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Scientia Horticulturae 179 (2014) 103–111

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l h om epage: www.elsev ier .com/ locate /sc ihor t i

he isohydric cv. Montepulciano (Vitis vinifera L.) does not improvets whole-plant water use efficiency when subjected to pre-veraison

ater stress

tefano Poni ∗, Marco Galbignani, Eugenio Magnanini, Fabio Bernizzoni, Alberto Vercesi,atteo Gatti, Maria Clara Merli

stituto di Frutti-Viticoltura, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy

r t i c l e i n f o

rticle history:eceived 14 June 2014eceived in revised form 1 September 2014ccepted 12 September 2014

eywords:as-exchangessimilation rateranspiration raterape compositioneaf-to-fruit ratio

a b s t r a c t

Understanding how water use efficiency (WUE) changes under drought is crucial for interpreting adap-tive responses of species and cultivars to such abiotic stress. Several recent papers have concluded thatin grapevine these responses and the guidelines stemming therefrom can differ, depending upon theparameter chosen to express WUE. In the present paper a complete set of WUE expressions, includingthe physiological and agronomical, were compared in potted, fruiting cv. Montepulciano (Vitis viniferaL.) grapevines which were either well watered (WW) or subjected to progressive pre-veraison drought(WS) by supplying decreasing fractions (i.e. 70%, 50% and 30% of daily vine transpiration, Tg) determinedgravimetrically before vines were fully rewatered. While single-leaf intrinsic water-use efficiency (WUEi)increased with water stress severity, seasonal and diurnal whole-canopy WUE were similar at pre-stress,70% Tg, and upon rewatering but dropped in WS during severe water stress. Agronomic WUE calculatedas mass of dry weight stored in annual biomass (leaves, canes and bunches) per L of water used, was

also lower in WS, whereas WS had similar must composition with WW despite a 37% reduction in theyield per vine. Results warn that whole-canopy WUE is a much better index than any single-leaf basedWUE parameter for extrapolation to agronomic WUE and actual grape composition. Under our specificcase study, it can be recommended that, to avoid significant profit loss, water supply to drought stressedMontepulciano at pre-veraison should not be lower than 70% of daily vine water use.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

There is a quite shared consensus that the large variation inerms of tolerance to drought observed within the Vitis vinifera. species is primarily dependent on changes in stomatal conduc-ance (Chaves and Oliveira, 2004; Chaves et al., 2010; Escalonat al., 1999; Flexas et al., 2002) as between-genotype variation inhotosynthetic rate seems to be minor under both well watered

nd water deficit conditions (Bota et al., 2001; Chaves et al., 1987;chultz, 1996; Soar et al., 2006).

Abbreviations: A, leaf assimilation rate; E, leaf transpiration rate; gs, leaf stomatalonductance; NCER, net CO2 exchange rate; Tc, canopy transpiration; Tg, gravimetricine water loss; WUEc, canopy WUE; WUEi, intrinsic water use efficiency; WUEinst,nstantaneous water use efficiency.∗ Corresponding author. Tel.: +39 0523599271; fax: +39 0523599268.

E-mail address: stefano.poni@unicatt.it (S. Poni).

ttp://dx.doi.org/10.1016/j.scienta.2014.09.021304-4238/© 2014 Elsevier B.V. All rights reserved.

V. vinifera has been classified as “drought avoiding” (Smartand Coombe, 1983) or as “pessimist” according to the ecologi-cal classification introduced by Jones (1980) where “pessimist”are those genotypes which, under drought, would adapt for max-imum preservation of their water status and more conservativeuse of future resources, whereas the “optimist” makes a moreluxurious use of the available water through a looser stomatalcontrol. Following the physiological classification envisaged fromTardieu and Simonneau (1998) identifying isohydric [i.e. capacityof maintaining a fairly constant midday leaf water potential (� leaf)regardless of soil water availability] and anisohydric behaviors (i.e.daytime leaf water potential significantly decreases with evapora-tive demand during the day and is typically lower in drought thanin well-watered plants), several papers investigating the issue inthe grapevines have led to conclude that some V. vinifera culti-

vars of different geographical origin can fall within either one ofthe two categories (Schultz, 2003). For instance, general evidenceexists for cultivars Grenache, Viognier, Tempranillo, Falanghina andLambrusco (Chaves et al., 2010; Giorio et al., 2007; Poni et al., 2009;

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ousa et al., 2006) to respond as iso- or near-isohydric, whereashiraz, Chardonnay, Cabernet Sauvignon and Riesling are catego-ized as aniso- or near anisohydric (Chaves et al., 2010; Lovisolot al., 2010; Schultz, 2003). An unresolved issue is also how cultivarslassified as iso- or anisohydric modify their water use efficiencyWUE) under water deficit as compared to well watered conditions.n a general basis, the behavior expected under drought from a

so-hydric cultivar (i.e. Grenache) that responds to the water short-ge with a pronounced stomatal sensitivity is a high intrinsic WUEimed at soil water conservation and plant survival and, conversely,igher susceptibility to thermal stress (i.e. chlorophyll bleachingnd PS-II down-regulation) and low assimilation leading, in turn, toow sugar content, anthocyanins, phenolics and wood maturationSchultz, 1996). On the other hand, a somewhat opposite behav-or is expected from a anisohydric genotype (i.e. Shiraz) based onts lower stomatal sensitivity. Unfortunately, Lovisolo et al. (2010)ooling data of 27 references reporting midday � leaf, stomatal con-uctance (gs) and intrinsic water use efficiency [WUEi calculated asssimilation(A)/gs] for isohydric or anisohydric grapevine speciesr cultivars fully irrigated or subjected to some degree of watertress showed that the two categories present very similar WUEinder both irrigation and water stress, and concluded that no clearattern of correlation between WUE and the iso- or anisohydricharacter was observed.

Indeed, differences in WUE between cultivars might also dependpon different techniques and ways of expression including sin-le leaf intrinsic and instantaneous WUE (Gómez del Campot al., 2004; Poni et al., 2007; Costa et al., 2012), whole-canopyUE (Palliotti et al., 2011, 2014), isotopic composition (13C/12C)

Gaudillère et al., 2002) or biomass accumulation per unit of watersed (Tomàs et al., 2012). For instance, Tomàs et al. (2012) com-aring the physiological performance of eight V. vinifera cultivarsither well watered or subjected to a water deficit, none of theeaf level estimates of WUE, i.e. WUEi(A/gs), WUEinst(A/E) or �13Chowed any significant and consistent correlation with whole plantiomass WUE. In addition, when data are extracted for three iso-ydric cultivars (i.e. Grenache, Manto Negro and Tempranillo),xpressing WUE on a whole canopy basis (WUEWP) does not solvendeed the problem as, when compared to single leaf WUE assess-

ent under drought, WUEWP was unchanged in Tempranillo, itlightly increased in Grenache and it dropped in Manto Negro.

Further insight into the issue can derive from assessment ofhole-canopy WUE that implies the use of tree enclosure systems,hich also allow for long-term and continuous measurement ofO2 and/or water vapor gas exchange (Poni et al., 2014).

The purposes of the present study were therefore to (a) com-ine single-leaf and whole-canopy WUE assessment (i.e. bothhysiological and agronomical expression) in potted, fruitingontepulciano vines subjected to pre-veraison water stress and

valuate their degree of interrelatedness, and (b) establish theegree of correlation of the various WUE expressions with vineerformance and grape composition in order to derive objectiveata about the critical level of water stress vines can withstand.

. Materials and methods

.1. Plant material and treatment layout

The experiment was conducted in 2013 at Piacenza (44◦55′N,◦44′E), Italy, on three-year-old cane-pruned cv. Montepulcianoines (V. vinifera L.). grafted on SO4 and grown outdoors in 40 L

ots. Canes were 1 m long with 8–10 dormant buds each. Eightines were arranged along a single, vertically shoot-positioned,5◦ NE-SW oriented row and hedgerow-trained. Each vine had a

m long fruiting cane having 8–10 dormant buds that was raised

rae 179 (2014) 103–111

90 cm from the ground with three pairs of surmounting catch wiresfor a canopy wall extending about 1.3 m above the main wire. Thepots were filled with a mixture of sand, loam and clay (65%, 20%and 15% by volume, respectively) and kept well watered until thebeginning of stress. Eight vines were randomly assigned to a well-watered (WW) and a water stressed (WS) treatment. For preciseaccounting of vine water use and, in turn, of water supply, as of budbreak (approximately 20 April) individual daily gravimetric vinewater loss (Tg) was continuously measured using a platform scale(ABC Bilance, Campogalliano, Italy) of 400 mm × 400 mm × 140 mm(length × width × height) having a maximum range of 150 kg and50 g accuracy placed underneath each pot. Each pot surface wascovered with a plastic sheet to prevent entrance of rain waterand to minimize losses due to soil evaporation. Each scale waswired to the data logger described hereafter with the whole-canopygas-exchange system and instantaneous pot weight recorded con-currently with environmental and gas-exchange parameters.

Starting on DOY 171, a progressive water deficit was imposedon the WS vines by reducing water supply to 70% of Tg (2.8 L/daydelivered in WS vs. 4 L day in WW) until DOY 179. On DOY 180 (29June) supply was reduced to 50% Tg (2 L/day in WS vs. 4 L/day inWW) until DOY 190 and then raised to 2.5 L/day in WW vs. 5 L/dayon DOYs 191 and 192. The increase to 100% Tg from 4 to 5 L/vine wasdue to canopy growth and increasing air VPD as the mid-summerseason approached. Beginning on DOY 193 maximum stress as 30%of Tg was applied (1.5 L/day vs. 5 L/day in WW) until rewatering onDOY 198 (17 July) to 100% of Tg. No sign of berry pigmentation wasseen at rewatering. To assure maximum accuracy, fractional waterrestitution was manually applied twice a day.

Progression of water stress was monitored by measuringpredawn and (� pd) and midday (� l) leaf water potential on DOY169, 179, 186, 189, 197, and 206. � pd measurements were takenbefore sunrise on three leaves per vine using a Scholander pressurechamber (3500 Model, Soilmoisture Equip. Corp., Santa Barbara,CA), whereas � l were recorded around solar noon on three wellexposed, mature leaves per vine inserted on the median portion ofthe main shoot.

2.2. Single-leaf gas exchange

Leaf net assimilation (A), transpiration (E) and stomatal conduc-tance (gs) rates of well-exposed, mature primary and lateral leaveswere measured on DOYs 169, 179, 186, 189, 197, and 206 using aCIRAS-2 portable photosynthesis system (PP Systems, Amesbury,MA, USA). At each measurement, three primary leaves inserted onthe basal, median and apical portions of each of two shoots pervine were sampled. The first or second fully expanded basal leaf ofthe lateral that had developed underneath the trimming cut wasmeasured as well. The two sampled shoots, usually the first basaland the apical on the cane, were not enclosed in the chamber inorder to allow free access and concurrent single-leaf and whole-canopy readings on the same vines. Readings were performed inthe morning hours (10:00–12:00) under constant saturating light[∼=1500 �mol/(m2 s)] imposed with an additional external lampmounted on top of the leaf chamber. Measurements were taken atambient relative humidity and the flow fed to the broad-leaf cham-ber (4.5 cm2 window size) was 200 mL/min. To ensure stability ofthe inlet reference CO2 concentration [CO2], a mini CO2 cartridgewas used to provide automatic control of inlet (CO2) at 380 mmol/L.

2.3. Whole-canopy gas exchange

Whole-canopy net CO2 exchange rate (NCER) measurementswere taken using the improved multi-chamber system reportedin Poni et al. (2014). The flow rate fed to the chambers was set at10.4 L/s and kept constant throughout the whole measuring season.

ticulturae 179 (2014) 103–111 105

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S. Poni et al. / Scientia Hor

hole-canopy NCER (�mol CO2/s) and whole-canopy transpira-ion (Tc) were calculated from flow rates, CO2 and H2O differentialsfter Long and Hallgren (1985). Whole-canopy water-use efficiencyWUE) was calculated as NCER/Tc and given as mmol CO2/mol H2O.

The chambers were set up on each vine and continuously oper-ted 24 h per day from DOY 157 (6 June, 14 days prior to stress)ntil DOY 205 (24 July, one week after rewatering). Ambient (inlet)ir temperature and the air temperature at each chamber’s outletere measured by shielded 1/0.2 mm diameter PFA –Teflon insu-

ated type-T thermocouples (Omega Eng. Inc., Stamford, CT), andirect and diffuse radiation were measured with a BF2 sunshineensor (Delta-T Devices, Ltd., Cambridge, England) placed horizon-ally on top of a support stake next to the chambers enclosing theanopies.

.4. Vegetative growth, yield, dry matter partitioning and grapeomposition

The day after harvest (9 September) the vines were entirelyefoliated and the fresh weight (FW) of main and lateral leaf frac-ions recorded separately. The surface of each blade was then

easured in the laboratory with a LA meter (LI-3000A, LI-COR Bio-ciences, Lincoln, NB, USA). Concurrently, two leaf samples per vineormed by 15 main and 5 lateral leaves were randomly taken, theirW recorded and then oven-dried at 70 ◦C until dehydration to con-tant dry weight (DW). Soon after defoliation, total cane numberer vine was recorded and then vines were pruned to fully removehe one-year-old wood. Each wood sample was then weighed andut in oven at 85 ◦C until dehydration to constant weight.

Each chambered vine was individually picked at harvest, andll bunches were counted and weighed. A sample of four buncheser vine was then randomly taken, their FW individually recordednd then, on each bunch, the numbers of normal and “chicken”erries were counted. Each rachis was immediately weighed andhen oven-dried at 70 ◦C until constant weight. The same procedureas applied to the pomace resulting from the berry crushing. For

he resulting juice, it was assumed that DW could approximate theotal sugar calculated from juice weight by soluble solid concen-ration (◦Brix) determined by a temperature-compensating Atagoefractometer (RX-5000 Atago Co., Ltd., Tokyo, Japan).

At harvest, two 50-berry samples per vine were taken tonsure that the positions within the bunch (top, mid, bottom) andxposures (internal or external berries) were represented. Theseamples were then weighed and stored at −20 ◦C for subsequentnalyses. All the remaining crop per vine was crushed and, besidesust soluble solids concentration (◦Brix), titratable acidity (TA) waseasured by titration with 0.1 N NaOH to a pH 8.2 end point and

xpressed as g/L of tartaric acid equivalents. Tartrate was assessedn must via the colorimetric method based on silver nitrate andmmonium vanadate reactions (Lipka and Tanner, 1974). Malateas determined with a kit (Megazyme Int., Bray, Ireland) that uses

-malic dehydrogenase to catalyze the reaction between malatend NAD+ to oxaloacetate and NADH. The reaction products wereeasured spectrophotometrically by the change in absorbance

t 340 nm from the reduction of NAD+ to NADH. Potassium (K+)oncentration was measured in the must using an ion-selectivelectrode (Model 96-61, Crison, Carpi, Italy).

Anthocyanins and phenolic substances were determined afterland (1988) using one of the 50-berry samples left to thaw and thenomogenized at high speed (7602 × g) with an Ultra-Turrax (Rosecientific Ltd., Edmonton, AB, Canada) homogenizer for 1 min. Tworams of the homogenate were transferred to a pre-tared centrifuge

ube, enriched with 10 mL aqueous ethanol (50%, pH 5.0), cappednd mixed periodically for 1 h before centrifugation at 959 × g for

min. A portion of the extract (0.5 mL) was added to 10 mL 1 MCL, mixed and let stand for 3 h; then the absorbance values were

and water stressed (©, �) Montepulciano vines during the trial. Duration of eachwater deficit level (70%, 50% and 30% of Tg) is shown by the horizontal bar. Verticalbars indicate SE (n = 12). Arrow indicates re-watering.

measured at 520 nm and 280 nm on a Kontron spectrophotometer(Tri-M Systems and Engineering, Inc., Port Coquitlam, BC, Canada).Anthocyanins and phenolic substances were expressed as concen-tration (mg/g of FM) and content (mg/berry).

The second 50-berry sample was used for determination of thegrowth of single berry organs (skin, flesh and seeds). Upon thawing,each berry was sliced in half with a razor blade, the seeds and fleshcarefully removed from each berry half using a small metal spatulawithout rupturing any pigmented hypodermal cells and the seedsthen carefully separated by hand from the flesh. Both skins andseeds were rinsed in de-ionized water, blotted dry and weighed.

2.5. Statistical treatment

One-way analysis of variance was carried out and, in case ofsignificance of F test, mean separation was performed by the t testat P < 0.05 and 0.01. Degree of variation around means is given asstandard error.

3. Results

3.1. Leaf-water potentials and single-leaf gas exchange

Predawn leaf-water potential (� pd) progressively decreasedalong with a gradual reduction of the Tg fraction supplied to thevines, reaching the lowest value (∼=−0.90 MPa) at the end of the 30%Tg restitution period (Fig. 1). A week after the rewatering on DOY199, � pd of previously stressed vines recovered to the same levelof WW pots (−0.2 MPa). Midday leaf water potential (� l) did notdiffer between WW and WS for data taken during the 30% and 50%restitution periods, whereas � l was significantly lower in WS at theend of the most severe water deficit (Fig. 1). In both treatments, � lshowed quite ample variation across different dates with lowestvalues (∼=−1.1 MPa) reached on DOY 189. Rewatering reported � lof WS to a level similar to that recorded in WW. Visual observa-tions carried out on DOY 192 (11 July, last day of 50% Tg regime)indicated that WS leaves had a moderate tendency toward a verti-cal orientation during the warmest part of the day, while no initialyellowing was displayed by the basal leaves.

Reducing water supply by 30% of Tg did not significantly affect Aand E rates as compared to pre-treatment rates (Fig. 2A and B). Con-versely, gs was already significantly reduced in WS leading, in turn,

to increased WUEi (Fig. 2C), However, data taken on DOY 179 (i.e.last day of the 70% Tg supply) showed that while A rates remainedclose to optimal (e.g. around 13–15 �mol/m2 s), both E and gs weremuch lower than rates recorded the previous date (Fig. 2B) due

106 S. Poni et al. / Scientia Horticultu

Fig. 2. Assimilation rate (A), transpiration rate and stomatal conductance (B) andintrinsic and instantaneous water use efficiency (C) recorded on single primary andlateral leaves throughout the experimental period on Montepulciano vines eitherwell-watered (�, �) or water stressed (©, �). In each panel, duration of each waterdeficit level (70%, 50% and 30% of Tg) is shown by the horizontal bar. Vertical barsindicate SE (n = 32). Arrow indicates re-watering. All data taken on well-exposed,healthy mature leaves at ambient relative humidity from 10:00 to 12:00.

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o an unseasonal cool day with air temperature and VPD recordeduring the measurement time window ranging across 24 ◦C and.45 kPa, respectively. A, E and gs were drastically reduced duringeriods of 50% and 30% Tg restitutions, although imposing the mostevere stress level did not seem to further limit gas-exchange. In

S, severe stomatal closure was testified by gs values that on DOY86 and 197 were below the threshold of 50 mmol/m2 s. Data taken

days after rewatering (DOY 206) indicated full recovery in WS forll the gas-exchange parameters. While WUEi in WS increased pro-

ressively with severity of the imposed water deficit reaching theaximum value of 123 mmol CO2/mol H2O on DOY 197, the same

id not apply to WUEinst that was increased in WS vines only at thend of the 30% Tg restitution period (Fig. 2C).

rae 179 (2014) 103–111

3.2. Whole-canopy gas exchange

The multi-chamber system ran uninterruptedly from DOY 157to 205, and most of the 49 measuring days were marked byclear skies (i.e. PAR ≥ 800 �mol/m2 s) and ambient VPD fluctuat-ing between 1.5 and 2.2 kPa. Lower VPD (≤1.5 kPa) was recordedonly a few days prior to stress and before imposition of the 50%Tg stress level (Fig. 3A). Mean seasonal daily averaged values of airtemperature recorded at chamber inlet, outlets WW and outletsWS were 26.9, 28.7 (+1.8 ◦C) and 29.3 (+2.4 ◦C) respectively (datanot shown).

Despite day-to-day fluctuations due to climate variability, initialpre-stress NCER/canopy rates were very close between vines allo-cated to treatments while a tendency to show higher NCER/canopywas manifested even before the water deficit was imposed dueto some within-treatment differences in canopy size and devel-opment (Fig. 3B). Upon imposition of the different fractions ofsupplied water, averaged NCER rates per canopy were 60.9%, 44.1%and 32.5% of rates measured in WW plants for Tg levels of 70%,50% and 30%, respectively. After re-watering, mean NCER/canopyin WS was 61.5% of rates recorded in WW. Canopy transpiration(Tc) averaged over the respective length of each water supplylevel was 74.8%, 62.0% and 54.2% of rates recorded in WW at70%, 50% and 30% restitution levels, respectively (Fig. 3C). Meanpost-rewatering Tc in WS was 63.4% of weight loss measured inWW. Whole-canopy water-use efficiency expressed as NCER/Tc

ratio (�mol CO2/mmol H2O) did not differ significantly betweentreatments at pre-stress and 70% Tg restitution. Conversely, WUEmarkedly decreased in WS during most of 50% and 30% Tg restitu-tion periods (Fig. 3D), with an average reduction of 18.6% and 34.1%,respectively, as compared to WW. Upon rewatering, whole-canopyWUE of WS promptly recovered to the WW level. Analyzing theabove data in a regression mode (Fig. 4) highlighted that, on a frac-tional basis, both NCER and Tc linearly decreased with fraction ofwater supply, although regression slopes were different suggestingthat, at 50% and 30% Tg, NCER was more limited than Tc.

Diurnal NCER/vine trends recorded in pre-stress (DOY 168)showed maximum rates between 10:00 and 12:00 when incom-ing PAR was ≥1000 �mol/m2 s and air VPD ranged between 1 and2 kPa (Fig. 5A and B). Daily Tc mirrored quite closely the diurnalPAR (Fig. 5C). Despite an expected high variation of canopy WUEearly in the day due to low system sensitivity at still low NCER andTc rates, it was apparent that canopy WUE progressively decreasedthroughout the day (Fig. 5D). Data taken on DOY 176 (5th day at70% water supply) showed mild limitation for NCER and Tc in themorning hours (Fig. 5F and G) and a stronger one for both param-eters later in the day despite some temporary recovery after thesecond water supply at 14:00. Overall, canopy WUE during the daywas similar between the two treatments (Fig. 5H).

The level of stress reached 6 days after lowering water supplyto 50% Tg (DOY 186) was quite severe: mean daily NCER was only23.8% of WW, dropped to nil around solar noon and showed mildrecovery after the afternoon irrigation (Fig. 5J). Conversely, Tc on thesame day was less limited (42.4% of Tc measured in WW) (Fig. 5K).As a result, WUE was much lower in WS during most of the day,with a tendency to catch up to WW levels in late afternoon (Fig. 5L).Data taken on DOY 195 (2nd day at 30% restitution) confirmed theoverall trends observed on DOY 186 (Fig. 5N and O). In fact, on adaily basis, NCER in WS was 22.2% of WW, whereas Tc was 40.4%of WW. Diurnal trends of canopy WUE (Fig. 5P) confirmed thatWUE drastically decreased in WS throughout the day, with max-imum limitation around solar noon. Rewatering restored a canopy

NCER that was 64.2% of WW (Fig. 5R), whereas mean Tc recordedover DOY 202 was 57.8% of WW rates. Notably, canopy WUE cal-culated at rewatering (Fig. 5T) showed no differences betweentreatments.

S. Poni et al. / Scientia Horticulturae 179 (2014) 103–111 107

Fig. 3. (A) Seasonal trends of air vapor pressure deficit (VPD) (�), direct (�) and diffuse (�) photosynthetically active radiation (PAR), (B) whole-canopy net CO2 exchanger efficis 30% ofr

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ate (NCER), (C) whole-canopy transpiration (Tc), and (D) whole-canopy water usetressed (©). In panels B, C and D, duration of each water deficit level (70%, 50% and

e-watering.

.3. Vegetative growth, yield, dry matter partitioning and grapeomposition

As expected, both treatments shared a very similar shoot andunch number per vine (Table 1). The imposed stress had no sig-ificant effect on total leaf area and no yellowing or premature

hedding of older basal leaves was observed. Conversely, some yieldomponents and the end-season leaf-to-fruit ratio were modifiedy treatments (Table 1). In more details, yield per vine was reducedy 37% in WS plants due to either lower berry number per bunch

ig. 4. Relationship between fractional variation of NCER (�) and Tc (�) recordedn WS and the fraction of water supply. Data are given as percent of WW. Regres-ion equations for NCER and Tc are, respectively: y = 0.8008x + 3.0967, R2 = 0.99 and

= 0.5785x + 31.966, R2 = 0.99. Slopes of single regressions run on WW and WS dataid not differ according to the test of equality of slopes (P = 0.05). Fraction of WUEc

n WS (percent of WW) calculated as grand mean ratio of NCER/Tc is also shown (�).

ency (WUEc) measured on Montepulciano vines either well-watered (�) or water Tg) is shown by the horizontal bar. Vertical bars indicate SE (n = 4). Arrow indicates

and a much higher incidence of “chicken” berries. Quite interest-ingly, due to the relative variation in growth and yield componentsin WS, the leaf area-to-yield ratio at harvest was higher than in WW(Table 1).

A look at berry growth components shows that normal berriesdid not differ in weight between WW and WS nor did skin andflesh growth regardless if expressed on an absolute or a relativebasis (Table 2). By contrast, WS significantly limited total and singleseed weight per berry (Table 2).

Fresh and dry weight of leaves and canes were not significantlyaffected by water deficit (Table 3), whereas, as expected, totalabove-ground dry biomass (excluding annual trunk and cordongrowth) in WS had a lower value than in WW (−25.4%). SeasonalWUE calculated as the ratio between above ground dry matter andcumulated water use was significantly lower in WS (3.35 g/L) thanin WW (3.86 g/L).

Of the must composition parameters determined at harvest, TAand tartaric acid were lower in WS that also showed higher pHthan WW (Table 4). The remaining parameters including total sol-uble solids, total anthocyanins and phenolics were not affected bytreatments.

4. Discussion

Calibrating the amount of water supplied to the vines on thebasis of a continuous recording of gravimetric vine water loss madeit possible to attain a rather fine control of the level of imposed

water stress. As a matter of fact, � pd was about −0.4 MPa at theend of the 70% Tg restitution period, which according to Ojeda et al.(2001) defines a moderate water stress. According to the sameauthors, a “severe” water stress takes place once � pd decreases

108 S. Poni et al. / Scientia Horticulturae 179 (2014) 103–111

Fig. 5. (A, E, I, M and Q) Diurnal trends (dawn to dusk) of air vapor pressure deficit (VPD) (�), direct (�) and diffuse (�) photosynthetically active radiation (PAR); (B, F, J,N and R) whole-canopy net CO2 exchange rate (NCER); (C, G, K, O and S) whole-canopy transpiration (Tc), and (D, H, L, P and T) whole-canopy water use efficiency (WUE)measured on Montepulciano vines either well-watered (�) or water stressed (©) in representative clear days of each water stress status (pre-stress – DOY 168; 70% Tg –DOY 176; 50% Tg – DOY 186; 30% Tg – DOY 195; re-watering – DOY 202). Panels C, G, K, O and S also report whole-vine gravimetric water loss (Tg) for well-watered (�) andwater stressed (�) vines. Vertical bars indicate SE (n = 4). In panels B, F, J, N and R arrows indicate time of manual water supply.

Table 1Vegetative growth, yield components and leaf-to-fruit ratio (vine basis) recorded on Montepulciano grapevines either well-watered (WW) or subjected to a progressivepre-veraison water deficit (WS). * and ** denote significant differences between treatments at P < 0.05 and 0.01 according to within column mean separation performed withStudent–Newman–Keuls (SNK) test. ns = not significant.

Shoots/vine

Total LA(m2)

Primary LA Lateral LA Bunches/vine

Bunchweight (g)

Totalberries/buncha

Chicken berries(% of total)

Yield/vine (kg)

LA/yield(m2/kg)

WW 9.7 2.90 2.17 0.59 10.0 361 150 3 3.603 0.77WS 9.0 2.76 2.24 0.66 10.0 226 133 31 2.270 1.28Sig. ns ns ns ns ns ** * ** ** *

a Summation of “hen” and “chicken” berries.

Table 2Berry growth components (absolute and relative basis) and seed number recorded on Montepulciano vines either well-watered (WW) or subjected to a progressive pre-veraison water deficit (WS). * and ** denote significant differences between treatments at P < 0.05 and 0.01 according to within column mean separation performed withStudent–Newman–Keuls (SNK) test. ns = not significant.

Berryweight (g)

Berry skinweight (g)

Total seedweight (g)

Seeds/berry Single seedweight (g)

Flesh berryweight (g)

Skin-to-berryratio (%)

Skin-to-fleshratio (%)

WW 2.480 0.240 0.0742 1.88 0.0400 2.166 9.9 11.6WS 2.549 0.271 0.0662 1.86 0.0360 2.227 10.9 13.0Sig. ns * * ns * ns ns ns

Table 3Fresh (FW) and dry weight (DW) of current-year above ground biomass (leaves, canes and bunches), seasonal water use and calculated agronomic water use efficiency (gDW/L) recorded on Montepulciano vines either well-watered (WW) or subjected to a progressive pre-veraison water deficit (WS). * and ** denote significant differencesbetween treatments at P < 0.05 and 0.01 according to within column mean separation performed with Student–Newman–Keuls (SNK) test. ns = not significant.

Leaves Canes Bunches Total above ground biomassa Seasonal wateruseb (L)

WUE (g DW/L)

FW DW FW DW FW DW FW DW %

WW 676 252 280 133 3603 778 4559 1162 25.5 301.0 3.86WS 626 239 231 111 2207 517 3127 867 27.8 258.5 3.35Sig. ns ns ns ns * * * ** ns ** *

a Current year root, trunk and cordon biomass increment not included in calculations.b Calculated from cumulated daily gravimetric water loss.

S. Poni et al. / Scientia Horticulturae 179 (2014) 103–111 109

Table 4Must composition recorded on Montepulciano vines either well-watered (WW) or subjected to a progressive pre-veraison water deficit (WS). * and ** denote significantdifferences between treatments at P < 0.05 and 0.01 according to within column mean separation performed with Student–Newman–Keuls (SNK) test. ns = not significant.

Soluble solids(◦Brix)

pH Titratableacidity (g/L)

Tartaric acid(g/L)

Malic acid (g/L) K+ (ppm) Total anthocyanins Total phenolics

mg/berry mg/g mg/berry mg/g

WW 21.4 3.18 6.3 5.5 1.2 1234 3.82 1.45 7.57 2.931.1

ns

bsl

icssrluadw1aa(

vhVlecilWaocdwlbfguiowaac

sbbrp(vatts

WS 22.0 3.28 5.4 5.1

Sig. ns * ** *

elow −0.6 MPa, a condition which in our study was almost reachedix days after the beginning of the 50% water supply (DOY 186) andasted for 11 days prior to rewatering.

Seasonal trends of � pd and � l recorded in this study uponmposing at pre-veraison a soil water deficit of increasing severityonfirmed the iso-hydric nature of Montepulciano as previouslyuggested by Palliotti et al. (2009), as at most dates during thetress � l in WS was very similar to that measured in WW; though,eadings taken the day before vines were rewatered showed someoosening of stomatal control since � l in WS was lower than val-es measured in WW. Noteworthy, Montepulciano seems to holdnother feature which has been reported especially tight in isohy-ric genotypes, that is stronger stomatal sensitivity to increasedater pressure deficit (Soar et al., 2006). In fact, data taken on DOY

79 at the end of the 70% Tg restitution period show much less neg-tive values that those taken pre-stress (DOY 169) quite likely as

result of much different evaporative demand over the two daysi.e. 2.13 kPa on DOY 169 vs. 0.44 kPa on DOY 179).

There is a quite shared consensus in literature that isohydricarieties are better adapted to drought environments than aniso-ydric ones (Pou et al., 2012; Schultz, 2003; Soar et al., 2006;andeleur et al., 2009; Tombesi et al., 2014) primarily due to (i)

arger increase in WUEi, (ii) higher stomatal sensitivity to changingvaporative demand and (iii) lower hydraulic conductivity and sus-eptibility to cavitation. While the latter feature was not assessedn this study, items (i) and (ii) were both confirmed. In particu-ar, high stomatal sensitivity of Montepulciano was ascertained as

UEi was higher in WS at any date after the beginning of stressnd re-adjusted to WW level after rewatering. On a general basis,ur increased WUEi can be added to data reported in a recent studyarried out for three seasons on potted vines representative of eightifferent V. vinifera genotypes: their WUEi measured under droughtas in all cases consistently higher than that recorded under non-

imiting water supply (Tomàs et al., 2012). As already pointed outy Schultz and Stoll (2010), though, while WUEi being independentrom leaf-to-air VPD is the appropriate WUE expression when theoal is to compare performance of different species and varietiesnder different experimental and growing conditions, the “work-

ng” expression of leaf WUE is WUEinst, i.e. the carbon gain per unitf water loss. Indeed, calculated WUEinst under moderate to severeater deficit leads to a multitude of different responses embracing

n increase (Intrieri et al., 1998), no change (Tomàs et al., 2012) or decrease (Schultz and Stoll, 2010) as compared to well wateredonditions.

The above scenario showed a further and sharp change in ourtudy when physiological WUE was evaluated on a whole-canopyasis by relating moles of CO2 assimilated to moles of water losty transpiration. In fact, seasonal and selected diurnal trends rep-esentative of each water supply regime provide a very clear-cuticture suggesting that as the water shortage became more severei.e. � pd lower than −0.6 MPa), canopy WUE of Montepulcianoines markedly decreased. Moreover, data taken during DOYs 186

nd 195 (Fig. 5L and P) consistently showed that, except for dataaken early in the morning or late in the afternoon under light andemperature limited gas exchange rates, canopy WUE was con-tantly lower during most part of the day, reaching a minimum

1313 3.74 1.50 7.91 3.10ns ns ns ns ns

around solar noon and before the second daily irrigation was sup-plied. Very few analogous comparisons are available in literature: intwo instances, single-leaf vs. whole-canopy WUE comparison per-formed in potted (Poni et al., 2009) and field grown vines (Tararaet al., 2011) confirmed that, at the canopy level, no gain in WUE wasseen under drought, whereas in another case (Palliotti et al., 2014)data taken on cvs. Montepulciano and Sangiovese grown in pots andsubjected to prolonged water stress between fruit-set and veraisonshowed that stressed vines maintained a higher WUE even at thecanopy level. This discrepancy, however, may be due to differencesin rootstocks and canopy geometry: while our vines were grafted onthe rather drought sensitive SO4 rootstock (Serra et al., 2014), Pal-liotti et al. worked with 1103P, which Samson and Casteran (1971)describe as resistant. We also worked on a traditional VSP trel-lis with alternance of row-side illumination throughout the day,whereas Palliotti et al. (2014) used potted vines having a goblet-like canopy type with obvious differences in terms of leaf exposureand dynamics of light interception during the day.

From a physiological standpoint, it is of paramount importancethat while the single-leaf sampling performed in the present trial,and in many previous approaches using well exposed leaves usuallymeasured during the morning hours, can maintain validity if usedfor comparison purposes (i.e. genotype WUE variation as recentlyinvestigated by Tomàs et al., 2012), it seems to have a very poorrelationship with whole-canopy response. Indeed, a whole-canopyapproach is able to provide assessment of the entire leaf commu-nity without any artificial perturbation of natural leaf position; inthis connection it has to be pointed out that Schultz and Stoll (2010)have shown that for readings taken on leaves in their natural posi-tion there is no increase in WUEinst for non-irrigated ‘Riesling’ vinesas compared to those well-watered.

A hint to explain discrepancy between single leaf and wholecanopy WUE in this study is offered by evaluating which condi-tions led to maximum gap between these two parameters which,quite systematically, occurred at a water supply ≤50% of Tg (Fig. 3D)and, specifically, during the central part of the day at high PARand evaporative demand. It is very enlightening to note thatrelatively high WUE of WS vines recorded on DOYs 184 and191 matches partial cloudiness (average daily PAR was, respec-tively, 577 and 686 �mol/m2 s vs. over 800 �mol/m2 s measuredthe other days), lower VPD and a higher diffuse-to-direct PARratio than the remaining days. It thus seems that, the level ofsoil-water stress being equal, canopy WUE is quite sensitive toenvironmental conditions and, especially, to evaporative demand.When daily diffuse-to-direct PAR ratio was plotted against canopyWUE in WW, a significant positive linear relationship occurred(y = 0.0572x + 1.8448; r = 0.83), thereby confirming that higher dif-fuse light conditions can significantly contribute to raise WUEunder severe water stress. This outcome agrees with Petrie et al.(2009), who reported that under diffuse light (cloudy) conditionschambered vines appeared to be more efficient as they photosyn-thesized at a higher rate per unit of light intercepted. It is quite

conceivable that, as hazy days have a higher fraction of diffuselight than clear days, they would still saturate photosynthesis atthe exterior of the canopy while increasing the photosyntheticcontribution of internal, mostly shaded leaves; transpiration is

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10 S. Poni et al. / Scientia Hor

oncurrently lowered by the lower evaporative demand and WUEenefits from the balance.

Overall, the hypothesis made in this study to explain lowereasonal and canopy WUE of WS under severe stress is that, forhose leaves either partially or fully shaded, while assimilation waseverely limited by both low light and severe stomatal closure, tran-piration was proportionally less limited as leaf-to-air VPD wouldot change as much depending on leaf location in the canopy.

n agreement with this assumption, Medrano et al. (2012) in anlegant work aimed at evaluating WUEinst of undisturbed leavesn different locations of a VSP trained canopy (top, side-exteriornd internal) demonstrated that, regardless of severity of watertress, the leaf-to-air pressure deficit was not significantly differentmong leaf positions.

Moreover, at the canopy level, adjustments showed from theontepulciano stressed vines help further at explaining their lowUE. Due to its isohydric (i.e. “pessimist”) strategy, Montepul-

iano is expected to react to the developing stress with mechanismimed at reducing water loss such as faster cessation of growth,arlier yellowing and drop of basal leaves, tendency of leaves tossume a predominantly vertical orientation during the warmestart of the day to limit transpiration (Schultz, 1996). According toata reported in Table 1 and based on visual observations, none ofhe above mechanism took place suggesting great caution whenhe isohydric character is somehow automatically associated tohis type of responses. Beside, caution is also advised from liter-ture findings: recent surveys conducted on the anisohydric cv.angiovese have shown its very pronounced tendency to verticaleaf orientation and early basal leaf shedding when subjected to

ater stress (Palliotti et al., 2011, 2014). In addition, a compari-on carried out on potted cvs. Grenache, Chardonnay and Shirazrapevines either well watered or water stress has shown that aefinitely milder limitation in leaf area development occurred inhe isohydric Grenache as compared to the anisohydric ChardonnayPou et al., 2012).

Upon rewatering, canopy WUE of previously stressed vinesacked up to the same levels of WW (Fig. 3D). However, this bal-nce was reached in WS at absolute NCER and Tc values that,n a fractional basis, were about 20% lower than the WS/WWatios calculated pre-stress. Quite interestingly, the recovery apti-ude of Montepulciano is different depending upon the scale ofssessment: single leaf assimilation measurements indicated fullecovery of both A and E one week after rewatering, whereas whole-anopy gas exchange suggest that this was not the case.

A second major issue is whether a given canopy WUE undertress then correlates to the balance between the plant’s dry mat-er production and its water consumption (i.e. agronomic WUE).ctually, in the case of the perennial grapevine, the target isore complex since desirable traits are both adequate dry mat-

er partitioning to the economically viable organs (i.e. bunches) andequired grape composition. Overall, in agreement with Tomàs et al.2014) our study confirms that while no correlation exists betweeningle-leaf WUE assessment and agronomic WUE, seasonal canopy

UE seems to agree with lower end-of-season agronomic WUE inS (Table 3) and similar grape composition at a yield level reduced

y 37% in WS (Table 4).The progressive pre-veraison water stress imposed on the vines

ad no impact on total leaf area per vine (Table 1). Detailed leafounting and area processing performed after harvest showed thathile lateral leaves did not differ in number and size between

reatments, WS had fewer primary leaves than WW (199 vs. 237, = 0.05) as a likely effect of temporary water shortage, yet final

ize of primary leaves in WS was larger than WW (114 vs. 89 cm2,

= 0.05) due to a pronounced growth compensation upon rewater-ng and during the remainder of the season. On the other hand, its remarkable that the temporary drought stress was effective at

rae 179 (2014) 103–111

sharply reducing yield per vine via an effect entirely attributableto increased millerandage and, to a lesser extent, to reducedfruit-set (Table 1). Millerandage defines normal fruit despite inad-equate seed development in a portion of the fertilized berries andresults in bunches having the appearance of “hens and chickens”(Fougère-Rifot et al., 1995; May, 2004). While the term “hens” refersto normal size berries, “chickens” describes small berries withtiny degenerated seeds often lacking in endosperm (May, 2004).Although the expansion of “chickens” is halted within approxi-mately three weeks after flowering due to seeds abortion, theseberries apparently ripen normally and, due to their much smallersize, their concentration in soluble solids is often much higher thanthat recorded in “hens” berries. In the present study, it is quite likelythat a water stress that started at fruit-set and extended through-out most of the lag-phase of berry growth during which the embryogrows rapidly and the seed enters the maturation stage, caused asharp increase in the fraction of “chicken” berries, hence heavilyreducing yield potential of the WS vines. That there was a directlink between the imposed water stress and seed development isalso shown from data reported in Table 2 showing that total seedweight per berry was lower in WS plants.

A further issue deserving discussion is why in WS, although theleaf-to-fruit ratio was improved in WS as compared to WW andyield was curtailed by 36%, must composition was overall unaf-fected. It is speculated that the severe source limitation recordedover most of the 50% Tg and during 30% Tg restitution periodsconstrained the incipient sugar and color accumulation and offsetadvantages that were expected based on the more favorable leaf to-fruit ratio. Ojeda et al. (2001) have shown in field-grown CabernetS. vines that a severe water deficit applied before veraison did notimprove the biosynthesis of proanthocyanins and anthocyanins.Lastly, it has to be noted that the imposed stress had no effects on“hens” berry size nor on the relative skin growth (Table 2) thereforeminimizing the chance of a positive effect on berry pigmentation.

5. Conclusions

Our overall results show for the isohydric cv. Montepulcianono relationship between different expression of single-leaf WUEvs. whole-canopy assessment, confirming the inadequacy of WUEiat assessing adaptive responses of this variety to water stress.Conversely, an evaluation of such an adaptive response based onseasonal and diurnal whole-canopy CO2 exchange and water losswas quite nicely linked to a decrease of agronomic WUE (i.e. g DM/L)in WS and unimproved grape composition despite a drastic reduc-tion in yield per vine.

In terms of recommendations, our data indicate that restituting<70% of actual vine water use over a stress imposed on Mon-tepulciano at pre-veraison is not advisable as it would result in asignificant profit loss. Work is in progress to determine whetherinterrelationships of the parameters used to express WUE andtheir correlation with vine performance and grape composition willchange when a progressive water supply is applied after veraison.

Acknowledgements

The research leading to these results has received fundingfrom the European Community’s Seventh Framework Program(FP7/2007-2013) under the grant agreement no. FP7-311775,Project INNOVINE.

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