (Fig 1f) was higher in root-restricted vines than in controlsduring the first 50 days following root removal The RPI vinesshowed the highest levels of WUE of all studied treatments

A linear decay function was fitted to data relating gmax toyPD

(Fig 2a) The fitted curves were significantly different at thePlt 001 level Analysis of the relationship established betweengmax and yPD indicated gmax decreased more rapidly in root-restricted plants than in intact plants as water stress increased(Fig 2a) In other words the same level of available soil water asindicated byyPD gmax was lower in root-restricted plants than inintact plants Factors other than soil water availability appearedto play an important role in this relationship Nonetheless therelationship between gmax and yStem showed that the decrease ingmax with increasing water stress during the day was equivalent(same slope) for intact versus root-removed plants (Fig 2b)A curvilinear decay function was fitted to data relating Amax and

Stomatal behaviour and root removal Functional Plant Biology 1021

yPD (data not shown) At low levels of water stress(yPD gt ndash03MPa) Amax was greater for intact plants than root-restricted plants In contrast the adjusted levels of Amax for the

root-pruned vines remained not significantly different as waterstress increased during the first 25 days following root pruningthen decreased uniformly for all treatments as leaf senescenceensued at the end of the season (Fig 1e)

Stomatal sensitivity and response to VPD

Significant linear relationshipswere obtainedbetween the [A(RHCa)] and gs for both root pruned (pooled) and non-root prunedcontrols (Fig 3) The proposed stomatal sensitivity factor k washigher in vines without root removal than for root-pruned vines

The relationships between VPD andA gs andE are presentedin Fig 4 and are representative for diurnal observations made on25 June 9 July and 16 August Observations made over these3 days with light-saturating conditions indicated that A and gsdecreased as VPD increased (Fig 4a b) independent of rootpruning treatment The decrease in gs in relation to VPD wasbest characterised by a decreasing linear function in the root-restricted plants as well as in intact plants (Fig 4a) gs varied withVPD between the treatments for the same VPD where the root-restricted plants systematically showed lowergs values than intactplantsApositive correlationwasobservedbetweenVPDand leaf

00

ndash01

ndash02

ndash03

ndash04

ndash05

ndash06

ndash03

ndash06

ndash09

ndash12

ndash15

ndash03

ndash06

ndash09

ndash12

ndash15

ndash18

300

250

200

150

100

50

20

15

10

5

0

80

70

60

50

40160 180 200 220 240 260 160 180 200 220 240 260

ControlMidday Mid-morning

Mid-morning

Mid-morning

(c)

Midday(b)

Night(a) (d)

(e)

(f)

RPIRPD

F V H F

Day of year

ΨLe

af (

MP

a)Ψ

PD (

MP

a)Ψ

Ste

m (

MP

a)

g max

(mm

ol H

2O m

ndash2 s

ndash1)

Am

ax

(μm

ol C

O2

mndash2

sndash1

)in

trin

sic

WU

E (

Ag

s)(μ

mol

CO

2m

ol H

2O)

V H

Fig 1 Changes in (a) pre-dawnwater potential (yPD) (b) stemwater potential (yStem) (c) leafwater potential (yleaf) (d)maximumstomatal conductance (gmax) (e)maximumphotosyntheticrate (Amax) and (f) intrinsicwateruseefficiency (WUE)during the2004seasonwithdifferent rootpruning treatments 8 hours following the first pre-dawn leaf water potential measurement(JulianDay 152) 2ndash3 framework roots were removed from the quadrant of the vine trunkwherethe drip irrigation emitter was located (root-pruning irrigated RPI) or from the opposite sidequadrant (root-pruningdryRPD)ThefirstyStemmeasurementwas taken5daysbefore the root-pruning treatments (Julian day 147) and the first yleaf measurement 3 days following rootpruning (Julian day 155) F flowering V veraison H harvest Means se for nine leavesReprinted with permission of the American Journal of Enology and Viticulture

ΨPD (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14

g max

(m

mol

mndash2

sndash1

)

0

50

100

150

200

250

300

ΨStem midday (MPa)

(a) (b)

Control RPIRPD

R2=064

R2=077

R2=075

R2=087

Fig 2 Relationship between the maximal stomatal conductance (gmax) and(a) pre-dawn water potential (yPD) (b) and stem water potential (yStem) forcontrol vines (solid line) and root-pruning treatments together (dashed line)Data se for measurements on nine fully-exposed leaves per treatment

1022 Functional Plant Biology V Zufferey and D R Smart

transpiration E (Fig 4c) In comparison with the root-restrictedplants intact plants showed greater E values particularly at highlevels of VPD E values tended to increase more in intact plantswith increasing evaporative demand (greater slope) than in root-pruned plants

Leaf specific hydraulic conductance

Differences in stomatal behaviour were related to plant hydraulicproperties (Tyree and Sperry 1988) The leaf specific hydraulicconductanceKplant was estimated from the relationship of singleleaf transpiration rate to the soil-xylem potential difference sincethe soil water potential (ys) can be assumed to be very close to thepre-dawn leaf water potential (yPD) (Breda et al 1995) We noteexceptions from desert environments (Donovan et al 2003) andsoils with extreme heterogeneity in available water (Ameacuteglioet al 1999) Fig 5 shows the relationship between diurnal gs asa function of Kplant or yleaf for 3 different days Although norelationshipwas found to exist between gs andyleaf (Fig 5dndashf) gsand Kplant were found to be closely linked (Fig 5andashc) in all thestudy treatments analysed The lowest gs rates were observed onleaves of root-restrictedplants (RPI) andwere associatedwith thelowest values observed for Kplant

These data indicate that differences in stomatal behaviouramong the various treatments were linked to whole-plant

hydraulics Moreover this relationship existed despite of thefact that moderate differences were recorded in leaf areabetween the treatments at 113 22m2 vinendash1 (mean sen= 3) for the control 86 02m2 vinendash1 RPI and 9507m2 vinendash1 RPD (Smart et al 2006) Only the control andRPI treatments differed significantly and represented about a24 difference in leaf area It should be noted that treatmentswith the lowest stomatal sensitivity factor (root-restricted plants)also had the smallest leaf area per plant (Smart et al 2006)and consequently the lowest rate of soil water consumptionIn addition intact plants and root-removed plants showed acommon relationship between Kplant and the degree of waterstress as estimated either by yPD or by yStem (Fig 6)Nonetheless since E Kplant and gs do not represent completelyindependent variables some degree of auto-correlation may beimplicit in the relationships presented (see Schultz 2003)

Discussion

Partial root removal provided an experimental opportunity tomanipulate liquid as well as vapour phase resistances along

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300pre-veraison

Control RPIRPD

05 10 15 20 25 300

50

100

150

200

250

300post-veraison

A (hsCa)

k = 85

k = 62

k = 90

k = 55

Fig 3 Relationship between the [A (RHCa] and the stomatalconductance (gs) for adult leaves on Syrah vines at pre- and post-veraisonin 2004 Pre-veraison data is before Julian day 200 when fruit colouringwas first observed (Fig 1) Control vines (solid line) and root pruningtreatments together (dashed line) k is termed the stomatal sensitivityfactor (and represent the slope in the equation gs = g0 + k A (RHCa)see lsquoMaterials and methodsrsquo)

VPDa (kPa)

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300

A (

μmol

CO

2 m

ndash2 s

ndash1)

0

5

10

15

(a)

0 1 2 3 4

E (

mm

ol H

2O m

ndash2 s

ndash1)

0

2

4

6

8

(b)

(c)

Control RPIRPD

R2 = 079

R2 = 072

R2 = 050

R2 = 082

R2 = 068

R2 = 066

Fig 4 Relationship between (a) stomatal conductance (gs) (b) netphotosynthesis (A) and (c) transpiration (E) and the air vapour pressuredeficit (VPDa) during the course of 3 days (25 June 26 July and 16 August)yPDgt ndash03MPa by control plantsyPDgt ndash045MPa by root-restricted plantsLight-saturating conditions

Stomatal behaviour and root removal Functional Plant Biology 1023

the pathway of water movement in the soilndashplantndashatmospherecontinuum Removal of ~30 of the root system resulted in animmediate (24 h) and substantial decrease in gs and gas exchangeby photosynthesis and (Fig 1b d) Plant water status in contrastlagged slightly behind these changes (Fig 1a c e) These results

may concur with the hypothesis of a lag between an increase inroot water absorption to offset changes in diurnal transpirationdemand (Kramer and Boyer 1995) but suggests an inabilityof remaining roots to improve water uptake Axial waterredistribution can serve to rehydrate the entire vine (Smartet al 2005 Bauerle et al 2008) but in this case where rootswere left intact in irrigated soil and pruned from non-irrigatedsoil axial redistribution or an increase in water uptake couldonly apparently compensate for a portion of the water supplyLeaf wilting symptoms taken together with very negative valuesofyStem andyleaf were indicative of an increase in the hydraulicresistances between the soil and leaves during periods of highevaporative demand The accelerated decline in gmax and Amax atthe end of the season was consistent among treatments and waslikely a consequence of leaf aging (Fig 1b d)

Stomata respond to a variety of moisture related or watertransport factors under light-saturating conditions includinghumidity transpiration soil moisture and critical levels ofwater potential along the soilndashplantndashatmosphere continuum(Sperry et al 2002) Ball et al (1987) proposed the BWBindex as a means of evaluating the combined effects of theclimatic variables of air humidity and external Ca on gsA linear relationship was noted between the BWB index andgs (Fig 3) where apparent stomatal sensitivity was representedby the slope (k) of gs with ARHCa (Leuning 1990 Harleyand Baldocchi 1995) In all cases in the current investigationsensitivity of stomata was greater (higher k values) in theintact plants as compared with the root-restricted plants Thisindicated that internal signals in this case imposed by rootpruning treatments that caused immediate stomatal closure(Fig 1b) removed a portion of the stomatesrsquo ability torespond to external environmental factors of humidity and Ca

as empirically described by the BWB model Nonethelessstomates of both the intact and root pruned vines appearedequally sensitive to VPD throughout the season

The present results show that stomatal closure occurredfollowing an increase in VPD and was linked to a rise in thetranspiration rate (E) of the whole leaf in both intact and root-restricted plants Results of other studies have suggested thatthe closure of stomata with rising VPD appears to be more of afeedback response linked to some other aspect of transpiration(Shackel and Brinckmann 1985 Mott and Franks 2001) and leafwater loss rather than to a direct response to humidity at the leafsurface (Mott and Parkhurst 1991Monteith 1995) Neverthelessunder higher VPD leaf transpiration of root-pruned plants waslower than control plants An explanation for this phenomenonmay lie in part in the decrease ofyleaf in the root-restricted vinesand consequent fall in whole-plant water flux FurthermoreCi ofleaves of root-restricted plants was somewhat lower than intactplants (data not shown) and there was a and similar correlationbetween gs and A This confirmed in part that patchy stomatalclosure was of little significance in the present study and inconditions of progressive developing water deficit (Mott andBuckley 1998)

The observed relationship between stomatal behaviour VPD(Fig 4a) and plant water loss (Fig 4c) further suggests that theresponse to VPD was related to plant hydraulics and that itmay play a role in regulating leaf water potential (Oren et al2001)Underwater deficits progressive closure of stomata canbe

Kplant (mmol MPandash1 mndash2 sndash1)

g s (m

mol

mndash2

sndash1

)

50

100

150

200

250

30025 June

50

100

150

200

250

30026 July

0 2 4 6 8 10 120

50

100

150

200

250

30016 August

(a) (d)

(b) (e)

(c) (f)

Ψleaf (MPa)

ndash18ndash15ndash12ndash09ndash06ndash03

Control RPI

RPD

R2 = 087

R2 = 056

R2 = 071

Fig 5 (andashc) Stomatal conductance (gs) as a function of leaf specificconductance (Kplant) and (dndashf) of leaf water potential (yleaf) for differentroot pruning treatments during 3 days in 2004

ΨPD (MPa) ΨStem (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14 ndash16

Kpl

ant (

mm

ol M

Pandash1

mndash2

sndash1

)

0

2

4

6

8

10(a) (b)

Control RPIRPD

R2 = 075R2 = 077

Fig 6 Relationship between mid-morning leaf specific hydraulicconductance Kplant and (a) pre-dawn water potential (yPD) and (b)midday stem water potential (yStem) for different root pruning treatments

1024 Functional Plant Biology V Zufferey and D R Smart

observed when VPD increases and hydraulic conductance alongthe soil-to-leaf pathway (Kplant) decreases yleaf then remainsabove a critical threshold preventing xylem cavitation andhydraulic failure (Tyree and Sperry 1988) A positivecorrelation between hydraulic and stomatal conductances(Saliendra et al 1995 Comstock 2000 Addington et al2004) but a relatively constant yleaf suggests a feedback linkbetween gs and some form of hydraulic signal (Fuchs andLivingston 1996 Nardini et al 2001) Experimentallyreducing xylem-hydraulic conductance by various techniques(Sperry et al 1993 Hubbard et al 1999 2001) including rootpruning (Teskey et al 1983 Meinzer and Grantz 1990) bringsabout a decrease in gs thereby suggesting the hydraulicconductance for transpiration flux forms an integral anddynamic part of the stomatal control mechanism (Franks2004) Our observations from partial root removal in irrigatedversusnon-irrigated soils supports this contention in asmuchasgsand yleaf in RPI and RPD vines were shifted to different levelswith RPI having lower gs and yleaf

According to Meinzer (2002) true feed-forward control of gswith respect toyleaf would allow stomata to limit transpiration inanticipation of potential reductions in yleaf resulting fromrestricted available water in the soil environment or increasedsoil-to-leaf hydraulic resistance Experiments by Teskey et al(1983) on Abies amabilis and by Meinzer and Grantz (1990) insugarcane demonstrated that gs decreased in response to rootremoval with no change inyleaf This response can be interpretedas generating a hydraulic signal in the form of diminished leaf-specific hydraulic conductance following partial root pruning Ifstomata did not respond by restricting transpiration yleaf woulddecline In the present experiments gs of root-restricted plantsdecreased immediately asKplant fell during the day This lead to arapid drop in yleaf (and leaf wilting) in root-pruned plantscompared with intact plants This response differed from theapparent feed-forward stomatal behaviour in response to rootpruning where stomata appeared to overcompensate and causeyleaf to be noticeably less negative following root removal thanbefore (Meinzer and Grantz 1990)

Feed-forward behaviour of stomata with respect to regulationof yleaf has been attributed to the presence of chemical signalsthat modify stomatal opening and which are brought to theleaf in the transpiration flow (Davies et al 1994) In drought-avoiding plants (isohydric) the leaf water potential is unaffectedby soil water deficits and isohydric behaviour is linked to aninteraction between both chemical and hydraulic information(Tardieu and Simonneau 1998) In drought-tolerant species(anisohydric) a controlling influence of leaf water status on gsneed not be invoked Rather leaf water status is likely to vary as aconsequence of water flux through the plant which is controlledby stomatal conductance Schultz (2003) observed that thegrapevine cultivar lsquoSyrahrsquo behaves in an anisohydric mannerand that the differences in thewater-conducting capacity of stemsand especially petioles may be the origin of the isohydric andanisohydric signal regulating gs in grapevine Results from thepresent study showed that yleaf of lsquoSyrahrsquo fell sharply asevaporative demand rose During the daytime and when waterstress was accentuated as a result of partial root removal morenegative leaf water potentials were realised compared withwell-watered intact plants This provided some confirmation of

anisohydric behaviour but also suggested root and other signalsin the plant hydraulic path have a strong influence on stomatalbehaviour Other contradictory studies have been reported inthe literature (see review by Chaves et al 2010) showingthat the same cultivar could behave differently dependingon experimental conditions (eg intensity and duration ofwater deficits VPD and root system) and physiological oranatomical adjustments (eg cell osmotic adjustment wallelasticity vessel conductivity)

However no recovery of Kplant was observed during theseason in root-restricted plants in comparison with the intactcontrols An increase in the total root surface area for absorptionby the growth of new roots after partial root pruning couldimprove Kplant (Sperry et al 2002) and Alsina and co-workersverified this hypothesis for grape (Alsina et al 2011) It is notcertain that roots produced during a period of water stress(drought) will have sufficient water conductivity (Ewers et al2000)Atgt60 cmdepth inmoist soils root conductivity increasedbut gs did not increase substantially Either the failure to producenew roots or lower conductivity would help to explain whyKplant

in root-restricted vines did not regain hydraulic conductancelevels approaching those of the intact non-root pruned vinesduring the growing season Moreover it should be noted thatdiurnal measurements of Kplant conducted in the field may giverise to errors if they were conducted under non-steady-stateconditions (Hubbard et al 2001) Non-steady-state conditionsmight arise as a consequence of significant capacitance (Schultzand Matthews 1997 Smart et al 2005) or rapid changes inenvironmental conditions over time

Partial root removal may also have had an influence onembolism development as a consequence of rapid changes inwater potential and as a consequence may have contributed tostomatal closure by an embolism related hydraulic signal(Lovisolo and Schubert 1998) Salleo et al (2000) suggestedthat even cavitation could act as a hydraulicmodulator for stomataopening The impact of cavitation on total hydraulic conductanceand finally on gs would depend on the position of cavitation inthe root-leaf pathway and on the distribution of hydraulicresistances between roots stems petioles and leaves (Meinzer2002 Zufferey et al 2011) Root systems often constitute a largepart of the total plant hydraulic resistance (Tyree et al 1998) andthe relative contributions of stem and leaf resistance to theremaining shoot resistance can vary substantially (Choat et al2010) Leaf wilting symptoms together with the very low gsvalues observed in this study for root-restricted vines andparticularly in root-pruned plants in wet irrigated soils may beattributed in part to an increased vulnerability of xylem tocavitation coupled with a high evaporative demand Thesecavitation events may act in this way as a signal for stomatalclosure (Nardini et al 2001) or stomatal closure would occur toprevent cavitation (Cochard et al 2002)

Significant decreases in gs recorded in root-restrictedplants were closely related to a reduction in leaf-specifichydraulic conductance (Fig 5) However other factors suchas hormonal control or even a combination of chemical andhydraulic messages which various authors (Tardieu andDavies 1993 Tardieu and Simonneau 1998 reviewed byHolbrook et al 2002) have brought to light cannot be entirelyruled out and have not been directly evaluated in this

Stomatal behaviour and root removal Functional Plant Biology 1025

investigationTheobservation thatgmaxwasdifferent between theroot-restricted plants and intact plants at similaryPD representingdifferent soilwater status prospectedby the roots andpresumablytriggering ABA synthesis (or other hormonal factors such ascytokinins) suggests that either the signals were different innature or concentration or the receptor site sensitivity variedbetween the different root pruning treatments We also notethat yPD changed within 48 h following root pruning Thisindicated that root pruned vines were not able to refill xylemand recharge hydraulic status to the same extent as the intactcontrols despite of the fact that roots of RPD vines had rootsexposed to the same irrigation source as the controls This hasshown that axial redistribution and reverse flow of water occursto such an extent that rehydration of lateral roots occurs (Smartet al 2005 Burgess and Bleby 2006) This raises questionswith respect to root signalling If pruning of roughly 30 ofthe root system occurs then it might be a reasonable assumptionthat removal of 30 of fine roots and fine root tips occurs whichmay be the primary source of the root signals that influence gs(Bauerle et al 2008) Further this suggests that root signallingwas of lesser importance in the initial response of gs and A to rootpruning

References

AddingtonRNMitchell RJOrenRDonovanLA (2004)Stomatal sensitivityto vapor pressure deficit and its relationship to hydraulic conductance inPinus palustris Tree Physiology 24 561ndash569 doi101093treephys245561

AlsinaMMSmartDRBauerleTdeHerraldeFBielCStockertCMNegronCSaveR (2011)Seasonal changes ofwhole root systemconductanceby adrought tolerant grape root system Journal of Experimental Botany 6299ndash109 doi101093jxberq247

Ameacuteglio TArcher P CohenMValancogneCDaudet FDayauSCruiziat P(1999) Significance and limits in the use of predawn leaf water potentialfor tree irrigation Plant and Soil 207 155ndash167 doi101023A1026415302759

Ball JT Woodrow IE Berry JA (1987) A model predicting stomatalconductance and its contribution to the control of photosynthesis underdifferent environmental conditions In lsquoProgress in photosynthesisresearchrsquo (Ed J Biggins) pp 221ndash234 (Martinus Nijhoff DordrechtThe Netherlands)

Bauerle TL Richards JH Smart DR Eissenstat DM (2008) Importance ofinternal hydraulic redistribution for prolonging the lifespan of roots in drysoil Plant Cell amp Environment 31 177ndash186

Breda N Granier A Barataud F Moyne C (1995) Soil water dynamics in anoak stand I Soil moisture water potentials and water uptake by rootsPlant and Soil 172 17ndash27

Brodribb TJ Holbrook NM (2003) Stomatal closure during leafdehydratation correlation with other leaf physiological traits PlantPhysiology 132 2166ndash2173 doi101104pp103023879

Burgess SSO Bleby TM (2006) Redistribution of soil water to lateral rootsmediatedby stem tissuesJournalofExperimentalBotany57 3283ndash3291doi101093jxberl085

ChavesMMTenhunen JDHarley P LangeOL (1987)Gas exchange studiesin two Portuguese grapevine cultivars Physiologia Plantarum 70639ndash647 doi101111j1399-30541987tb04318x

Chaves MM Zarrouk O Fransisco R Costa JM Santos T Regalado APRodriguesMLLopesCM (2010)Grapevine under deficit irrigation hintsfrom physiological and molecular data Annals of Botany 105 661ndash676doi101093aobmcq030

Choat B DraytonWMBrodersen CRMatthewsMA Shackel KAWadaHMcElrone AJ (2010) Measurement of vulnerability to water stress-induced cavitation in grapevine a comparison of four techniquesapplied to long-vesseled species Plant Cell amp Environment 331502ndash1512

Cochard H Coll L Le Roux X Ameglio T (2002) Unraveling the effects ofplant hydraulics on stomatal closure during water stress in walnut PlantPhysiology 128 282ndash290 doi101104pp010400

Comstock JP (2000) Variation in hydraulic architecture and gas exchange intwo desert sub-shrubs Hymenoclea salsola (TampG) and Ambrosiadumosa Oecologia 125 1ndash10 doi101007PL00008879

Correia MJ Pereira JS Chaves MM Rodrigues ML Pacheco CA (1995)ABA xylem concentrations determine maximum daily leaf conductanceof field-grown Vitis vinifera L plants Plant Cell amp Environment 18511ndash521 doi101111j1365-30401995tb00551x

Davies WJ Zhang J (1991) Root signals and the regulation of growth anddevelopment of plants in drying soil Annual Review of Plant Physiologyand Plant Molecular Biology 42 55ndash76 doi101146annurevpp42060191000415

Davies WJ Tardieu F Trejo CL (1994) How do chemical signals work inplants that grow in drying soil Plant Physiology 104 309ndash314

Donovan LA Richards JH LintonMJ (2003)Magnitude and mechanisms ofpredawn disequilibrium between predawn and plant soil water potentialsin desert shrubsEcology84 463ndash470 doi1018900012-9658(2003)084[0463MAMODB]20CO2

Ewers BEOrenR Sperry JS (2000) Influence of nutrient versuswater supplyon hydraulic architecture and water balance in Pinus taeda Plant Cell ampEnvironment 23 1055ndash1066 doi101046j1365-3040200000625x

Franks PJ (2004) Stomatal control and hydraulic conductance with specialreference to tall trees Tree Physiology 24 865ndash878 doi101093treephys248865

FuchsEELivingstonNJ (1996)Hydraulic control of stomatal conductance inDouglasfir (Pseudotsugamensiesii (Mirb) Franco) and alder (Alnus rubra(Bong)) seedlings Plant Cell amp Environment 19 1091ndash1098doi101111j1365-30401996tb00216x

Harley PC Baldocchi DD (1995) Scaling carbon dioxide and water vapourexchange from leaf to canopy in a deciduous forest I Leaf modelparametrization Plant Cell amp Environment 18 1146ndash1156doi101111j1365-30401995tb00625x

Holbrook NM Shashidhar VR James RAMunns R (2002) Stomatal controlin tomato with ABA-deficient roots response of grafted plants to soildrying Journal of Experimental Botany 53 1503ndash1514 doi101093jexbot533731503

Hubbard RM Bond BJ Ryan MG (1999) Evidence that hydraulicconductance limits photosynthesis in old Pinus ponderosa trees TreePhysiology 19 165ndash172 doi101093treephys193165

Hubbard RM Ryan MG Stiller V Sperry JS (2001) Stomatal conductanceand photosynthesis vary lineary with plant hydraulic conductance inPonderosa pine Plant Cell amp Environment 24 113ndash121 doi101046j1365-3040200100660x

Jarvis AJ Davies WJ (1998) Modeling stomatal responses to soil andatmospheric drought Journal of Experimental Botany 49(SpecialIssue) 399ndash406 doi101093jxb49Special_Issue399

Jones HG (1998) Stomatal control of photosynthesis and transpirationJournal of Experimental Botany 49(Special Issue) 387ndash398

Kramer PJ Boyer JS (1995) lsquoWater relations of plants and soilsrsquo (AcademicPress San Diego CA)

Leuning R (1990) Modeling stomatal behaviour and photosynthesis ofEucalyptus grandis Australian Journal of Plant Physiology 17159ndash175 doi101071PP9900159

Loveys BR (1991) How useful is a knowledge of ABA physiology for cropimprovement In lsquoAbscisic acid physiology and biochemistryrsquo (Eds WJDavies HG Jones) pp 245ndash260 (Bios Scientific Publishers Oxford)

1026 Functional Plant Biology V Zufferey and D R Smart

yPD (data not shown) At low levels of water stress(yPD gt ndash03MPa) Amax was greater for intact plants than root-restricted plants In contrast the adjusted levels of Amax for the

root-pruned vines remained not significantly different as waterstress increased during the first 25 days following root pruningthen decreased uniformly for all treatments as leaf senescenceensued at the end of the season (Fig 1e)

Stomatal sensitivity and response to VPD

Significant linear relationshipswere obtainedbetween the [A(RHCa)] and gs for both root pruned (pooled) and non-root prunedcontrols (Fig 3) The proposed stomatal sensitivity factor k washigher in vines without root removal than for root-pruned vines

The relationships between VPD andA gs andE are presentedin Fig 4 and are representative for diurnal observations made on25 June 9 July and 16 August Observations made over these3 days with light-saturating conditions indicated that A and gsdecreased as VPD increased (Fig 4a b) independent of rootpruning treatment The decrease in gs in relation to VPD wasbest characterised by a decreasing linear function in the root-restricted plants as well as in intact plants (Fig 4a) gs varied withVPD between the treatments for the same VPD where the root-restricted plants systematically showed lowergs values than intactplantsApositive correlationwasobservedbetweenVPDand leaf

00

ndash01

ndash02

ndash03

ndash04

ndash05

ndash06

ndash03

ndash06

ndash09

ndash12

ndash15

ndash03

ndash06

ndash09

ndash12

ndash15

ndash18

300

250

200

150

100

50

20

15

10

5

0

80

70

60

50

40160 180 200 220 240 260 160 180 200 220 240 260

ControlMidday Mid-morning

Mid-morning

Mid-morning

(c)

Midday(b)

Night(a) (d)

(e)

(f)

RPIRPD

F V H F

Day of year

ΨLe

af (

MP

a)Ψ

PD (

MP

a)Ψ

Ste

m (

MP

a)

g max

(mm

ol H

2O m

ndash2 s

ndash1)

Am

ax

(μm

ol C

O2

mndash2

sndash1

)in

trin

sic

WU

E (

Ag

s)(μ

mol

CO

2m

ol H

2O)

V H

Fig 1 Changes in (a) pre-dawnwater potential (yPD) (b) stemwater potential (yStem) (c) leafwater potential (yleaf) (d)maximumstomatal conductance (gmax) (e)maximumphotosyntheticrate (Amax) and (f) intrinsicwateruseefficiency (WUE)during the2004seasonwithdifferent rootpruning treatments 8 hours following the first pre-dawn leaf water potential measurement(JulianDay 152) 2ndash3 framework roots were removed from the quadrant of the vine trunkwherethe drip irrigation emitter was located (root-pruning irrigated RPI) or from the opposite sidequadrant (root-pruningdryRPD)ThefirstyStemmeasurementwas taken5daysbefore the root-pruning treatments (Julian day 147) and the first yleaf measurement 3 days following rootpruning (Julian day 155) F flowering V veraison H harvest Means se for nine leavesReprinted with permission of the American Journal of Enology and Viticulture

ΨPD (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14

g max

(m

mol

mndash2

sndash1

)

0

50

100

150

200

250

300

ΨStem midday (MPa)

(a) (b)

Control RPIRPD

R2=064

R2=077

R2=075

R2=087

Fig 2 Relationship between the maximal stomatal conductance (gmax) and(a) pre-dawn water potential (yPD) (b) and stem water potential (yStem) forcontrol vines (solid line) and root-pruning treatments together (dashed line)Data se for measurements on nine fully-exposed leaves per treatment

1022 Functional Plant Biology V Zufferey and D R Smart

transpiration E (Fig 4c) In comparison with the root-restrictedplants intact plants showed greater E values particularly at highlevels of VPD E values tended to increase more in intact plantswith increasing evaporative demand (greater slope) than in root-pruned plants

Leaf specific hydraulic conductance

Differences in stomatal behaviour were related to plant hydraulicproperties (Tyree and Sperry 1988) The leaf specific hydraulicconductanceKplant was estimated from the relationship of singleleaf transpiration rate to the soil-xylem potential difference sincethe soil water potential (ys) can be assumed to be very close to thepre-dawn leaf water potential (yPD) (Breda et al 1995) We noteexceptions from desert environments (Donovan et al 2003) andsoils with extreme heterogeneity in available water (Ameacuteglioet al 1999) Fig 5 shows the relationship between diurnal gs asa function of Kplant or yleaf for 3 different days Although norelationshipwas found to exist between gs andyleaf (Fig 5dndashf) gsand Kplant were found to be closely linked (Fig 5andashc) in all thestudy treatments analysed The lowest gs rates were observed onleaves of root-restrictedplants (RPI) andwere associatedwith thelowest values observed for Kplant

These data indicate that differences in stomatal behaviouramong the various treatments were linked to whole-plant

hydraulics Moreover this relationship existed despite of thefact that moderate differences were recorded in leaf areabetween the treatments at 113 22m2 vinendash1 (mean sen= 3) for the control 86 02m2 vinendash1 RPI and 9507m2 vinendash1 RPD (Smart et al 2006) Only the control andRPI treatments differed significantly and represented about a24 difference in leaf area It should be noted that treatmentswith the lowest stomatal sensitivity factor (root-restricted plants)also had the smallest leaf area per plant (Smart et al 2006)and consequently the lowest rate of soil water consumptionIn addition intact plants and root-removed plants showed acommon relationship between Kplant and the degree of waterstress as estimated either by yPD or by yStem (Fig 6)Nonetheless since E Kplant and gs do not represent completelyindependent variables some degree of auto-correlation may beimplicit in the relationships presented (see Schultz 2003)

Discussion

Partial root removal provided an experimental opportunity tomanipulate liquid as well as vapour phase resistances along

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300pre-veraison

Control RPIRPD

05 10 15 20 25 300

50

100

150

200

250

300post-veraison

A (hsCa)

k = 85

k = 62

k = 90

k = 55

Fig 3 Relationship between the [A (RHCa] and the stomatalconductance (gs) for adult leaves on Syrah vines at pre- and post-veraisonin 2004 Pre-veraison data is before Julian day 200 when fruit colouringwas first observed (Fig 1) Control vines (solid line) and root pruningtreatments together (dashed line) k is termed the stomatal sensitivityfactor (and represent the slope in the equation gs = g0 + k A (RHCa)see lsquoMaterials and methodsrsquo)

VPDa (kPa)

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300

A (

μmol

CO

2 m

ndash2 s

ndash1)

0

5

10

15

(a)

0 1 2 3 4

E (

mm

ol H

2O m

ndash2 s

ndash1)

0

2

4

6

8

(b)

(c)

Control RPIRPD

R2 = 079

R2 = 072

R2 = 050

R2 = 082

R2 = 068

R2 = 066

Fig 4 Relationship between (a) stomatal conductance (gs) (b) netphotosynthesis (A) and (c) transpiration (E) and the air vapour pressuredeficit (VPDa) during the course of 3 days (25 June 26 July and 16 August)yPDgt ndash03MPa by control plantsyPDgt ndash045MPa by root-restricted plantsLight-saturating conditions

Stomatal behaviour and root removal Functional Plant Biology 1023

the pathway of water movement in the soilndashplantndashatmospherecontinuum Removal of ~30 of the root system resulted in animmediate (24 h) and substantial decrease in gs and gas exchangeby photosynthesis and (Fig 1b d) Plant water status in contrastlagged slightly behind these changes (Fig 1a c e) These results

may concur with the hypothesis of a lag between an increase inroot water absorption to offset changes in diurnal transpirationdemand (Kramer and Boyer 1995) but suggests an inabilityof remaining roots to improve water uptake Axial waterredistribution can serve to rehydrate the entire vine (Smartet al 2005 Bauerle et al 2008) but in this case where rootswere left intact in irrigated soil and pruned from non-irrigatedsoil axial redistribution or an increase in water uptake couldonly apparently compensate for a portion of the water supplyLeaf wilting symptoms taken together with very negative valuesofyStem andyleaf were indicative of an increase in the hydraulicresistances between the soil and leaves during periods of highevaporative demand The accelerated decline in gmax and Amax atthe end of the season was consistent among treatments and waslikely a consequence of leaf aging (Fig 1b d)

Stomata respond to a variety of moisture related or watertransport factors under light-saturating conditions includinghumidity transpiration soil moisture and critical levels ofwater potential along the soilndashplantndashatmosphere continuum(Sperry et al 2002) Ball et al (1987) proposed the BWBindex as a means of evaluating the combined effects of theclimatic variables of air humidity and external Ca on gsA linear relationship was noted between the BWB index andgs (Fig 3) where apparent stomatal sensitivity was representedby the slope (k) of gs with ARHCa (Leuning 1990 Harleyand Baldocchi 1995) In all cases in the current investigationsensitivity of stomata was greater (higher k values) in theintact plants as compared with the root-restricted plants Thisindicated that internal signals in this case imposed by rootpruning treatments that caused immediate stomatal closure(Fig 1b) removed a portion of the stomatesrsquo ability torespond to external environmental factors of humidity and Ca

as empirically described by the BWB model Nonethelessstomates of both the intact and root pruned vines appearedequally sensitive to VPD throughout the season

The present results show that stomatal closure occurredfollowing an increase in VPD and was linked to a rise in thetranspiration rate (E) of the whole leaf in both intact and root-restricted plants Results of other studies have suggested thatthe closure of stomata with rising VPD appears to be more of afeedback response linked to some other aspect of transpiration(Shackel and Brinckmann 1985 Mott and Franks 2001) and leafwater loss rather than to a direct response to humidity at the leafsurface (Mott and Parkhurst 1991Monteith 1995) Neverthelessunder higher VPD leaf transpiration of root-pruned plants waslower than control plants An explanation for this phenomenonmay lie in part in the decrease ofyleaf in the root-restricted vinesand consequent fall in whole-plant water flux FurthermoreCi ofleaves of root-restricted plants was somewhat lower than intactplants (data not shown) and there was a and similar correlationbetween gs and A This confirmed in part that patchy stomatalclosure was of little significance in the present study and inconditions of progressive developing water deficit (Mott andBuckley 1998)

The observed relationship between stomatal behaviour VPD(Fig 4a) and plant water loss (Fig 4c) further suggests that theresponse to VPD was related to plant hydraulics and that itmay play a role in regulating leaf water potential (Oren et al2001)Underwater deficits progressive closure of stomata canbe

Kplant (mmol MPandash1 mndash2 sndash1)

g s (m

mol

mndash2

sndash1

)

50

100

150

200

250

30025 June

50

100

150

200

250

30026 July

0 2 4 6 8 10 120

50

100

150

200

250

30016 August

(a) (d)

(b) (e)

(c) (f)

Ψleaf (MPa)

ndash18ndash15ndash12ndash09ndash06ndash03

Control RPI

RPD

R2 = 087

R2 = 056

R2 = 071

Fig 5 (andashc) Stomatal conductance (gs) as a function of leaf specificconductance (Kplant) and (dndashf) of leaf water potential (yleaf) for differentroot pruning treatments during 3 days in 2004

ΨPD (MPa) ΨStem (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14 ndash16

Kpl

ant (

mm

ol M

Pandash1

mndash2

sndash1

)

0

2

4

6

8

10(a) (b)

Control RPIRPD

R2 = 075R2 = 077

Fig 6 Relationship between mid-morning leaf specific hydraulicconductance Kplant and (a) pre-dawn water potential (yPD) and (b)midday stem water potential (yStem) for different root pruning treatments

1024 Functional Plant Biology V Zufferey and D R Smart

observed when VPD increases and hydraulic conductance alongthe soil-to-leaf pathway (Kplant) decreases yleaf then remainsabove a critical threshold preventing xylem cavitation andhydraulic failure (Tyree and Sperry 1988) A positivecorrelation between hydraulic and stomatal conductances(Saliendra et al 1995 Comstock 2000 Addington et al2004) but a relatively constant yleaf suggests a feedback linkbetween gs and some form of hydraulic signal (Fuchs andLivingston 1996 Nardini et al 2001) Experimentallyreducing xylem-hydraulic conductance by various techniques(Sperry et al 1993 Hubbard et al 1999 2001) including rootpruning (Teskey et al 1983 Meinzer and Grantz 1990) bringsabout a decrease in gs thereby suggesting the hydraulicconductance for transpiration flux forms an integral anddynamic part of the stomatal control mechanism (Franks2004) Our observations from partial root removal in irrigatedversusnon-irrigated soils supports this contention in asmuchasgsand yleaf in RPI and RPD vines were shifted to different levelswith RPI having lower gs and yleaf

According to Meinzer (2002) true feed-forward control of gswith respect toyleaf would allow stomata to limit transpiration inanticipation of potential reductions in yleaf resulting fromrestricted available water in the soil environment or increasedsoil-to-leaf hydraulic resistance Experiments by Teskey et al(1983) on Abies amabilis and by Meinzer and Grantz (1990) insugarcane demonstrated that gs decreased in response to rootremoval with no change inyleaf This response can be interpretedas generating a hydraulic signal in the form of diminished leaf-specific hydraulic conductance following partial root pruning Ifstomata did not respond by restricting transpiration yleaf woulddecline In the present experiments gs of root-restricted plantsdecreased immediately asKplant fell during the day This lead to arapid drop in yleaf (and leaf wilting) in root-pruned plantscompared with intact plants This response differed from theapparent feed-forward stomatal behaviour in response to rootpruning where stomata appeared to overcompensate and causeyleaf to be noticeably less negative following root removal thanbefore (Meinzer and Grantz 1990)

Feed-forward behaviour of stomata with respect to regulationof yleaf has been attributed to the presence of chemical signalsthat modify stomatal opening and which are brought to theleaf in the transpiration flow (Davies et al 1994) In drought-avoiding plants (isohydric) the leaf water potential is unaffectedby soil water deficits and isohydric behaviour is linked to aninteraction between both chemical and hydraulic information(Tardieu and Simonneau 1998) In drought-tolerant species(anisohydric) a controlling influence of leaf water status on gsneed not be invoked Rather leaf water status is likely to vary as aconsequence of water flux through the plant which is controlledby stomatal conductance Schultz (2003) observed that thegrapevine cultivar lsquoSyrahrsquo behaves in an anisohydric mannerand that the differences in thewater-conducting capacity of stemsand especially petioles may be the origin of the isohydric andanisohydric signal regulating gs in grapevine Results from thepresent study showed that yleaf of lsquoSyrahrsquo fell sharply asevaporative demand rose During the daytime and when waterstress was accentuated as a result of partial root removal morenegative leaf water potentials were realised compared withwell-watered intact plants This provided some confirmation of

anisohydric behaviour but also suggested root and other signalsin the plant hydraulic path have a strong influence on stomatalbehaviour Other contradictory studies have been reported inthe literature (see review by Chaves et al 2010) showingthat the same cultivar could behave differently dependingon experimental conditions (eg intensity and duration ofwater deficits VPD and root system) and physiological oranatomical adjustments (eg cell osmotic adjustment wallelasticity vessel conductivity)

However no recovery of Kplant was observed during theseason in root-restricted plants in comparison with the intactcontrols An increase in the total root surface area for absorptionby the growth of new roots after partial root pruning couldimprove Kplant (Sperry et al 2002) and Alsina and co-workersverified this hypothesis for grape (Alsina et al 2011) It is notcertain that roots produced during a period of water stress(drought) will have sufficient water conductivity (Ewers et al2000)Atgt60 cmdepth inmoist soils root conductivity increasedbut gs did not increase substantially Either the failure to producenew roots or lower conductivity would help to explain whyKplant

in root-restricted vines did not regain hydraulic conductancelevels approaching those of the intact non-root pruned vinesduring the growing season Moreover it should be noted thatdiurnal measurements of Kplant conducted in the field may giverise to errors if they were conducted under non-steady-stateconditions (Hubbard et al 2001) Non-steady-state conditionsmight arise as a consequence of significant capacitance (Schultzand Matthews 1997 Smart et al 2005) or rapid changes inenvironmental conditions over time

Partial root removal may also have had an influence onembolism development as a consequence of rapid changes inwater potential and as a consequence may have contributed tostomatal closure by an embolism related hydraulic signal(Lovisolo and Schubert 1998) Salleo et al (2000) suggestedthat even cavitation could act as a hydraulicmodulator for stomataopening The impact of cavitation on total hydraulic conductanceand finally on gs would depend on the position of cavitation inthe root-leaf pathway and on the distribution of hydraulicresistances between roots stems petioles and leaves (Meinzer2002 Zufferey et al 2011) Root systems often constitute a largepart of the total plant hydraulic resistance (Tyree et al 1998) andthe relative contributions of stem and leaf resistance to theremaining shoot resistance can vary substantially (Choat et al2010) Leaf wilting symptoms together with the very low gsvalues observed in this study for root-restricted vines andparticularly in root-pruned plants in wet irrigated soils may beattributed in part to an increased vulnerability of xylem tocavitation coupled with a high evaporative demand Thesecavitation events may act in this way as a signal for stomatalclosure (Nardini et al 2001) or stomatal closure would occur toprevent cavitation (Cochard et al 2002)

Significant decreases in gs recorded in root-restrictedplants were closely related to a reduction in leaf-specifichydraulic conductance (Fig 5) However other factors suchas hormonal control or even a combination of chemical andhydraulic messages which various authors (Tardieu andDavies 1993 Tardieu and Simonneau 1998 reviewed byHolbrook et al 2002) have brought to light cannot be entirelyruled out and have not been directly evaluated in this

Stomatal behaviour and root removal Functional Plant Biology 1025

investigationTheobservation thatgmaxwasdifferent between theroot-restricted plants and intact plants at similaryPD representingdifferent soilwater status prospectedby the roots andpresumablytriggering ABA synthesis (or other hormonal factors such ascytokinins) suggests that either the signals were different innature or concentration or the receptor site sensitivity variedbetween the different root pruning treatments We also notethat yPD changed within 48 h following root pruning Thisindicated that root pruned vines were not able to refill xylemand recharge hydraulic status to the same extent as the intactcontrols despite of the fact that roots of RPD vines had rootsexposed to the same irrigation source as the controls This hasshown that axial redistribution and reverse flow of water occursto such an extent that rehydration of lateral roots occurs (Smartet al 2005 Burgess and Bleby 2006) This raises questionswith respect to root signalling If pruning of roughly 30 ofthe root system occurs then it might be a reasonable assumptionthat removal of 30 of fine roots and fine root tips occurs whichmay be the primary source of the root signals that influence gs(Bauerle et al 2008) Further this suggests that root signallingwas of lesser importance in the initial response of gs and A to rootpruning

References

AddingtonRNMitchell RJOrenRDonovanLA (2004)Stomatal sensitivityto vapor pressure deficit and its relationship to hydraulic conductance inPinus palustris Tree Physiology 24 561ndash569 doi101093treephys245561

AlsinaMMSmartDRBauerleTdeHerraldeFBielCStockertCMNegronCSaveR (2011)Seasonal changes ofwhole root systemconductanceby adrought tolerant grape root system Journal of Experimental Botany 6299ndash109 doi101093jxberq247

Ameacuteglio TArcher P CohenMValancogneCDaudet FDayauSCruiziat P(1999) Significance and limits in the use of predawn leaf water potentialfor tree irrigation Plant and Soil 207 155ndash167 doi101023A1026415302759

Ball JT Woodrow IE Berry JA (1987) A model predicting stomatalconductance and its contribution to the control of photosynthesis underdifferent environmental conditions In lsquoProgress in photosynthesisresearchrsquo (Ed J Biggins) pp 221ndash234 (Martinus Nijhoff DordrechtThe Netherlands)

Bauerle TL Richards JH Smart DR Eissenstat DM (2008) Importance ofinternal hydraulic redistribution for prolonging the lifespan of roots in drysoil Plant Cell amp Environment 31 177ndash186

Breda N Granier A Barataud F Moyne C (1995) Soil water dynamics in anoak stand I Soil moisture water potentials and water uptake by rootsPlant and Soil 172 17ndash27

Brodribb TJ Holbrook NM (2003) Stomatal closure during leafdehydratation correlation with other leaf physiological traits PlantPhysiology 132 2166ndash2173 doi101104pp103023879

Burgess SSO Bleby TM (2006) Redistribution of soil water to lateral rootsmediatedby stem tissuesJournalofExperimentalBotany57 3283ndash3291doi101093jxberl085

ChavesMMTenhunen JDHarley P LangeOL (1987)Gas exchange studiesin two Portuguese grapevine cultivars Physiologia Plantarum 70639ndash647 doi101111j1399-30541987tb04318x

Chaves MM Zarrouk O Fransisco R Costa JM Santos T Regalado APRodriguesMLLopesCM (2010)Grapevine under deficit irrigation hintsfrom physiological and molecular data Annals of Botany 105 661ndash676doi101093aobmcq030

Choat B DraytonWMBrodersen CRMatthewsMA Shackel KAWadaHMcElrone AJ (2010) Measurement of vulnerability to water stress-induced cavitation in grapevine a comparison of four techniquesapplied to long-vesseled species Plant Cell amp Environment 331502ndash1512

Cochard H Coll L Le Roux X Ameglio T (2002) Unraveling the effects ofplant hydraulics on stomatal closure during water stress in walnut PlantPhysiology 128 282ndash290 doi101104pp010400

Comstock JP (2000) Variation in hydraulic architecture and gas exchange intwo desert sub-shrubs Hymenoclea salsola (TampG) and Ambrosiadumosa Oecologia 125 1ndash10 doi101007PL00008879

Correia MJ Pereira JS Chaves MM Rodrigues ML Pacheco CA (1995)ABA xylem concentrations determine maximum daily leaf conductanceof field-grown Vitis vinifera L plants Plant Cell amp Environment 18511ndash521 doi101111j1365-30401995tb00551x

Davies WJ Zhang J (1991) Root signals and the regulation of growth anddevelopment of plants in drying soil Annual Review of Plant Physiologyand Plant Molecular Biology 42 55ndash76 doi101146annurevpp42060191000415

Davies WJ Tardieu F Trejo CL (1994) How do chemical signals work inplants that grow in drying soil Plant Physiology 104 309ndash314

Donovan LA Richards JH LintonMJ (2003)Magnitude and mechanisms ofpredawn disequilibrium between predawn and plant soil water potentialsin desert shrubsEcology84 463ndash470 doi1018900012-9658(2003)084[0463MAMODB]20CO2

Ewers BEOrenR Sperry JS (2000) Influence of nutrient versuswater supplyon hydraulic architecture and water balance in Pinus taeda Plant Cell ampEnvironment 23 1055ndash1066 doi101046j1365-3040200000625x

Franks PJ (2004) Stomatal control and hydraulic conductance with specialreference to tall trees Tree Physiology 24 865ndash878 doi101093treephys248865

FuchsEELivingstonNJ (1996)Hydraulic control of stomatal conductance inDouglasfir (Pseudotsugamensiesii (Mirb) Franco) and alder (Alnus rubra(Bong)) seedlings Plant Cell amp Environment 19 1091ndash1098doi101111j1365-30401996tb00216x

Harley PC Baldocchi DD (1995) Scaling carbon dioxide and water vapourexchange from leaf to canopy in a deciduous forest I Leaf modelparametrization Plant Cell amp Environment 18 1146ndash1156doi101111j1365-30401995tb00625x

Holbrook NM Shashidhar VR James RAMunns R (2002) Stomatal controlin tomato with ABA-deficient roots response of grafted plants to soildrying Journal of Experimental Botany 53 1503ndash1514 doi101093jexbot533731503

Hubbard RM Bond BJ Ryan MG (1999) Evidence that hydraulicconductance limits photosynthesis in old Pinus ponderosa trees TreePhysiology 19 165ndash172 doi101093treephys193165

Hubbard RM Ryan MG Stiller V Sperry JS (2001) Stomatal conductanceand photosynthesis vary lineary with plant hydraulic conductance inPonderosa pine Plant Cell amp Environment 24 113ndash121 doi101046j1365-3040200100660x

Jarvis AJ Davies WJ (1998) Modeling stomatal responses to soil andatmospheric drought Journal of Experimental Botany 49(SpecialIssue) 399ndash406 doi101093jxb49Special_Issue399

Jones HG (1998) Stomatal control of photosynthesis and transpirationJournal of Experimental Botany 49(Special Issue) 387ndash398

Kramer PJ Boyer JS (1995) lsquoWater relations of plants and soilsrsquo (AcademicPress San Diego CA)

Leuning R (1990) Modeling stomatal behaviour and photosynthesis ofEucalyptus grandis Australian Journal of Plant Physiology 17159ndash175 doi101071PP9900159

Loveys BR (1991) How useful is a knowledge of ABA physiology for cropimprovement In lsquoAbscisic acid physiology and biochemistryrsquo (Eds WJDavies HG Jones) pp 245ndash260 (Bios Scientific Publishers Oxford)

1026 Functional Plant Biology V Zufferey and D R Smart

transpiration E (Fig 4c) In comparison with the root-restrictedplants intact plants showed greater E values particularly at highlevels of VPD E values tended to increase more in intact plantswith increasing evaporative demand (greater slope) than in root-pruned plants

Leaf specific hydraulic conductance

Differences in stomatal behaviour were related to plant hydraulicproperties (Tyree and Sperry 1988) The leaf specific hydraulicconductanceKplant was estimated from the relationship of singleleaf transpiration rate to the soil-xylem potential difference sincethe soil water potential (ys) can be assumed to be very close to thepre-dawn leaf water potential (yPD) (Breda et al 1995) We noteexceptions from desert environments (Donovan et al 2003) andsoils with extreme heterogeneity in available water (Ameacuteglioet al 1999) Fig 5 shows the relationship between diurnal gs asa function of Kplant or yleaf for 3 different days Although norelationshipwas found to exist between gs andyleaf (Fig 5dndashf) gsand Kplant were found to be closely linked (Fig 5andashc) in all thestudy treatments analysed The lowest gs rates were observed onleaves of root-restrictedplants (RPI) andwere associatedwith thelowest values observed for Kplant

These data indicate that differences in stomatal behaviouramong the various treatments were linked to whole-plant

hydraulics Moreover this relationship existed despite of thefact that moderate differences were recorded in leaf areabetween the treatments at 113 22m2 vinendash1 (mean sen= 3) for the control 86 02m2 vinendash1 RPI and 9507m2 vinendash1 RPD (Smart et al 2006) Only the control andRPI treatments differed significantly and represented about a24 difference in leaf area It should be noted that treatmentswith the lowest stomatal sensitivity factor (root-restricted plants)also had the smallest leaf area per plant (Smart et al 2006)and consequently the lowest rate of soil water consumptionIn addition intact plants and root-removed plants showed acommon relationship between Kplant and the degree of waterstress as estimated either by yPD or by yStem (Fig 6)Nonetheless since E Kplant and gs do not represent completelyindependent variables some degree of auto-correlation may beimplicit in the relationships presented (see Schultz 2003)

Discussion

Partial root removal provided an experimental opportunity tomanipulate liquid as well as vapour phase resistances along

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300pre-veraison

Control RPIRPD

05 10 15 20 25 300

50

100

150

200

250

300post-veraison

A (hsCa)

k = 85

k = 62

k = 90

k = 55

Fig 3 Relationship between the [A (RHCa] and the stomatalconductance (gs) for adult leaves on Syrah vines at pre- and post-veraisonin 2004 Pre-veraison data is before Julian day 200 when fruit colouringwas first observed (Fig 1) Control vines (solid line) and root pruningtreatments together (dashed line) k is termed the stomatal sensitivityfactor (and represent the slope in the equation gs = g0 + k A (RHCa)see lsquoMaterials and methodsrsquo)

VPDa (kPa)

g s (

mm

ol m

ndash2 s

ndash1)

0

50

100

150

200

250

300

A (

μmol

CO

2 m

ndash2 s

ndash1)

0

5

10

15

(a)

0 1 2 3 4

E (

mm

ol H

2O m

ndash2 s

ndash1)

0

2

4

6

8

(b)

(c)

Control RPIRPD

R2 = 079

R2 = 072

R2 = 050

R2 = 082

R2 = 068

R2 = 066

Fig 4 Relationship between (a) stomatal conductance (gs) (b) netphotosynthesis (A) and (c) transpiration (E) and the air vapour pressuredeficit (VPDa) during the course of 3 days (25 June 26 July and 16 August)yPDgt ndash03MPa by control plantsyPDgt ndash045MPa by root-restricted plantsLight-saturating conditions

Stomatal behaviour and root removal Functional Plant Biology 1023

the pathway of water movement in the soilndashplantndashatmospherecontinuum Removal of ~30 of the root system resulted in animmediate (24 h) and substantial decrease in gs and gas exchangeby photosynthesis and (Fig 1b d) Plant water status in contrastlagged slightly behind these changes (Fig 1a c e) These results

may concur with the hypothesis of a lag between an increase inroot water absorption to offset changes in diurnal transpirationdemand (Kramer and Boyer 1995) but suggests an inabilityof remaining roots to improve water uptake Axial waterredistribution can serve to rehydrate the entire vine (Smartet al 2005 Bauerle et al 2008) but in this case where rootswere left intact in irrigated soil and pruned from non-irrigatedsoil axial redistribution or an increase in water uptake couldonly apparently compensate for a portion of the water supplyLeaf wilting symptoms taken together with very negative valuesofyStem andyleaf were indicative of an increase in the hydraulicresistances between the soil and leaves during periods of highevaporative demand The accelerated decline in gmax and Amax atthe end of the season was consistent among treatments and waslikely a consequence of leaf aging (Fig 1b d)

Stomata respond to a variety of moisture related or watertransport factors under light-saturating conditions includinghumidity transpiration soil moisture and critical levels ofwater potential along the soilndashplantndashatmosphere continuum(Sperry et al 2002) Ball et al (1987) proposed the BWBindex as a means of evaluating the combined effects of theclimatic variables of air humidity and external Ca on gsA linear relationship was noted between the BWB index andgs (Fig 3) where apparent stomatal sensitivity was representedby the slope (k) of gs with ARHCa (Leuning 1990 Harleyand Baldocchi 1995) In all cases in the current investigationsensitivity of stomata was greater (higher k values) in theintact plants as compared with the root-restricted plants Thisindicated that internal signals in this case imposed by rootpruning treatments that caused immediate stomatal closure(Fig 1b) removed a portion of the stomatesrsquo ability torespond to external environmental factors of humidity and Ca

as empirically described by the BWB model Nonethelessstomates of both the intact and root pruned vines appearedequally sensitive to VPD throughout the season

The present results show that stomatal closure occurredfollowing an increase in VPD and was linked to a rise in thetranspiration rate (E) of the whole leaf in both intact and root-restricted plants Results of other studies have suggested thatthe closure of stomata with rising VPD appears to be more of afeedback response linked to some other aspect of transpiration(Shackel and Brinckmann 1985 Mott and Franks 2001) and leafwater loss rather than to a direct response to humidity at the leafsurface (Mott and Parkhurst 1991Monteith 1995) Neverthelessunder higher VPD leaf transpiration of root-pruned plants waslower than control plants An explanation for this phenomenonmay lie in part in the decrease ofyleaf in the root-restricted vinesand consequent fall in whole-plant water flux FurthermoreCi ofleaves of root-restricted plants was somewhat lower than intactplants (data not shown) and there was a and similar correlationbetween gs and A This confirmed in part that patchy stomatalclosure was of little significance in the present study and inconditions of progressive developing water deficit (Mott andBuckley 1998)

The observed relationship between stomatal behaviour VPD(Fig 4a) and plant water loss (Fig 4c) further suggests that theresponse to VPD was related to plant hydraulics and that itmay play a role in regulating leaf water potential (Oren et al2001)Underwater deficits progressive closure of stomata canbe

Kplant (mmol MPandash1 mndash2 sndash1)

g s (m

mol

mndash2

sndash1

)

50

100

150

200

250

30025 June

50

100

150

200

250

30026 July

0 2 4 6 8 10 120

50

100

150

200

250

30016 August

(a) (d)

(b) (e)

(c) (f)

Ψleaf (MPa)

ndash18ndash15ndash12ndash09ndash06ndash03

Control RPI

RPD

R2 = 087

R2 = 056

R2 = 071

Fig 5 (andashc) Stomatal conductance (gs) as a function of leaf specificconductance (Kplant) and (dndashf) of leaf water potential (yleaf) for differentroot pruning treatments during 3 days in 2004

ΨPD (MPa) ΨStem (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14 ndash16

Kpl

ant (

mm

ol M

Pandash1

mndash2

sndash1

)

0

2

4

6

8

10(a) (b)

Control RPIRPD

R2 = 075R2 = 077

Fig 6 Relationship between mid-morning leaf specific hydraulicconductance Kplant and (a) pre-dawn water potential (yPD) and (b)midday stem water potential (yStem) for different root pruning treatments

1024 Functional Plant Biology V Zufferey and D R Smart

observed when VPD increases and hydraulic conductance alongthe soil-to-leaf pathway (Kplant) decreases yleaf then remainsabove a critical threshold preventing xylem cavitation andhydraulic failure (Tyree and Sperry 1988) A positivecorrelation between hydraulic and stomatal conductances(Saliendra et al 1995 Comstock 2000 Addington et al2004) but a relatively constant yleaf suggests a feedback linkbetween gs and some form of hydraulic signal (Fuchs andLivingston 1996 Nardini et al 2001) Experimentallyreducing xylem-hydraulic conductance by various techniques(Sperry et al 1993 Hubbard et al 1999 2001) including rootpruning (Teskey et al 1983 Meinzer and Grantz 1990) bringsabout a decrease in gs thereby suggesting the hydraulicconductance for transpiration flux forms an integral anddynamic part of the stomatal control mechanism (Franks2004) Our observations from partial root removal in irrigatedversusnon-irrigated soils supports this contention in asmuchasgsand yleaf in RPI and RPD vines were shifted to different levelswith RPI having lower gs and yleaf

According to Meinzer (2002) true feed-forward control of gswith respect toyleaf would allow stomata to limit transpiration inanticipation of potential reductions in yleaf resulting fromrestricted available water in the soil environment or increasedsoil-to-leaf hydraulic resistance Experiments by Teskey et al(1983) on Abies amabilis and by Meinzer and Grantz (1990) insugarcane demonstrated that gs decreased in response to rootremoval with no change inyleaf This response can be interpretedas generating a hydraulic signal in the form of diminished leaf-specific hydraulic conductance following partial root pruning Ifstomata did not respond by restricting transpiration yleaf woulddecline In the present experiments gs of root-restricted plantsdecreased immediately asKplant fell during the day This lead to arapid drop in yleaf (and leaf wilting) in root-pruned plantscompared with intact plants This response differed from theapparent feed-forward stomatal behaviour in response to rootpruning where stomata appeared to overcompensate and causeyleaf to be noticeably less negative following root removal thanbefore (Meinzer and Grantz 1990)

Feed-forward behaviour of stomata with respect to regulationof yleaf has been attributed to the presence of chemical signalsthat modify stomatal opening and which are brought to theleaf in the transpiration flow (Davies et al 1994) In drought-avoiding plants (isohydric) the leaf water potential is unaffectedby soil water deficits and isohydric behaviour is linked to aninteraction between both chemical and hydraulic information(Tardieu and Simonneau 1998) In drought-tolerant species(anisohydric) a controlling influence of leaf water status on gsneed not be invoked Rather leaf water status is likely to vary as aconsequence of water flux through the plant which is controlledby stomatal conductance Schultz (2003) observed that thegrapevine cultivar lsquoSyrahrsquo behaves in an anisohydric mannerand that the differences in thewater-conducting capacity of stemsand especially petioles may be the origin of the isohydric andanisohydric signal regulating gs in grapevine Results from thepresent study showed that yleaf of lsquoSyrahrsquo fell sharply asevaporative demand rose During the daytime and when waterstress was accentuated as a result of partial root removal morenegative leaf water potentials were realised compared withwell-watered intact plants This provided some confirmation of

anisohydric behaviour but also suggested root and other signalsin the plant hydraulic path have a strong influence on stomatalbehaviour Other contradictory studies have been reported inthe literature (see review by Chaves et al 2010) showingthat the same cultivar could behave differently dependingon experimental conditions (eg intensity and duration ofwater deficits VPD and root system) and physiological oranatomical adjustments (eg cell osmotic adjustment wallelasticity vessel conductivity)

However no recovery of Kplant was observed during theseason in root-restricted plants in comparison with the intactcontrols An increase in the total root surface area for absorptionby the growth of new roots after partial root pruning couldimprove Kplant (Sperry et al 2002) and Alsina and co-workersverified this hypothesis for grape (Alsina et al 2011) It is notcertain that roots produced during a period of water stress(drought) will have sufficient water conductivity (Ewers et al2000)Atgt60 cmdepth inmoist soils root conductivity increasedbut gs did not increase substantially Either the failure to producenew roots or lower conductivity would help to explain whyKplant

in root-restricted vines did not regain hydraulic conductancelevels approaching those of the intact non-root pruned vinesduring the growing season Moreover it should be noted thatdiurnal measurements of Kplant conducted in the field may giverise to errors if they were conducted under non-steady-stateconditions (Hubbard et al 2001) Non-steady-state conditionsmight arise as a consequence of significant capacitance (Schultzand Matthews 1997 Smart et al 2005) or rapid changes inenvironmental conditions over time

Partial root removal may also have had an influence onembolism development as a consequence of rapid changes inwater potential and as a consequence may have contributed tostomatal closure by an embolism related hydraulic signal(Lovisolo and Schubert 1998) Salleo et al (2000) suggestedthat even cavitation could act as a hydraulicmodulator for stomataopening The impact of cavitation on total hydraulic conductanceand finally on gs would depend on the position of cavitation inthe root-leaf pathway and on the distribution of hydraulicresistances between roots stems petioles and leaves (Meinzer2002 Zufferey et al 2011) Root systems often constitute a largepart of the total plant hydraulic resistance (Tyree et al 1998) andthe relative contributions of stem and leaf resistance to theremaining shoot resistance can vary substantially (Choat et al2010) Leaf wilting symptoms together with the very low gsvalues observed in this study for root-restricted vines andparticularly in root-pruned plants in wet irrigated soils may beattributed in part to an increased vulnerability of xylem tocavitation coupled with a high evaporative demand Thesecavitation events may act in this way as a signal for stomatalclosure (Nardini et al 2001) or stomatal closure would occur toprevent cavitation (Cochard et al 2002)

Significant decreases in gs recorded in root-restrictedplants were closely related to a reduction in leaf-specifichydraulic conductance (Fig 5) However other factors suchas hormonal control or even a combination of chemical andhydraulic messages which various authors (Tardieu andDavies 1993 Tardieu and Simonneau 1998 reviewed byHolbrook et al 2002) have brought to light cannot be entirelyruled out and have not been directly evaluated in this

Stomatal behaviour and root removal Functional Plant Biology 1025

investigationTheobservation thatgmaxwasdifferent between theroot-restricted plants and intact plants at similaryPD representingdifferent soilwater status prospectedby the roots andpresumablytriggering ABA synthesis (or other hormonal factors such ascytokinins) suggests that either the signals were different innature or concentration or the receptor site sensitivity variedbetween the different root pruning treatments We also notethat yPD changed within 48 h following root pruning Thisindicated that root pruned vines were not able to refill xylemand recharge hydraulic status to the same extent as the intactcontrols despite of the fact that roots of RPD vines had rootsexposed to the same irrigation source as the controls This hasshown that axial redistribution and reverse flow of water occursto such an extent that rehydration of lateral roots occurs (Smartet al 2005 Burgess and Bleby 2006) This raises questionswith respect to root signalling If pruning of roughly 30 ofthe root system occurs then it might be a reasonable assumptionthat removal of 30 of fine roots and fine root tips occurs whichmay be the primary source of the root signals that influence gs(Bauerle et al 2008) Further this suggests that root signallingwas of lesser importance in the initial response of gs and A to rootpruning

References

AddingtonRNMitchell RJOrenRDonovanLA (2004)Stomatal sensitivityto vapor pressure deficit and its relationship to hydraulic conductance inPinus palustris Tree Physiology 24 561ndash569 doi101093treephys245561

AlsinaMMSmartDRBauerleTdeHerraldeFBielCStockertCMNegronCSaveR (2011)Seasonal changes ofwhole root systemconductanceby adrought tolerant grape root system Journal of Experimental Botany 6299ndash109 doi101093jxberq247

Ameacuteglio TArcher P CohenMValancogneCDaudet FDayauSCruiziat P(1999) Significance and limits in the use of predawn leaf water potentialfor tree irrigation Plant and Soil 207 155ndash167 doi101023A1026415302759

Ball JT Woodrow IE Berry JA (1987) A model predicting stomatalconductance and its contribution to the control of photosynthesis underdifferent environmental conditions In lsquoProgress in photosynthesisresearchrsquo (Ed J Biggins) pp 221ndash234 (Martinus Nijhoff DordrechtThe Netherlands)

Bauerle TL Richards JH Smart DR Eissenstat DM (2008) Importance ofinternal hydraulic redistribution for prolonging the lifespan of roots in drysoil Plant Cell amp Environment 31 177ndash186

Breda N Granier A Barataud F Moyne C (1995) Soil water dynamics in anoak stand I Soil moisture water potentials and water uptake by rootsPlant and Soil 172 17ndash27

Brodribb TJ Holbrook NM (2003) Stomatal closure during leafdehydratation correlation with other leaf physiological traits PlantPhysiology 132 2166ndash2173 doi101104pp103023879

Burgess SSO Bleby TM (2006) Redistribution of soil water to lateral rootsmediatedby stem tissuesJournalofExperimentalBotany57 3283ndash3291doi101093jxberl085

ChavesMMTenhunen JDHarley P LangeOL (1987)Gas exchange studiesin two Portuguese grapevine cultivars Physiologia Plantarum 70639ndash647 doi101111j1399-30541987tb04318x

Chaves MM Zarrouk O Fransisco R Costa JM Santos T Regalado APRodriguesMLLopesCM (2010)Grapevine under deficit irrigation hintsfrom physiological and molecular data Annals of Botany 105 661ndash676doi101093aobmcq030

Choat B DraytonWMBrodersen CRMatthewsMA Shackel KAWadaHMcElrone AJ (2010) Measurement of vulnerability to water stress-induced cavitation in grapevine a comparison of four techniquesapplied to long-vesseled species Plant Cell amp Environment 331502ndash1512

Cochard H Coll L Le Roux X Ameglio T (2002) Unraveling the effects ofplant hydraulics on stomatal closure during water stress in walnut PlantPhysiology 128 282ndash290 doi101104pp010400

Comstock JP (2000) Variation in hydraulic architecture and gas exchange intwo desert sub-shrubs Hymenoclea salsola (TampG) and Ambrosiadumosa Oecologia 125 1ndash10 doi101007PL00008879

Correia MJ Pereira JS Chaves MM Rodrigues ML Pacheco CA (1995)ABA xylem concentrations determine maximum daily leaf conductanceof field-grown Vitis vinifera L plants Plant Cell amp Environment 18511ndash521 doi101111j1365-30401995tb00551x

Davies WJ Zhang J (1991) Root signals and the regulation of growth anddevelopment of plants in drying soil Annual Review of Plant Physiologyand Plant Molecular Biology 42 55ndash76 doi101146annurevpp42060191000415

Davies WJ Tardieu F Trejo CL (1994) How do chemical signals work inplants that grow in drying soil Plant Physiology 104 309ndash314

Donovan LA Richards JH LintonMJ (2003)Magnitude and mechanisms ofpredawn disequilibrium between predawn and plant soil water potentialsin desert shrubsEcology84 463ndash470 doi1018900012-9658(2003)084[0463MAMODB]20CO2

Ewers BEOrenR Sperry JS (2000) Influence of nutrient versuswater supplyon hydraulic architecture and water balance in Pinus taeda Plant Cell ampEnvironment 23 1055ndash1066 doi101046j1365-3040200000625x

Franks PJ (2004) Stomatal control and hydraulic conductance with specialreference to tall trees Tree Physiology 24 865ndash878 doi101093treephys248865

FuchsEELivingstonNJ (1996)Hydraulic control of stomatal conductance inDouglasfir (Pseudotsugamensiesii (Mirb) Franco) and alder (Alnus rubra(Bong)) seedlings Plant Cell amp Environment 19 1091ndash1098doi101111j1365-30401996tb00216x

Harley PC Baldocchi DD (1995) Scaling carbon dioxide and water vapourexchange from leaf to canopy in a deciduous forest I Leaf modelparametrization Plant Cell amp Environment 18 1146ndash1156doi101111j1365-30401995tb00625x

Holbrook NM Shashidhar VR James RAMunns R (2002) Stomatal controlin tomato with ABA-deficient roots response of grafted plants to soildrying Journal of Experimental Botany 53 1503ndash1514 doi101093jexbot533731503

Hubbard RM Bond BJ Ryan MG (1999) Evidence that hydraulicconductance limits photosynthesis in old Pinus ponderosa trees TreePhysiology 19 165ndash172 doi101093treephys193165

Hubbard RM Ryan MG Stiller V Sperry JS (2001) Stomatal conductanceand photosynthesis vary lineary with plant hydraulic conductance inPonderosa pine Plant Cell amp Environment 24 113ndash121 doi101046j1365-3040200100660x

Jarvis AJ Davies WJ (1998) Modeling stomatal responses to soil andatmospheric drought Journal of Experimental Botany 49(SpecialIssue) 399ndash406 doi101093jxb49Special_Issue399

Jones HG (1998) Stomatal control of photosynthesis and transpirationJournal of Experimental Botany 49(Special Issue) 387ndash398

Kramer PJ Boyer JS (1995) lsquoWater relations of plants and soilsrsquo (AcademicPress San Diego CA)

Leuning R (1990) Modeling stomatal behaviour and photosynthesis ofEucalyptus grandis Australian Journal of Plant Physiology 17159ndash175 doi101071PP9900159

Loveys BR (1991) How useful is a knowledge of ABA physiology for cropimprovement In lsquoAbscisic acid physiology and biochemistryrsquo (Eds WJDavies HG Jones) pp 245ndash260 (Bios Scientific Publishers Oxford)

1026 Functional Plant Biology V Zufferey and D R Smart

the pathway of water movement in the soilndashplantndashatmospherecontinuum Removal of ~30 of the root system resulted in animmediate (24 h) and substantial decrease in gs and gas exchangeby photosynthesis and (Fig 1b d) Plant water status in contrastlagged slightly behind these changes (Fig 1a c e) These results

may concur with the hypothesis of a lag between an increase inroot water absorption to offset changes in diurnal transpirationdemand (Kramer and Boyer 1995) but suggests an inabilityof remaining roots to improve water uptake Axial waterredistribution can serve to rehydrate the entire vine (Smartet al 2005 Bauerle et al 2008) but in this case where rootswere left intact in irrigated soil and pruned from non-irrigatedsoil axial redistribution or an increase in water uptake couldonly apparently compensate for a portion of the water supplyLeaf wilting symptoms taken together with very negative valuesofyStem andyleaf were indicative of an increase in the hydraulicresistances between the soil and leaves during periods of highevaporative demand The accelerated decline in gmax and Amax atthe end of the season was consistent among treatments and waslikely a consequence of leaf aging (Fig 1b d)

Stomata respond to a variety of moisture related or watertransport factors under light-saturating conditions includinghumidity transpiration soil moisture and critical levels ofwater potential along the soilndashplantndashatmosphere continuum(Sperry et al 2002) Ball et al (1987) proposed the BWBindex as a means of evaluating the combined effects of theclimatic variables of air humidity and external Ca on gsA linear relationship was noted between the BWB index andgs (Fig 3) where apparent stomatal sensitivity was representedby the slope (k) of gs with ARHCa (Leuning 1990 Harleyand Baldocchi 1995) In all cases in the current investigationsensitivity of stomata was greater (higher k values) in theintact plants as compared with the root-restricted plants Thisindicated that internal signals in this case imposed by rootpruning treatments that caused immediate stomatal closure(Fig 1b) removed a portion of the stomatesrsquo ability torespond to external environmental factors of humidity and Ca

as empirically described by the BWB model Nonethelessstomates of both the intact and root pruned vines appearedequally sensitive to VPD throughout the season

The present results show that stomatal closure occurredfollowing an increase in VPD and was linked to a rise in thetranspiration rate (E) of the whole leaf in both intact and root-restricted plants Results of other studies have suggested thatthe closure of stomata with rising VPD appears to be more of afeedback response linked to some other aspect of transpiration(Shackel and Brinckmann 1985 Mott and Franks 2001) and leafwater loss rather than to a direct response to humidity at the leafsurface (Mott and Parkhurst 1991Monteith 1995) Neverthelessunder higher VPD leaf transpiration of root-pruned plants waslower than control plants An explanation for this phenomenonmay lie in part in the decrease ofyleaf in the root-restricted vinesand consequent fall in whole-plant water flux FurthermoreCi ofleaves of root-restricted plants was somewhat lower than intactplants (data not shown) and there was a and similar correlationbetween gs and A This confirmed in part that patchy stomatalclosure was of little significance in the present study and inconditions of progressive developing water deficit (Mott andBuckley 1998)

The observed relationship between stomatal behaviour VPD(Fig 4a) and plant water loss (Fig 4c) further suggests that theresponse to VPD was related to plant hydraulics and that itmay play a role in regulating leaf water potential (Oren et al2001)Underwater deficits progressive closure of stomata canbe

Kplant (mmol MPandash1 mndash2 sndash1)

g s (m

mol

mndash2

sndash1

)

50

100

150

200

250

30025 June

50

100

150

200

250

30026 July

0 2 4 6 8 10 120

50

100

150

200

250

30016 August

(a) (d)

(b) (e)

(c) (f)

Ψleaf (MPa)

ndash18ndash15ndash12ndash09ndash06ndash03

Control RPI

RPD

R2 = 087

R2 = 056

R2 = 071

Fig 5 (andashc) Stomatal conductance (gs) as a function of leaf specificconductance (Kplant) and (dndashf) of leaf water potential (yleaf) for differentroot pruning treatments during 3 days in 2004

ΨPD (MPa) ΨStem (MPa)

00 ndash01 ndash02 ndash03 ndash04 ndash05 ndash06 ndash08 ndash10 ndash12 ndash14 ndash16

Kpl

ant (

mm

ol M

Pandash1

mndash2

sndash1

)

0

2

4

6

8

10(a) (b)

Control RPIRPD

R2 = 075R2 = 077

Fig 6 Relationship between mid-morning leaf specific hydraulicconductance Kplant and (a) pre-dawn water potential (yPD) and (b)midday stem water potential (yStem) for different root pruning treatments

1024 Functional Plant Biology V Zufferey and D R Smart

observed when VPD increases and hydraulic conductance alongthe soil-to-leaf pathway (Kplant) decreases yleaf then remainsabove a critical threshold preventing xylem cavitation andhydraulic failure (Tyree and Sperry 1988) A positivecorrelation between hydraulic and stomatal conductances(Saliendra et al 1995 Comstock 2000 Addington et al2004) but a relatively constant yleaf suggests a feedback linkbetween gs and some form of hydraulic signal (Fuchs andLivingston 1996 Nardini et al 2001) Experimentallyreducing xylem-hydraulic conductance by various techniques(Sperry et al 1993 Hubbard et al 1999 2001) including rootpruning (Teskey et al 1983 Meinzer and Grantz 1990) bringsabout a decrease in gs thereby suggesting the hydraulicconductance for transpiration flux forms an integral anddynamic part of the stomatal control mechanism (Franks2004) Our observations from partial root removal in irrigatedversusnon-irrigated soils supports this contention in asmuchasgsand yleaf in RPI and RPD vines were shifted to different levelswith RPI having lower gs and yleaf

According to Meinzer (2002) true feed-forward control of gswith respect toyleaf would allow stomata to limit transpiration inanticipation of potential reductions in yleaf resulting fromrestricted available water in the soil environment or increasedsoil-to-leaf hydraulic resistance Experiments by Teskey et al(1983) on Abies amabilis and by Meinzer and Grantz (1990) insugarcane demonstrated that gs decreased in response to rootremoval with no change inyleaf This response can be interpretedas generating a hydraulic signal in the form of diminished leaf-specific hydraulic conductance following partial root pruning Ifstomata did not respond by restricting transpiration yleaf woulddecline In the present experiments gs of root-restricted plantsdecreased immediately asKplant fell during the day This lead to arapid drop in yleaf (and leaf wilting) in root-pruned plantscompared with intact plants This response differed from theapparent feed-forward stomatal behaviour in response to rootpruning where stomata appeared to overcompensate and causeyleaf to be noticeably less negative following root removal thanbefore (Meinzer and Grantz 1990)

Feed-forward behaviour of stomata with respect to regulationof yleaf has been attributed to the presence of chemical signalsthat modify stomatal opening and which are brought to theleaf in the transpiration flow (Davies et al 1994) In drought-avoiding plants (isohydric) the leaf water potential is unaffectedby soil water deficits and isohydric behaviour is linked to aninteraction between both chemical and hydraulic information(Tardieu and Simonneau 1998) In drought-tolerant species(anisohydric) a controlling influence of leaf water status on gsneed not be invoked Rather leaf water status is likely to vary as aconsequence of water flux through the plant which is controlledby stomatal conductance Schultz (2003) observed that thegrapevine cultivar lsquoSyrahrsquo behaves in an anisohydric mannerand that the differences in thewater-conducting capacity of stemsand especially petioles may be the origin of the isohydric andanisohydric signal regulating gs in grapevine Results from thepresent study showed that yleaf of lsquoSyrahrsquo fell sharply asevaporative demand rose During the daytime and when waterstress was accentuated as a result of partial root removal morenegative leaf water potentials were realised compared withwell-watered intact plants This provided some confirmation of

anisohydric behaviour but also suggested root and other signalsin the plant hydraulic path have a strong influence on stomatalbehaviour Other contradictory studies have been reported inthe literature (see review by Chaves et al 2010) showingthat the same cultivar could behave differently dependingon experimental conditions (eg intensity and duration ofwater deficits VPD and root system) and physiological oranatomical adjustments (eg cell osmotic adjustment wallelasticity vessel conductivity)

However no recovery of Kplant was observed during theseason in root-restricted plants in comparison with the intactcontrols An increase in the total root surface area for absorptionby the growth of new roots after partial root pruning couldimprove Kplant (Sperry et al 2002) and Alsina and co-workersverified this hypothesis for grape (Alsina et al 2011) It is notcertain that roots produced during a period of water stress(drought) will have sufficient water conductivity (Ewers et al2000)Atgt60 cmdepth inmoist soils root conductivity increasedbut gs did not increase substantially Either the failure to producenew roots or lower conductivity would help to explain whyKplant

in root-restricted vines did not regain hydraulic conductancelevels approaching those of the intact non-root pruned vinesduring the growing season Moreover it should be noted thatdiurnal measurements of Kplant conducted in the field may giverise to errors if they were conducted under non-steady-stateconditions (Hubbard et al 2001) Non-steady-state conditionsmight arise as a consequence of significant capacitance (Schultzand Matthews 1997 Smart et al 2005) or rapid changes inenvironmental conditions over time

Partial root removal may also have had an influence onembolism development as a consequence of rapid changes inwater potential and as a consequence may have contributed tostomatal closure by an embolism related hydraulic signal(Lovisolo and Schubert 1998) Salleo et al (2000) suggestedthat even cavitation could act as a hydraulicmodulator for stomataopening The impact of cavitation on total hydraulic conductanceand finally on gs would depend on the position of cavitation inthe root-leaf pathway and on the distribution of hydraulicresistances between roots stems petioles and leaves (Meinzer2002 Zufferey et al 2011) Root systems often constitute a largepart of the total plant hydraulic resistance (Tyree et al 1998) andthe relative contributions of stem and leaf resistance to theremaining shoot resistance can vary substantially (Choat et al2010) Leaf wilting symptoms together with the very low gsvalues observed in this study for root-restricted vines andparticularly in root-pruned plants in wet irrigated soils may beattributed in part to an increased vulnerability of xylem tocavitation coupled with a high evaporative demand Thesecavitation events may act in this way as a signal for stomatalclosure (Nardini et al 2001) or stomatal closure would occur toprevent cavitation (Cochard et al 2002)

Significant decreases in gs recorded in root-restrictedplants were closely related to a reduction in leaf-specifichydraulic conductance (Fig 5) However other factors suchas hormonal control or even a combination of chemical andhydraulic messages which various authors (Tardieu andDavies 1993 Tardieu and Simonneau 1998 reviewed byHolbrook et al 2002) have brought to light cannot be entirelyruled out and have not been directly evaluated in this

Stomatal behaviour and root removal Functional Plant Biology 1025

investigationTheobservation thatgmaxwasdifferent between theroot-restricted plants and intact plants at similaryPD representingdifferent soilwater status prospectedby the roots andpresumablytriggering ABA synthesis (or other hormonal factors such ascytokinins) suggests that either the signals were different innature or concentration or the receptor site sensitivity variedbetween the different root pruning treatments We also notethat yPD changed within 48 h following root pruning Thisindicated that root pruned vines were not able to refill xylemand recharge hydraulic status to the same extent as the intactcontrols despite of the fact that roots of RPD vines had rootsexposed to the same irrigation source as the controls This hasshown that axial redistribution and reverse flow of water occursto such an extent that rehydration of lateral roots occurs (Smartet al 2005 Burgess and Bleby 2006) This raises questionswith respect to root signalling If pruning of roughly 30 ofthe root system occurs then it might be a reasonable assumptionthat removal of 30 of fine roots and fine root tips occurs whichmay be the primary source of the root signals that influence gs(Bauerle et al 2008) Further this suggests that root signallingwas of lesser importance in the initial response of gs and A to rootpruning

References

AddingtonRNMitchell RJOrenRDonovanLA (2004)Stomatal sensitivityto vapor pressure deficit and its relationship to hydraulic conductance inPinus palustris Tree Physiology 24 561ndash569 doi101093treephys245561

AlsinaMMSmartDRBauerleTdeHerraldeFBielCStockertCMNegronCSaveR (2011)Seasonal changes ofwhole root systemconductanceby adrought tolerant grape root system Journal of Experimental Botany 6299ndash109 doi101093jxberq247

Ameacuteglio TArcher P CohenMValancogneCDaudet FDayauSCruiziat P(1999) Significance and limits in the use of predawn leaf water potentialfor tree irrigation Plant and Soil 207 155ndash167 doi101023A1026415302759

Ball JT Woodrow IE Berry JA (1987) A model predicting stomatalconductance and its contribution to the control of photosynthesis underdifferent environmental conditions In lsquoProgress in photosynthesisresearchrsquo (Ed J Biggins) pp 221ndash234 (Martinus Nijhoff DordrechtThe Netherlands)

Bauerle TL Richards JH Smart DR Eissenstat DM (2008) Importance ofinternal hydraulic redistribution for prolonging the lifespan of roots in drysoil Plant Cell amp Environment 31 177ndash186

Breda N Granier A Barataud F Moyne C (1995) Soil water dynamics in anoak stand I Soil moisture water potentials and water uptake by rootsPlant and Soil 172 17ndash27

Brodribb TJ Holbrook NM (2003) Stomatal closure during leafdehydratation correlation with other leaf physiological traits PlantPhysiology 132 2166ndash2173 doi101104pp103023879

Burgess SSO Bleby TM (2006) Redistribution of soil water to lateral rootsmediatedby stem tissuesJournalofExperimentalBotany57 3283ndash3291doi101093jxberl085

ChavesMMTenhunen JDHarley P LangeOL (1987)Gas exchange studiesin two Portuguese grapevine cultivars Physiologia Plantarum 70639ndash647 doi101111j1399-30541987tb04318x

Chaves MM Zarrouk O Fransisco R Costa JM Santos T Regalado APRodriguesMLLopesCM (2010)Grapevine under deficit irrigation hintsfrom physiological and molecular data Annals of Botany 105 661ndash676doi101093aobmcq030

Choat B DraytonWMBrodersen CRMatthewsMA Shackel KAWadaHMcElrone AJ (2010) Measurement of vulnerability to water stress-induced cavitation in grapevine a comparison of four techniquesapplied to long-vesseled species Plant Cell amp Environment 331502ndash1512

Cochard H Coll L Le Roux X Ameglio T (2002) Unraveling the effects ofplant hydraulics on stomatal closure during water stress in walnut PlantPhysiology 128 282ndash290 doi101104pp010400

Comstock JP (2000) Variation in hydraulic architecture and gas exchange intwo desert sub-shrubs Hymenoclea salsola (TampG) and Ambrosiadumosa Oecologia 125 1ndash10 doi101007PL00008879

Correia MJ Pereira JS Chaves MM Rodrigues ML Pacheco CA (1995)ABA xylem concentrations determine maximum daily leaf conductanceof field-grown Vitis vinifera L plants Plant Cell amp Environment 18511ndash521 doi101111j1365-30401995tb00551x

Davies WJ Zhang J (1991) Root signals and the regulation of growth anddevelopment of plants in drying soil Annual Review of Plant Physiologyand Plant Molecular Biology 42 55ndash76 doi101146annurevpp42060191000415

Davies WJ Tardieu F Trejo CL (1994) How do chemical signals work inplants that grow in drying soil Plant Physiology 104 309ndash314

Donovan LA Richards JH LintonMJ (2003)Magnitude and mechanisms ofpredawn disequilibrium between predawn and plant soil water potentialsin desert shrubsEcology84 463ndash470 doi1018900012-9658(2003)084[0463MAMODB]20CO2

Ewers BEOrenR Sperry JS (2000) Influence of nutrient versuswater supplyon hydraulic architecture and water balance in Pinus taeda Plant Cell ampEnvironment 23 1055ndash1066 doi101046j1365-3040200000625x

Franks PJ (2004) Stomatal control and hydraulic conductance with specialreference to tall trees Tree Physiology 24 865ndash878 doi101093treephys248865

FuchsEELivingstonNJ (1996)Hydraulic control of stomatal conductance inDouglasfir (Pseudotsugamensiesii (Mirb) Franco) and alder (Alnus rubra(Bong)) seedlings Plant Cell amp Environment 19 1091ndash1098doi101111j1365-30401996tb00216x

Harley PC Baldocchi DD (1995) Scaling carbon dioxide and water vapourexchange from leaf to canopy in a deciduous forest I Leaf modelparametrization Plant Cell amp Environment 18 1146ndash1156doi101111j1365-30401995tb00625x

Holbrook NM Shashidhar VR James RAMunns R (2002) Stomatal controlin tomato with ABA-deficient roots response of grafted plants to soildrying Journal of Experimental Botany 53 1503ndash1514 doi101093jexbot533731503

Hubbard RM Bond BJ Ryan MG (1999) Evidence that hydraulicconductance limits photosynthesis in old Pinus ponderosa trees TreePhysiology 19 165ndash172 doi101093treephys193165

Hubbard RM Ryan MG Stiller V Sperry JS (2001) Stomatal conductanceand photosynthesis vary lineary with plant hydraulic conductance inPonderosa pine Plant Cell amp Environment 24 113ndash121 doi101046j1365-3040200100660x

Jarvis AJ Davies WJ (1998) Modeling stomatal responses to soil andatmospheric drought Journal of Experimental Botany 49(SpecialIssue) 399ndash406 doi101093jxb49Special_Issue399

Jones HG (1998) Stomatal control of photosynthesis and transpirationJournal of Experimental Botany 49(Special Issue) 387ndash398

Kramer PJ Boyer JS (1995) lsquoWater relations of plants and soilsrsquo (AcademicPress San Diego CA)

Leuning R (1990) Modeling stomatal behaviour and photosynthesis ofEucalyptus grandis Australian Journal of Plant Physiology 17159ndash175 doi101071PP9900159

Loveys BR (1991) How useful is a knowledge of ABA physiology for cropimprovement In lsquoAbscisic acid physiology and biochemistryrsquo (Eds WJDavies HG Jones) pp 245ndash260 (Bios Scientific Publishers Oxford)

1026 Functional Plant Biology V Zufferey and D R Smart

observed when VPD increases and hydraulic conductance alongthe soil-to-leaf pathway (Kplant) decreases yleaf then remainsabove a critical threshold preventing xylem cavitation andhydraulic failure (Tyree and Sperry 1988) A positivecorrelation between hydraulic and stomatal conductances(Saliendra et al 1995 Comstock 2000 Addington et al2004) but a relatively constant yleaf suggests a feedback linkbetween gs and some form of hydraulic signal (Fuchs andLivingston 1996 Nardini et al 2001) Experimentallyreducing xylem-hydraulic conductance by various techniques(Sperry et al 1993 Hubbard et al 1999 2001) including rootpruning (Teskey et al 1983 Meinzer and Grantz 1990) bringsabout a decrease in gs thereby suggesting the hydraulicconductance for transpiration flux forms an integral anddynamic part of the stomatal control mechanism (Franks2004) Our observations from partial root removal in irrigatedversusnon-irrigated soils supports this contention in asmuchasgsand yleaf in RPI and RPD vines were shifted to different levelswith RPI having lower gs and yleaf

According to Meinzer (2002) true feed-forward control of gswith respect toyleaf would allow stomata to limit transpiration inanticipation of potential reductions in yleaf resulting fromrestricted available water in the soil environment or increasedsoil-to-leaf hydraulic resistance Experiments by Teskey et al(1983) on Abies amabilis and by Meinzer and Grantz (1990) insugarcane demonstrated that gs decreased in response to rootremoval with no change inyleaf This response can be interpretedas generating a hydraulic signal in the form of diminished leaf-specific hydraulic conductance following partial root pruning Ifstomata did not respond by restricting transpiration yleaf woulddecline In the present experiments gs of root-restricted plantsdecreased immediately asKplant fell during the day This lead to arapid drop in yleaf (and leaf wilting) in root-pruned plantscompared with intact plants This response differed from theapparent feed-forward stomatal behaviour in response to rootpruning where stomata appeared to overcompensate and causeyleaf to be noticeably less negative following root removal thanbefore (Meinzer and Grantz 1990)

Feed-forward behaviour of stomata with respect to regulationof yleaf has been attributed to the presence of chemical signalsthat modify stomatal opening and which are brought to theleaf in the transpiration flow (Davies et al 1994) In drought-avoiding plants (isohydric) the leaf water potential is unaffectedby soil water deficits and isohydric behaviour is linked to aninteraction between both chemical and hydraulic information(Tardieu and Simonneau 1998) In drought-tolerant species(anisohydric) a controlling influence of leaf water status on gsneed not be invoked Rather leaf water status is likely to vary as aconsequence of water flux through the plant which is controlledby stomatal conductance Schultz (2003) observed that thegrapevine cultivar lsquoSyrahrsquo behaves in an anisohydric mannerand that the differences in thewater-conducting capacity of stemsand especially petioles may be the origin of the isohydric andanisohydric signal regulating gs in grapevine Results from thepresent study showed that yleaf of lsquoSyrahrsquo fell sharply asevaporative demand rose During the daytime and when waterstress was accentuated as a result of partial root removal morenegative leaf water potentials were realised compared withwell-watered intact plants This provided some confirmation of

anisohydric behaviour but also suggested root and other signalsin the plant hydraulic path have a strong influence on stomatalbehaviour Other contradictory studies have been reported inthe literature (see review by Chaves et al 2010) showingthat the same cultivar could behave differently dependingon experimental conditions (eg intensity and duration ofwater deficits VPD and root system) and physiological oranatomical adjustments (eg cell osmotic adjustment wallelasticity vessel conductivity)

However no recovery of Kplant was observed during theseason in root-restricted plants in comparison with the intactcontrols An increase in the total root surface area for absorptionby the growth of new roots after partial root pruning couldimprove Kplant (Sperry et al 2002) and Alsina and co-workersverified this hypothesis for grape (Alsina et al 2011) It is notcertain that roots produced during a period of water stress(drought) will have sufficient water conductivity (Ewers et al2000)Atgt60 cmdepth inmoist soils root conductivity increasedbut gs did not increase substantially Either the failure to producenew roots or lower conductivity would help to explain whyKplant

in root-restricted vines did not regain hydraulic conductancelevels approaching those of the intact non-root pruned vinesduring the growing season Moreover it should be noted thatdiurnal measurements of Kplant conducted in the field may giverise to errors if they were conducted under non-steady-stateconditions (Hubbard et al 2001) Non-steady-state conditionsmight arise as a consequence of significant capacitance (Schultzand Matthews 1997 Smart et al 2005) or rapid changes inenvironmental conditions over time

Partial root removal may also have had an influence onembolism development as a consequence of rapid changes inwater potential and as a consequence may have contributed tostomatal closure by an embolism related hydraulic signal(Lovisolo and Schubert 1998) Salleo et al (2000) suggestedthat even cavitation could act as a hydraulicmodulator for stomataopening The impact of cavitation on total hydraulic conductanceand finally on gs would depend on the position of cavitation inthe root-leaf pathway and on the distribution of hydraulicresistances between roots stems petioles and leaves (Meinzer2002 Zufferey et al 2011) Root systems often constitute a largepart of the total plant hydraulic resistance (Tyree et al 1998) andthe relative contributions of stem and leaf resistance to theremaining shoot resistance can vary substantially (Choat et al2010) Leaf wilting symptoms together with the very low gsvalues observed in this study for root-restricted vines andparticularly in root-pruned plants in wet irrigated soils may beattributed in part to an increased vulnerability of xylem tocavitation coupled with a high evaporative demand Thesecavitation events may act in this way as a signal for stomatalclosure (Nardini et al 2001) or stomatal closure would occur toprevent cavitation (Cochard et al 2002)

Significant decreases in gs recorded in root-restrictedplants were closely related to a reduction in leaf-specifichydraulic conductance (Fig 5) However other factors suchas hormonal control or even a combination of chemical andhydraulic messages which various authors (Tardieu andDavies 1993 Tardieu and Simonneau 1998 reviewed byHolbrook et al 2002) have brought to light cannot be entirelyruled out and have not been directly evaluated in this

Stomatal behaviour and root removal Functional Plant Biology 1025

investigationTheobservation thatgmaxwasdifferent between theroot-restricted plants and intact plants at similaryPD representingdifferent soilwater status prospectedby the roots andpresumablytriggering ABA synthesis (or other hormonal factors such ascytokinins) suggests that either the signals were different innature or concentration or the receptor site sensitivity variedbetween the different root pruning treatments We also notethat yPD changed within 48 h following root pruning Thisindicated that root pruned vines were not able to refill xylemand recharge hydraulic status to the same extent as the intactcontrols despite of the fact that roots of RPD vines had rootsexposed to the same irrigation source as the controls This hasshown that axial redistribution and reverse flow of water occursto such an extent that rehydration of lateral roots occurs (Smartet al 2005 Burgess and Bleby 2006) This raises questionswith respect to root signalling If pruning of roughly 30 ofthe root system occurs then it might be a reasonable assumptionthat removal of 30 of fine roots and fine root tips occurs whichmay be the primary source of the root signals that influence gs(Bauerle et al 2008) Further this suggests that root signallingwas of lesser importance in the initial response of gs and A to rootpruning

References

AddingtonRNMitchell RJOrenRDonovanLA (2004)Stomatal sensitivityto vapor pressure deficit and its relationship to hydraulic conductance inPinus palustris Tree Physiology 24 561ndash569 doi101093treephys245561

AlsinaMMSmartDRBauerleTdeHerraldeFBielCStockertCMNegronCSaveR (2011)Seasonal changes ofwhole root systemconductanceby adrought tolerant grape root system Journal of Experimental Botany 6299ndash109 doi101093jxberq247

Ameacuteglio TArcher P CohenMValancogneCDaudet FDayauSCruiziat P(1999) Significance and limits in the use of predawn leaf water potentialfor tree irrigation Plant and Soil 207 155ndash167 doi101023A1026415302759

Ball JT Woodrow IE Berry JA (1987) A model predicting stomatalconductance and its contribution to the control of photosynthesis underdifferent environmental conditions In lsquoProgress in photosynthesisresearchrsquo (Ed J Biggins) pp 221ndash234 (Martinus Nijhoff DordrechtThe Netherlands)

Bauerle TL Richards JH Smart DR Eissenstat DM (2008) Importance ofinternal hydraulic redistribution for prolonging the lifespan of roots in drysoil Plant Cell amp Environment 31 177ndash186

Breda N Granier A Barataud F Moyne C (1995) Soil water dynamics in anoak stand I Soil moisture water potentials and water uptake by rootsPlant and Soil 172 17ndash27

Brodribb TJ Holbrook NM (2003) Stomatal closure during leafdehydratation correlation with other leaf physiological traits PlantPhysiology 132 2166ndash2173 doi101104pp103023879

Burgess SSO Bleby TM (2006) Redistribution of soil water to lateral rootsmediatedby stem tissuesJournalofExperimentalBotany57 3283ndash3291doi101093jxberl085

ChavesMMTenhunen JDHarley P LangeOL (1987)Gas exchange studiesin two Portuguese grapevine cultivars Physiologia Plantarum 70639ndash647 doi101111j1399-30541987tb04318x

Chaves MM Zarrouk O Fransisco R Costa JM Santos T Regalado APRodriguesMLLopesCM (2010)Grapevine under deficit irrigation hintsfrom physiological and molecular data Annals of Botany 105 661ndash676doi101093aobmcq030

Choat B DraytonWMBrodersen CRMatthewsMA Shackel KAWadaHMcElrone AJ (2010) Measurement of vulnerability to water stress-induced cavitation in grapevine a comparison of four techniquesapplied to long-vesseled species Plant Cell amp Environment 331502ndash1512

Cochard H Coll L Le Roux X Ameglio T (2002) Unraveling the effects ofplant hydraulics on stomatal closure during water stress in walnut PlantPhysiology 128 282ndash290 doi101104pp010400

Comstock JP (2000) Variation in hydraulic architecture and gas exchange intwo desert sub-shrubs Hymenoclea salsola (TampG) and Ambrosiadumosa Oecologia 125 1ndash10 doi101007PL00008879

Correia MJ Pereira JS Chaves MM Rodrigues ML Pacheco CA (1995)ABA xylem concentrations determine maximum daily leaf conductanceof field-grown Vitis vinifera L plants Plant Cell amp Environment 18511ndash521 doi101111j1365-30401995tb00551x

Davies WJ Zhang J (1991) Root signals and the regulation of growth anddevelopment of plants in drying soil Annual Review of Plant Physiologyand Plant Molecular Biology 42 55ndash76 doi101146annurevpp42060191000415

Davies WJ Tardieu F Trejo CL (1994) How do chemical signals work inplants that grow in drying soil Plant Physiology 104 309ndash314

Donovan LA Richards JH LintonMJ (2003)Magnitude and mechanisms ofpredawn disequilibrium between predawn and plant soil water potentialsin desert shrubsEcology84 463ndash470 doi1018900012-9658(2003)084[0463MAMODB]20CO2

Ewers BEOrenR Sperry JS (2000) Influence of nutrient versuswater supplyon hydraulic architecture and water balance in Pinus taeda Plant Cell ampEnvironment 23 1055ndash1066 doi101046j1365-3040200000625x

Franks PJ (2004) Stomatal control and hydraulic conductance with specialreference to tall trees Tree Physiology 24 865ndash878 doi101093treephys248865

FuchsEELivingstonNJ (1996)Hydraulic control of stomatal conductance inDouglasfir (Pseudotsugamensiesii (Mirb) Franco) and alder (Alnus rubra(Bong)) seedlings Plant Cell amp Environment 19 1091ndash1098doi101111j1365-30401996tb00216x

Harley PC Baldocchi DD (1995) Scaling carbon dioxide and water vapourexchange from leaf to canopy in a deciduous forest I Leaf modelparametrization Plant Cell amp Environment 18 1146ndash1156doi101111j1365-30401995tb00625x

Holbrook NM Shashidhar VR James RAMunns R (2002) Stomatal controlin tomato with ABA-deficient roots response of grafted plants to soildrying Journal of Experimental Botany 53 1503ndash1514 doi101093jexbot533731503

Hubbard RM Bond BJ Ryan MG (1999) Evidence that hydraulicconductance limits photosynthesis in old Pinus ponderosa trees TreePhysiology 19 165ndash172 doi101093treephys193165

Hubbard RM Ryan MG Stiller V Sperry JS (2001) Stomatal conductanceand photosynthesis vary lineary with plant hydraulic conductance inPonderosa pine Plant Cell amp Environment 24 113ndash121 doi101046j1365-3040200100660x

Jarvis AJ Davies WJ (1998) Modeling stomatal responses to soil andatmospheric drought Journal of Experimental Botany 49(SpecialIssue) 399ndash406 doi101093jxb49Special_Issue399

Jones HG (1998) Stomatal control of photosynthesis and transpirationJournal of Experimental Botany 49(Special Issue) 387ndash398

Kramer PJ Boyer JS (1995) lsquoWater relations of plants and soilsrsquo (AcademicPress San Diego CA)

Leuning R (1990) Modeling stomatal behaviour and photosynthesis ofEucalyptus grandis Australian Journal of Plant Physiology 17159ndash175 doi101071PP9900159

Loveys BR (1991) How useful is a knowledge of ABA physiology for cropimprovement In lsquoAbscisic acid physiology and biochemistryrsquo (Eds WJDavies HG Jones) pp 245ndash260 (Bios Scientific Publishers Oxford)

1026 Functional Plant Biology V Zufferey and D R Smart

investigationTheobservation thatgmaxwasdifferent between theroot-restricted plants and intact plants at similaryPD representingdifferent soilwater status prospectedby the roots andpresumablytriggering ABA synthesis (or other hormonal factors such ascytokinins) suggests that either the signals were different innature or concentration or the receptor site sensitivity variedbetween the different root pruning treatments We also notethat yPD changed within 48 h following root pruning Thisindicated that root pruned vines were not able to refill xylemand recharge hydraulic status to the same extent as the intactcontrols despite of the fact that roots of RPD vines had rootsexposed to the same irrigation source as the controls This hasshown that axial redistribution and reverse flow of water occursto such an extent that rehydration of lateral roots occurs (Smartet al 2005 Burgess and Bleby 2006) This raises questionswith respect to root signalling If pruning of roughly 30 ofthe root system occurs then it might be a reasonable assumptionthat removal of 30 of fine roots and fine root tips occurs whichmay be the primary source of the root signals that influence gs(Bauerle et al 2008) Further this suggests that root signallingwas of lesser importance in the initial response of gs and A to rootpruning

References

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AlsinaMMSmartDRBauerleTdeHerraldeFBielCStockertCMNegronCSaveR (2011)Seasonal changes ofwhole root systemconductanceby adrought tolerant grape root system Journal of Experimental Botany 6299ndash109 doi101093jxberq247

Ameacuteglio TArcher P CohenMValancogneCDaudet FDayauSCruiziat P(1999) Significance and limits in the use of predawn leaf water potentialfor tree irrigation Plant and Soil 207 155ndash167 doi101023A1026415302759

Ball JT Woodrow IE Berry JA (1987) A model predicting stomatalconductance and its contribution to the control of photosynthesis underdifferent environmental conditions In lsquoProgress in photosynthesisresearchrsquo (Ed J Biggins) pp 221ndash234 (Martinus Nijhoff DordrechtThe Netherlands)

Bauerle TL Richards JH Smart DR Eissenstat DM (2008) Importance ofinternal hydraulic redistribution for prolonging the lifespan of roots in drysoil Plant Cell amp Environment 31 177ndash186

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ChavesMMTenhunen JDHarley P LangeOL (1987)Gas exchange studiesin two Portuguese grapevine cultivars Physiologia Plantarum 70639ndash647 doi101111j1399-30541987tb04318x

Chaves MM Zarrouk O Fransisco R Costa JM Santos T Regalado APRodriguesMLLopesCM (2010)Grapevine under deficit irrigation hintsfrom physiological and molecular data Annals of Botany 105 661ndash676doi101093aobmcq030

Choat B DraytonWMBrodersen CRMatthewsMA Shackel KAWadaHMcElrone AJ (2010) Measurement of vulnerability to water stress-induced cavitation in grapevine a comparison of four techniquesapplied to long-vesseled species Plant Cell amp Environment 331502ndash1512

Cochard H Coll L Le Roux X Ameglio T (2002) Unraveling the effects ofplant hydraulics on stomatal closure during water stress in walnut PlantPhysiology 128 282ndash290 doi101104pp010400

Comstock JP (2000) Variation in hydraulic architecture and gas exchange intwo desert sub-shrubs Hymenoclea salsola (TampG) and Ambrosiadumosa Oecologia 125 1ndash10 doi101007PL00008879

Correia MJ Pereira JS Chaves MM Rodrigues ML Pacheco CA (1995)ABA xylem concentrations determine maximum daily leaf conductanceof field-grown Vitis vinifera L plants Plant Cell amp Environment 18511ndash521 doi101111j1365-30401995tb00551x

Davies WJ Zhang J (1991) Root signals and the regulation of growth anddevelopment of plants in drying soil Annual Review of Plant Physiologyand Plant Molecular Biology 42 55ndash76 doi101146annurevpp42060191000415

Davies WJ Tardieu F Trejo CL (1994) How do chemical signals work inplants that grow in drying soil Plant Physiology 104 309ndash314

Donovan LA Richards JH LintonMJ (2003)Magnitude and mechanisms ofpredawn disequilibrium between predawn and plant soil water potentialsin desert shrubsEcology84 463ndash470 doi1018900012-9658(2003)084[0463MAMODB]20CO2

Ewers BEOrenR Sperry JS (2000) Influence of nutrient versuswater supplyon hydraulic architecture and water balance in Pinus taeda Plant Cell ampEnvironment 23 1055ndash1066 doi101046j1365-3040200000625x

Franks PJ (2004) Stomatal control and hydraulic conductance with specialreference to tall trees Tree Physiology 24 865ndash878 doi101093treephys248865

FuchsEELivingstonNJ (1996)Hydraulic control of stomatal conductance inDouglasfir (Pseudotsugamensiesii (Mirb) Franco) and alder (Alnus rubra(Bong)) seedlings Plant Cell amp Environment 19 1091ndash1098doi101111j1365-30401996tb00216x

Harley PC Baldocchi DD (1995) Scaling carbon dioxide and water vapourexchange from leaf to canopy in a deciduous forest I Leaf modelparametrization Plant Cell amp Environment 18 1146ndash1156doi101111j1365-30401995tb00625x

Holbrook NM Shashidhar VR James RAMunns R (2002) Stomatal controlin tomato with ABA-deficient roots response of grafted plants to soildrying Journal of Experimental Botany 53 1503ndash1514 doi101093jexbot533731503

Hubbard RM Bond BJ Ryan MG (1999) Evidence that hydraulicconductance limits photosynthesis in old Pinus ponderosa trees TreePhysiology 19 165ndash172 doi101093treephys193165

Hubbard RM Ryan MG Stiller V Sperry JS (2001) Stomatal conductanceand photosynthesis vary lineary with plant hydraulic conductance inPonderosa pine Plant Cell amp Environment 24 113ndash121 doi101046j1365-3040200100660x

Jarvis AJ Davies WJ (1998) Modeling stomatal responses to soil andatmospheric drought Journal of Experimental Botany 49(SpecialIssue) 399ndash406 doi101093jxb49Special_Issue399

Jones HG (1998) Stomatal control of photosynthesis and transpirationJournal of Experimental Botany 49(Special Issue) 387ndash398

Kramer PJ Boyer JS (1995) lsquoWater relations of plants and soilsrsquo (AcademicPress San Diego CA)

Leuning R (1990) Modeling stomatal behaviour and photosynthesis ofEucalyptus grandis Australian Journal of Plant Physiology 17159ndash175 doi101071PP9900159

Loveys BR (1991) How useful is a knowledge of ABA physiology for cropimprovement In lsquoAbscisic acid physiology and biochemistryrsquo (Eds WJDavies HG Jones) pp 245ndash260 (Bios Scientific Publishers Oxford)

1026 Functional Plant Biology V Zufferey and D R Smart

LovisoloC SchubertA (1998)Effects ofwater stress onvessel size andxylemhydraulic conductivity inVitis viniferaL Journal of ExperimentalBotany49 693ndash700

Lovisolo C Hartung W Schubert A (2002) Whole-plant hydraulicconductance and root-to-shoot flow of abscisic acid are independentlyaffected by stress in grapevinesFunctional Plant Biology29 1349ndash1356doi101071FP02079

Meinzer FC (2002) Co-ordination of vapor and liquid phase water transportproprieties in plants Plant Cell amp Environment 25 265ndash274doi101046j1365-3040200200781x

Meinzer FC Grantz DA (1990) Stomatal and hydraulic conductance ingrowing sugarcane stomatal adjustment to water transport capacityPlant Cell amp Environment 13 383ndash388 doi101111j1365-30401990tb02142x

Monteith JL (1995) A reinterpretation of stomatal responses to humidityPlant Cell amp Environment 18 357ndash364 doi101111j1365-30401995tb00371x

Morison JI (1987) Intercellular CO2 concentration and stomatal response toCO2 In lsquoStomatal functionrsquo (Eds E Zeiger GD Farquhar IR Cowan)pp 229ndash251 (Stanford University Press Stanford)

Mott KA (1988) Do stomata respond to CO2 concentrations other thanintercellular Plant Physiology 86 200ndash203 doi101104pp861200

Mott KA Buckley TN (1998) Stomatal heterogeneity Journal ofExperimental Botany 49 407ndash417

Mott KA Franks PJ (2001) The role of epidermal turgor in stomatalinteractions following a local perturbation in humidity Plant Cell ampEnvironment 24 657ndash662 doi101046j0016-8025200100705x

Mott KA Parkhurst DF (1991) Stomata response to humidity in air and heloxPlant Cell amp Environment 14 509ndash515 doi101111j1365-30401991tb01521x

Nardini A Tyree MT Salleo S (2001) Xylem cavitation in the leaf of Prunuslaurocerasus and its impact on leaf hydraulics Plant Physiology 1251700ndash1709 doi101104pp12541700

Oren R Sperry JS Ewers BE Pataki DE Phillips N Megonigal JP (2001)Sensitivity ofmean canopy stomatal conductance to vapor pressure deficitin a flooded Taxadium distichum L forest hydraulic and non-hydrauliceffects Oecologia 126 21ndash29 doi101007s004420000497

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Salleo S Nardini A Pitt F LoGulloM (2000)Xylem cavitation and hydrauliccontrol of stomatal conductance in Laurel (Laurus nobilis L) Plant Cellamp Environment 23 71ndash79 doi101046j1365-3040200000516x

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Schultz HR (2003) Differences in hydraulic architecture account fornear-isohydric and anisohydric behaviour of two field-grown Vitisvinifera L cultivars during drought Plant Cell amp Environment 261393ndash1405 doi101046j1365-3040200301064x

Schultz HR Matthews MA (1997) High vapour pressure deficit exacerbatesxylem cavitation and photoinhibition in shade-grown Piper auritumHBampK during prolonged sunflecks I Dynamics of plant waterrelations Oecologia 110 312ndash319 doi101007s004420050164

Shackel KA Brinckmann E (1985) In situ measurement of epidermal cellturgor leaf water potential and gas exchange in Tradescantia virginia LPlant Physiology 78 66ndash70 doi101104pp78166

Smart RE Combe B (1983) Water relations of grapevines In lsquoAdditionalwoody crop plants Water deficit and plant growth Vol VIIrsquo (Ed TTKozlowski) pp 138ndash196 (Academic Press New York)

SmartDRCarlisle EAlonsoB (2005)Transverse hydraulic redistribution byagrapevinePlantCellampEnvironment28 157ndash166 doi101111j1365-3040200401254x

Smart DR Breazeale A Zufferey V (2006) Physiological changes in planthydraulics induced by partial root removal of irrigated grapevine (Vitisvinifera cv Syrah) American Journal of Enology and Viticulture 57201ndash209

Sperry JS Pockman WT (1993) Limitation of transpiration by hydraulicconductance and xylem cavitation in Betula occidentalis Plant Cell ampEnvironment 16 279ndash287 doi101111j1365-30401993tb00870x

Sperry JS Alder NN Easlack SE (1993) The effect of reduced hydraulicconductance on stomatal conductance and xylem cavitation Journal ofExperimental Botany 44 1075ndash1082 doi101093jxb4461075

Sperry JS Hacke UG Oren R Comstock JP (2002) Water deficit andhydraulic limits to leaf water supply Plant Cell amp Environment 25251ndash263 doi101046j0016-8025200100799x

Stoll M Loveys B Dry P (2000) Hormonal changes induced by partialrootzone drying of irrigated grapevine Journal of Experimental Botany51 1627ndash1634 doi101093jexbot513501627

Tardieu F DaviesWJ (1993) Integration of hydraulic and chemical signalingin the control of stomatal conductance and water status of droughtedplants Plant Cell amp Environment 16 341ndash349 doi101111j1365-30401993tb00880x

Tardieu F Simonneau T (1998)Variability among species of stomatal controlunder fluctuating soil water status and evaporative demand modelingisohydric and anisohydric behaviours Journal of Experimental Botany49 419ndash432

Tardieu F Lafarge T Simonneau T (1996) Stomatal control by fed orendogenous xylem ABA in sunflower interpretation of observedcorrelations between leaf water potential and stomatal conductance inanisohydric species Plant Cell amp Environment 19 75ndash84 doi101111j1365-30401996tb00228x

Teskey RO Hincley TM Grier CC (1983) Effect of interruption of flow pathon stomatal conductance of Abies amabilis Journal of ExperimentalBotany 34 1251ndash1259 doi101093jxb34101251

Turner NC (1988) Measurement of plant water status by the pressurechamber technique Irrigation Science 9 289ndash308 doi101007BF00296704

Tyree MT Sperry JS (1988) Do woody plants operate near the pointof catastrophic xylem dysfunction caused by dynamic water stressAnswer from a model Plant Physiology 88 574ndash580 doi101104pp883574

Tyree MT Valez V Dalling JW (1998) Growth dynamics of root and shoothydraulic conductance in seedling of five neotropical tree species scalingto show possible adaptation to differing light regimes Oecologia 114293ndash298 doi101007s004420050450

von Caemmerer S Farquhar GD (1981) Some relationships between thebiochemistry of photosynthesis and the gas exchange of leaves Planta153 376ndash387 doi101007BF00384257

WongSCCowan IRFarquharGD(1985)Leaf conductance in relation to rateof CO2 assimilation I Influence of nitrogen nutrition phosphorousnutrition photon flux density and ambient partial pressure of CO2

during ontogeny Plant Physiology 78 821ndash825 doi101104pp784821

ZuffereyVCochardHAmeglioT Spring J-LViret O (2011)Diurnal cyclesof embolism formation and repair in petioles of grapevine (Vitis viniferacv Chasselas) Journal of Experimental Botany 62(11) 3885ndash3894doi101093jxberr081

Stomatal behaviour and root removal Functional Plant Biology 1027

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