Water stress improves whole-canopy water use efficiency and berry composition of cv. Sangiovese (Vitis vinifera L.) grapevines grafted on the new drought-tolerant rootstock M4

Agricultural Water Management 169 (2016) 106–114

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Water stress improves whole-canopy water use efficiency and berry composition of cv. Sangiovese (Vitis vinifera L.) grapevines grafted on the new drought-tolerant rootstock M4 M.C. Merli a , E. Magnanini a , M. Gatti a , F.J. Pirez a , I. Buesa Pueyo b , D.S. Intrigliolo c , S. Poni a,∗ a Dipartimento di Scienze delle Produzioni Vegetali Sostenibili, Facoltà di Scienze Agrarie, Alimentari e Ambientali, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy b Instituto Valenciano de Investigaciones Agrarias, Centro Desarrollo Agricultura Sostenible Apartado Oficial, 46113 Moncada, Spain c Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Departamento de Riego, Campus Universitario de Espinardo, 30100 Espinardo, Murcia, Spain

a r t i c l e

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Article history: Received 3 November 2015 Received in revised form 13 February 2016 Accepted 21 February 2016 Keywords: Gas exchange Leaf–water potential Rootstock Water-use efficiency Water stress

a b s t r a c t Testing of new rootstocks for drought tolerance targets traditionally rain-fed districts where supplemental irrigation is more frequently needed due to the pressures of global warming. A seasonal evaluation of whole-canopy gas exchange, water-use efficiency (WUEc ), yield components and compositional traits of Vitis vinifera cv. Sangiovese grafted to the new drought-tolerant genotype M4 against the commercial SO4 stock is reported. The experiment was conducted in 2015 on twelve four-year-old, fruiting potted Sangiovese grapevines grafted on M4 and SO4 stocks and assigned to SO4-WW (well-watered), SO4-WS (water-stressed), M4-WW and M4-WS treatments. Water deficit was imposed pre-veraison by reducing water supply to 50% of whole-canopy demand derived from concurrent measurements of transpiration in WW and maintained until three weeks after veraison prior to full rewatering. While WUEc was unchanged in WS-SO4 as compared to WW-SO4, WUEc in WS-M4 increased by 22% vs. WW-M4 over the whole water deficit period and such gain was partially maintained upon rewatering. Higher WUEc in WS-M4 resulted from an ability to maintain canopy photosynthesis similar to WS-SO4 at a reduced water use. Although yield per vine was similarly reduced in the two WS treatments (about 1 kg less than WW), overall grape composition was improved in WS-M4 and worsened in WS-SO4 when compared to the WW controls. Total soluble solids (◦ Brix) rose by 11% in WS-M4 vs. the respective control, whereas in WS-S04 there was a slight decrease (−0.6 Brix). Most notably, anthocyanins accumulation was largely limited in vines grafted on SO4 (−45% vs. WW-SO4 when given on a concentration basis), while in M4grafted plants berry pigmentation slightly improved vs. WW (+12.5%). Hypothesis is made that grafting onto different stocks can trigger differential gene regulation under water stress and high temperatures leading to different sensitivity in synthesis and/or degradation of already formed anthocyanins. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Viticulture areas around the world where the Daktulosphaira vitifoliae F. (phylloxera) is still an endemic pathogen are bound to the use of tolerant rootstocks having blood of North American species (Granett et al., 2001). The role the stock plays has gained even greater importance lately in association with changes related

∗ Corresponding author. E-mail address: [email protected] (S. Poni). http://dx.doi.org/10.1016/j.agwat.2016.02.025 0378-3774/© 2016 Elsevier B.V. All rights reserved.

to global warming as drivers of demand for new genotypes tolerant to drought and salinity (Serra et al., 2014). Drought tolerance is becoming especially important even in traditionally rain-fed areas that have been experiencing significant summer drought with increasing frequency over the last two decades. This is the case of most districts in central Italy. In fact, they account for the great majority of the top red cv. Sangiovese acreage, about 70,000 ha in 2013 according to the government’s ISTAT statistics bureau (ISTAT, 2015). Given that the assessment of drought tolerance should focus on the ability of the scion-rootstock combination to crop an acceptable yield of the desired quality under conditions of water deficit, drought-tolerant

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genotypes could relieve traditionally rain-fed districts from the use of supplemental irrigation. Supplemental irrigation has several major shortcomings when introduced in areas where growers have no previous experience of irrigation management: (i) over use is common and leads to obvious water waste and vine imbalances; (ii) matching vine-water requirements and vine response to the level of water deficit the plant can withstand without compromising crop yield and quality while achieving some restriction of an usually excessive vegetative growth is hard to gauge; (iii) in the absence of a solid background on water relations, decisions about if, when and how to irrigate are left to such empirical methods as visual observation of grapevine organs; and (iv) supplemental water supply in these areas is quite skeptically regarded as a forcing tool, implying an undesirable yield increase and, hence, loss of grape quality. Recently, Meggio et al. (2014) compared the newly selected genotype M4 [(Vitis vinifera × Vitis berlandieri) × V. berlandieri cv. Resseguier n.1] to the commercial rootstock 101–14 Millardet et De Grasset (Vitis riparia × Vitis rupestris) in a drying-down potted trial and reported that M4 retained higher leaf assimilation (A) than well-watered (WW) vines at a field capacity lower than 30%. Most notably, at the same soil-moisture deficit, M4 showed a remarkable A recovery starting 6 days after initial stress, with its A reaching 60% of WW on the last day of stress (10) vs. 20% of control measured in 101-14. This response, in association with the much better recovery of M4 upon re-watering—A rose to 80% of WW as compared to 35% in 101-14—led the authors to conclude that M4 shows much better acclimation to drought than 101-14 because of better preserved root-tissue integrity and functionality. This very promising scenario naturally poses the question whether these results are also retained in grafted vines since there is consolidated knowledge that the scion can change the overall picture (Swanepoel and Southey, 1989; Tandonnet et al., 2010; Serra et al., 2014). Generally speaking, the consensus is that the root system is less sensitive to water deficit than the canopy (Lovisolo et al., 2010). Moreover, roots exhibit compensatory effects such as the “water lift” assessed in partial root-drying studies where water is transferred at night from the wet to the dry portion of the root system (Bauerle et al., 2008a,b). Van Zyl (1984) also showed that grapevine root growth can become enhanced even under moderate water-deficit conditions. In addition, ample literature is available indicating that scion-rootstock interaction exerts a major influence on drought tolerance by affecting canopy structure and size and, hence, water use (Gambetta et al., 2012; Serra et al., 2014). Interestingly, both rootstocks and cultivars seem to employ specific strategies toward incipient water stress. Although information is much more limited for roostocks, it is known that ungrafted 110 Richter plants show a rather conservative response to soil–water shortage manifested as tight stomatal regulation, a mechanism that appears to be regulated by abscisic acid (ABA) and allows the vines to maintain a fairly high mid-day leaf water potential even at severe water stress and upon rewatering enables them to adjust their root-to-shoot hydraulic conductivity to facilitate resumption of good gas-exchange rates. By following the physiological classification of scions proposed by Tardieu and Simonneau (1998), who identify isohydric (capacity of maintaining a fairly constant midday leaf water potential, leaf , regardless of soil water availability) and anisohydric (leaf significantly decreases with evaporative demand during the day and is typically lower in drought than in well-watered plants) responses, several papers conclude that certain V. vinifera cultivars of differing geographical origin can fall within either of the two categories (Schultz, 2003). For instance, general evidence exists for cvs. Montepulciano, Grenache, Viognier, Tempranillo and Lambrusco a foglia frastagliata (Sousa et al., 2006; Poni et al., 2009; Chaves et al., 2010; Palliotti et al., 2014a) to respond as iso- or near-isohydric, whereas Sangiovese, Shiraz,

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Chardonnay, Cabernet Sauvignon and Riesling are categorized as anisoor near-anisohydric (Schultz, 2003; Poni et al., 2007; Chaves et al., 2010; Lovisolo et al., 2010). Thus a challenging issue is whether the water tolerance strategy of a given scion predominates over the strategy induced by the rootstock, or vice versa. Indeed, studies comparing all possible grafting combinations, i.e., grafted, ungrafted, homografted, between various Vitis genotypes have found that reciprocal influence does exist, with the stock influencing scion vigor by regulating water and nutrient uptake and transport and the scion affecting stock growth through photosynthetic capacity and dry-matter partitioning (Düring, 1994). A second item deserving of attention is which parameters are to be taken into account for a reliable evaluation of stock-induced drought tolerance. While no standard method for classifying stocks according to their drought tolerance is yet available, and different rankings for the same rootstock are a frequent occurrence, traditional indicators of drought tolerance in relation to crop level and stability have recently been coupled to or replaced by faster physiological indicators, including stomatal conductance (gs ), xylem ABA content, leaf-water potential, chlorophyll content and, especially, intrinsic water-use efficiency (WUEi ) defined as the assimilation (A)/stomatal conductance (gs ) ratio. Yet very recent work has raised serious doubts about WUEi ’s reliability for assessing true drought tolerance. For instance, Tomás et al. (2012) have shown that for all treatment combinations of eight grapevine cultivars either well-irrigated or water-stressed, WUEi did not correlate with whole-plant WUE expressed as g of dry matter per L of water transpired. Recent papers by Palliotti et al., (2014a), Merli et al. (2015), ˜ (2015) have confirmed that wholeand Tarara and Pérez-Pena canopy WUE evaluated via a plant-enclosure approach is more reliable than any single-leaf-based WUE parameter for extrapolation to agronomic WUE and actual grape composition. Since no data comparing physiological and agronomical performance of V. vinifera’s cultivars grafted on M4 rootstock are available yet, we designed the current study to meet the two following objectives: (a) assess gas exchange and agronomic responses of drought Sangiovese grapevines grafted on M4 and on the susceptible SO4 (V. berlandieri × V. riparia) stock and (b) determine if any change in whole-canopy water use efficiency links to yield and grape composition performances.

2. Materials and methods 2.1. Plant material and experimental layout The experiment was conducted in 2015 on four-year-old, fruiting cv. Sangiovese (V. vinifera L.) vines (clone VCR 23) grafted to SO4 (V. berlandieri × V. riparia) and M4 [(V. vinifera × V. berlandieri) × V. berlandieri cv. Resseguier n.1] stocks grown outside in 40-L pots filled with a loam soil (USDA soil textural classification) of 41% sand, 39% loam and 20% clay; pH and organic matter were 8.02 and 1.22%, respectively. Agro-hydrological parameters, calculated after Saxton and Willey (2005) based on soil texture and organic matter, were 27.3% (v:v) and 11.5% for field capacity and wilting point, respectively. Pots were painted white before the trial to limit radiation-induced overheating, and each vine was fertilized twice (i.e., one week before and two weeks after bud-break estimated around 1 April) with 4 g of Greenplant 15 (N) + 5 (P2 O5 ) + 25 (K2 O) + 2 (MgO) + micro (Green Has Italia S.p.A. Canale d’Alba, Italy). White hail net (10% incoming light attenuation) was used to protect the vines throughout the experiment. In late February 2015, each vine was Guyot-pruned to retain a single 1 m long horizontal cane which was placed on a

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Fig. 1. View of the whole-canopy gas exchange system employed in the trial.

supporting wire run at 90 cm from the ground. Shoots were allowed to grow upwards along three catch wires extending 120 cm above the supporting wire. Any secondary shoot bursting from the retained nodes was removed and shoots were left untrimmed during the season. Twelve vines were arranged along a single, vertically shoot positioned, 35◦ NE-SW oriented row and randomly assigned to well watered (WW)—SO4, water-stressed (WS)—SO4, well watered (WW)—M4 and water-stressed (WS)—M4 treatments. Thus, three vine replicates were assigned to each treatment. Prior to system set up (DOY 177, June 26) all vines were irrigated daily to pot water capacity to make sure that no water stress occurred at pre-treatment. Upon system assembling (DOY 177) and until the day before the water deficit was imposed DOY 184 (July 3), all vines were provided with an amount of water corresponding to actual mean transpiration (Tc ) measured by the whole-canopy system described hereafter (Fig. 1). Such procedure was decided to assure that the supplied WW level could be an almost exact match of pre-stress water use and to avoid that water supplied in excess could become available at a later stage through uptake from the under-pots. Decision of using chamber-derived measurements to define amounts of water to be delivered to the vines was made on the basis of the very high correlation (r = 0.95) that Poni et al. (2014) found, on the same cultivar, between gravimetric vine water loss and canopy water loss measured by the chambers. Thereafter, water was replenished at decreasing fractions of Tc through the automated water-supply system described in detail in Poni et al. (2015). In brief, the system is designed to deliver to each individual vine fractions of Tc derived from concurrent measurements performed by the whole-canopy gas-exchange system. This approach makes the process sensitive to large fluctuations in water use which can occur depending upon evaporative demand [e.g., cloudy days with low vapor pressure deficit (VPD) vs. clear days with high VPD] or be simply due to the development of new leaf area (LA) during seasonal vegetative growth. Starting on DOY 184, water stressed (WS) vines on both rootstocks began to receive 50% of the cumulated Tc measure daily by the system on their respective well watered (WW) controls. Water

stress was relieved DOY 209 (July 28) by replenishing in all pots a full Tc supply. On DOY 184 (beginning of stress) clusters were still in their lag-phase, berries were mostly hard and green and soluble solids concentration in a 25 berry sample taken from each treatment was never beyond 4.5 Brix. Initial berry coloring was spotted on all vines around DOY 188–189, indicating that the water shortage was maintained for approximately three weeks after the onset of veraison. During water stress, each pot surface was covered with a plastic film to prevent infiltration of rain water and to minimize losses due to evaporation from the soil surface. 2.2. Gas-exchange and vine growth Whole-canopy net CO2 exchange rate (NCER) and whole-canopy transpiration (Tc ) measurements were taken using the multichamber system described in detail in Poni et al. (2014). The system could monitor up to 12 chambers and the switching interval was set at 90 s. Ambient (inlet) air temperature and the air temperature at each chamber’s outlet were measured by shielded 1/0.2 mm diameter PFA-Teflon insulated type-T thermocouples (Omega Eng. INC., Stamford, Connecticut, USA), whereas total radiation in the 400–700 nm photosynthetic active radiation (PAR) window—was measured with a cosine corrected quantum sensor (Silimet, Modena, Italy) placed horizontally on top of a support stake next to the chambers enclosing the canopies. The chambers were set up on each vine and continuously operated 24 h a day from DOY 177 (26 June, 6 days prior to beginning of stress) until DOY 231 (19 August, 22 days after re-watering). The air flow rate fed to the chambers was set at 14.7 L/s and kept constant throughout the measuring season. Data were not available on DOY 202, 213, 222, 226 due to either system maintenance or temporary failure or electrical power which impaired data recording on that specific date. Whole-canopy NCER (␮mol CO2 /s) and Tc (mmol H2 O/s) were calculated from flow rates and CO2 –H2 O water–vapor differentials after Long and Hallgren (1985). Wholecanopy water-use efficiency (WUEc ) was calculated as NCER/Tc and given as mmol CO2 /mol H2 O. In order to express NCER data on a

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per leaf area unit basis and, therefore, compensate for differences in vine size, total leaf area per vine was estimated the day prior to chamber assembly (DOY 176) by counting the number of unfolded leaves; then a sample of 12 leaves per vine (basal, median and apical primary leaves from each of the four vine shoots) was taken and the surface of each leaf measured in laboratory with an LA meter (LI-3000A, LI-COR Biosciences, Lincoln, NB, USA). At system dismantling on DOY 233, the vines were entirely defoliated and the surface of each primary and lateral leaf was measured with the same equipment. Wherever a significant increase in LA/vine occurred between the two time points (DOY 176–231), the LA increase/day was calculated and the derived daily total LA values used to express seasonal NCER/LA (␮mol/m2 s). On the same day, the number of basal primary leaves that had shed or showed yellowing was also counted. Severity of water stress was quantified by measuring pre-dawn leaf-water potential (pd ) on DOY 181, 185, 189, 192, 196, 205, 209, 211 and 217. Measurements were taken before sunrise on three leaves per vine sampled from basal nodes using a Scholander pressure chamber (3500 Model, Soilmosture Equip. Corp., Santa Barbara, CA). Chambers were snipped for quick access to the foliage and then immediately resealed with transparent tape. Soil moisture, as volumetric content, was also monitored by a TDR portable meter (TRASE System 1 6050 × 1, Soil Moisture Equipment Corp., USA, with 2% accuracy) throughout the trial period. Readings were taken the day preceding the pd readings. To assure maximum uniformity, readings were taken on each pot at the same time of day (usually around 6 PM) using 30-cm long waveguides. The latter were buried vertically in the soil to the bottom of the pot and, hence, the 30 cm readings integrate the entire soil-moisture profile.

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Fig. 2. Seasonal trends of incoming photosynthetically active radiation (PAR, 䊉), air vapour pressure deficit (VPD,→) (top), inlet chamber air temperature (T, ), outlet chambers air temperature for well-watered (T, 䊉), outlet chambers air temperature for water-stressed (T, ) and ambient CO2 concentration (䊐) (bottom) measured at the trial site. Values are daily means averaged from dawn to dusk.

2.3. Yield components and grape composition Each chambered vine was individually picked at harvest (DOY 232, August 20), and all clusters were counted and weighed. Two 50-berry samples per vine were taken to ensure that the positions within the cluster (top, mid, bottom) and exposures (internal or external berries) were represented. These samples were then weighed and stored at −20◦ C for subsequent analyses. All the remaining crop per vine was crushed and soluble solid concentration (◦ Brix) determined by a temperature-compensating Atago refractometer (RX-5000 Atago Co., Ltd, Tokyo, Japan). Titratable acidity (TA) was measured by titration with 0.1 N NaOH to a pH 8.2 end point and expressed as g/L of tartaric acid equivalents. Tartrate was assessed on must via the colorimetric method based on silver nitrate and ammonium vanadate reactions (Lipka and Tanner, 1974). Malate was determined with a kit (Megazyme Int., Bray, Ireland) that uses L-malic dehydrogenase to catalyze the reaction between malate and NAD+ to oxaloacetate and NADH. The reaction products were measured spectrophotometrically by the change in absorbance at 340 nm from the reduction of NAD+ to NADH. Potassium (K+ ) concentration was measured in the must using an ion-selective electrode (Model 96–61, Crison, Carpi, Italy). Anthocyanins and phenolic substances were determined according to Iland (1988) using one of the 50-berry samples left to thaw and then homogenized at high speed (7602 g) with an Ultra-Turrax (Rose Scientific Ltd., Edmonton, AB, Canada) homogenizer for 1 min. Two grams of the homogenate were transferred to a pre-tarred centrifuge tube, enriched with 10 mL aqueous ethanol (50%, pH 5.0), capped and mixed periodically for 1 h before centrifugation at 959 × g for 5 min. A portion of the extract (0.5 mL) was added to 10 mL 1 M HCl, mixed and let stand for 3 h; then the absorbance values were measured at 520 nm and 280 nm on a Jasco V-530 spectrophotometer (Jasco International Co., Ltd., Tokyo, Japan). Anthocyanins and phenolic substances were expressed as mg/g of fresh mass (FM) and mg/berry.

The second 50-berry sample was used for determination of the growth of single berry organs (skin, flesh and seeds). Upon thawing, each berry was sliced in half with a razor blade, the seeds and flesh carefully removed from each berry half using a small metal spatula without rupturing any pigmented hypodermal cells and the seeds then carefully separated by hand from the flesh. Both skins and seeds were rinsed in de-ionized water, blotted dry and weighed. 2.4. Statistical treatment One-way analysis of variance was carried out and, in case of significance of F test, mean separation was performed by the Student-Newman–Keuls test at p < 0.05. Degree of variation around means is given as standard error (SE) defined as the ratio between sample standard deviation (s) and the square root of sample size (n). Differences in slopes and intercepts for data sets of NCER/LA and Tc /LA (Fig. 7) were assesses according to analysis of covariance (ANCOVA) as described in Zar (1999). 3. Results 3.1. Climate and soil water status The weather pattern during the measuring period was marked by a large prevalence of clear and warm days pre-stress and during the water shortage (Fig. 2). Mean inlet ambient temperatures were quite often above 30 ◦ C with daily peaks of up to 40 ◦ C, whereas maximum air-to-leaf VPD (approximately 2.5 kPa) was recorded from DOY 198 to DOY 205 and later around DOY 220. Notably, differences between inlet and outlets T of the WW chambers were negligible, whereas outlet T in WS chambers clearly reflected the water stress status with a maximum overheating vs. inlet T of + 2.9 ◦ C reached on DOY 195. (Fig. 2).

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Fig. 3. Variation of pre-dawn leaf water potential (pd ) (top) and volumetric soil water content (% as v:v) (bottom) in well-watered and water-stressed Sangiovese grapevines grafted on SO4 (䊉, ) and M4 (, ) during the trial. Duration of the water deficit period (50% of canopy transpiration) is shown by the horizontal bar. Vertical bars indicate standard error (SE, n = 9 for top panel, n = 3 for bottom panel). Solid arrow indicates rewatering.

The dynamics of soil–water deficit markedly differed between the two stocks: while SO4 showed a gradual decrease of pd since the beginning of water limitation (−0.23 MPa on day 7 of stress down to a maximum stress of −0.39 MPa on DOY 205) (Fig. 3), M4 retained relatively high pd until DOY 193 and dropped to the least values (−0.29 MPa) three days later without showing any stress worsening hereafter. Noteworthy, at any date within the stress period, M4 had significantly higher pd than SO4 and the latter rootstock was also slower at reaching full rehydration upon rewatering (Fig. 3). General trends on soil water content overall mirrored the predawn leaf water potential patterns (Fig. 3), albeit showing higher within treatment variability especially during the stress period. At the first date of measurement upon onset of water deficit, soil water content of WS-M4 was higher than WS-SO4, whereas non significant differences between the two WS treatments were found for the remainder of the season. Nonetheless, when mean soil water content was correlated, within rootstock, with pd values (data pooled over dates and water supply, y = soil water content, x = pd ) the linear fit was very close: y = 0.157x − 5.5075, R2 = 0.92 for SO4 and y = 0.139x − 4.8776, R2 = 0.88 for M4.

Fig. 4. Seasonal trends of daily mean whole-canopy net CO2 exchange rate (NCER) per unit leaf area (LA) measured in well-watered and water-stressed Sangiovese grapevines grafted on SO4 (䊉, ) and M4 (, ). Duration of the water deficit period (50% of vine transpiration) is shown by the horizontal bar. Vertical bars indicate standard error (n = 3). Arrow indicates date of re-watering.

Cluster weight was consistently reduced in WS as compared to WW (−22% and −18% for SO4 and M4, respectively) leading to an almost equivalent decrease in yield per vine (−26% and −24% for SO4 and M4, respectively). Source-sink balance expressed either as LA/yield ratio and carbon/fruit mass (Table 1) showed that while no significant changes were shown within SO4, for M4 both expressions indicated that in WS the supply/demand function significantly increased. Berry growth components showed an expected limitation in total and flesh berry mass in both WS treatments as compared to the respective WW controls (Table 2). However, the latter response was not strong enough to induce a significant increase in the relative skin growth. Must composition showed large differences among rootstocks (Table 3). On vines grafted on SO4, the water deficit reduced total anthocyanins and phenolics (the latter on a whole berry basis only) as compared to WW; conversely, overall must composition was improved in WS Sangiovese vine grafted on M4 as higher total soluble solids and phenolic concentration, and lower titratable acidity.

3.2. Vegetative growth, yield components and grape composition

3.3. Whole-canopy gas exchange and WUE

Pre-stress shoot number and total leaf area (LA) per vine were similar among treatments (Table 1) as well as final total leaf area also assessed as primary leaves and laterals contribution. The LA area increment from pre-stress until defoliation was similar between WW and WS for M4, whereas it was lower in WS—SO4 grafted vines vs. WW-SO4. Fraction of leaves that were shed or showed yellowing at defoliation was similar in WS treatments (about 26% of total). Although vines grafted on M4 were slightly less fruitful than SO4 vines, clusters/vine did not differ within rootstock.

Seasonal trends of net CO2 exchange rate per unit leaf area (NCER/LA) for both stocks are shown in Fig. 4. Pre-stress NCER/LA was fairly similar in WW and WS Sangiovese vines grafted onto SO4 (7.0 and 7.6 ␮mol m−2 s−1 , respectively) and reducing water supply by 50% significantly lowered NCER/LA in WS at any date. NCER/LA averaged over the whole water deficit period (DOY 184 through DOY 208) were 6.2 and 3.2 ␮mol m−2 s−1 for WW and WS, respectively, meaning that fractional NCER/LA during stress was 49.6% of WW. Upon rewatering performed DOY 209, WS did promptly

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Table 1 Vegetative growth, yield components and source-sink balance indices (vine basis) recorded on Sangiovese grapevines either well-watered (WW) or subjected to a 50% canopy transpiration (Tc ) water stress (WS) from DOY 184 until DOY 208. * and ** denote significant differences between treatments at p < 0.05 and 0.01 according to within column mean separation performed with SNK-test. ns = not significant. LA = leaf area.

WW-SO4 WS-SO4 WW-M4 WS-M4 sig.

Shoots/vine

Pre-stress total LA (m2 )

Total final LA (m2 )

Primary LA (m2 )

Lateral LA (m2 )

Shed and yellow leaves/vine (% of total)

Clusters/vine Cluster weight (g)

Yield/vine (kg)

LA/yield (m2 kg−1 )

Carbon/fruit mass (nmol s−1 g−1 )

7.67 7.33 8.33 6.67 ns

1.79 1.58 1.88 2.08 ns

3.35 2.72 3.45 3.63 ns

1.54 1.13 1.57 1.73 ns

1.81 1.59 1.88 1.90 ns

16.6 25.4 16.1 25.6 ns

12.3 11.7 10.3 8.3 ns

3.50a 2.60ab 2.81ab 1.86b *

1.07b 1.22b 1.40b 2.25a **

4.447ab 3.733b 5.247ab 6.427a *

284a 223b 272a 223b **

Table 2 Berry growth components (absolute and relative basis) and seed number recorded on Sangiovese grapevines either well-watered (WW) or subjected to a 50% canopy transpiration (Tc ) water stress (WS) from DOY 184 until DOY 208. ** denotes significant differences between treatments at p < 0.01 according to within column mean separation performed with SNK test. ns = not significant.

WW-SO4 WS-SO4 WW-M4. WS-M4 sig.

Berry weight (g)

Skin weight (g)

Total seed weight (g)

Seeds/berry

Single seed weight (mg)

Flesh weight (g)

Skin-toflesh ratio (%)

Skin-toberry ratio (%)

Flesh-toberry ratio (%)

Seed-to-berry-ratio (%)

2.77a 2.34b 2.63a 2.45b **

0.172 0.166 0.169 0.188 ns

0.113 0.112 0.104 0.105 ns

2.92 3.08 2.72 2.64 ns

39.4 36.9 38.9 40.4 ns

2.49a 2.06b 2.35a 2.16b **

7.06 8.45 7.29 9.07 ns

6.31 7.36 6.51 7.91 ns

89.65 87.77 89.57 87.91 ns

4.04 4.88 3.92 4.17 ns

Table 3 Must composition recorded on Sangiovese grapevines either well-watered (WW) or subjected to a 50% canopy transpiration (Tc ) water stress (WS) from DOY 184 until DOY 208. * and ** denote significant differences between treatments at p < 0.05 and 0.01 according to within column mean separation performed with SNK-test. ns = not significant.

WW-SO4 WS-SO4 WW-M4 WS-M4 sig.

Total soluble solids (◦ Brix)

pH

Titratable acidity (g L−1 )

Malic acid K+ (ppm) Tartaric acid (g L−1 ) (g L−1 )

Total anthocyanins mg berry−1 mg g−1

Total phenolics mg berry−1 mg g−1

18.7b 18.1b 18.6b 20.4a **

3.17b 3.19ab 3.27ab 3.32a **

7.41a 7.22a 7.54a 6.38b **

8.69a 8.30b 7.90c 7.61c **

1.27a 0.61b 1.09ab 1.27a **

4.83a 4.19b 4.85a 5.33a **

2.36b 1.98b 3.01a 2.21ab **

1689b 1750ab 1776ab 1809a *

recover although mean NCER/LA rate for WS over the whole postrewatering period was 84% of the rate measured in WW (Fig. 4). Mirroring behavior shown in SO4, vines grafted on M4 showed very close pre-stress NCER/LA rate (7.4 vs. 7.6 ␮mol m−2 s−1 ) in WW and WS, respectively (Fig. 4). Mean percent reduction of NCER/LA throughout the whole water deficit period was 46.2% as compared to WW, whereas, upon rewatering, vines grafted on M4 showed capacity for a gradual, yet steady recovery which was almost complete at about 10 days after rehydration. The actual amount of water daily supplied from the automated system to each vine during the drought period (i.e., since DOY 184 till rewatering) was 2.059 ± 0.313 L and 1.084 ± 0.056 L in WWSO4 and WS-SO4, respectively; 2.225 ± 0.295 L and 1.013 ± 0.056 L in WW-M4 and WS-M4, in the order. Daily mean vine water supply averaged over the post-rewatering period (i.e., since DOY 209 till DOY 232) was 1.887 ± 0.158, 2.022 ± 0.126, 1.873 ± 0.207 and 1.726 ± 0.158 L for WW-SO4, WS-SO4, WW-M4 and WS-M4, respectively. Water use given as Tc /LA pre-stress was very similar among vines grafted on SO4 (1.22 and 1.32 mmol m−2 s−1 for WW and WS, respectively) and halving the water supply on DOY 184 determined an almost correspondent reduction of Tc (49.1%) as compared to WW over the whole stress period (Fig. 5). Recovery of Tc /LA after rewatering was prompter and higher than that showed in SO4 by NCER/LA, since Tc /LA of WS stressed vines rose to 91.8% of WW and most dates after replenishment of full water supply Tc /LA of both treatments did not differ.

0.45a 0.25b 0.39ab 0.49b *

1.73b 1.72b 1.73b 2.07a **

Canopy transpiration/LA in vines grafted on M4 was again comparable between treatment pre-stress (1.34 vs. 1.25 mmol m−2 s−1 ) in WW and WS, whereas limitation of Tc /LA due to the 50% reduction in water supply was 56.5% of rates measured in WW for the whole stress period (Fig. 5). Dynamic of Tc /LA resumption upon rewatering showed two distinct phases: little Tc /LA recover was seen until DOY 218; hereafter, transpiration rates between the two treatments were very similar. Mean Tc /LA calculated for WS over the whole recovering period was 0.802 mmol m−2 s−1 , i.e., 82.5% of the WW rates. Whole-canopy water-use efficiency given as the NCER/Tc ratio (mmol CO2 /mol H2 O) showed a quite distinct pattern between the two rootstocks (Fig. 6). Overall, vines grafted on SO4 and subjected to the water deficit did not change their WUE either throughout the period of reduced water supply and after rewatering. Conversely, WUEc calculated for vines grafted on M4 showed the same values pre-stress (5.6 mmol CO2 /mol H2 O in both WW and WS) and a significant improvement in WS during stress (5.4 mmolCO2 /mol H2 O in WS vs. 4.4 mmol CO2 /mol H2 O in WW, p < 0.01). Such higher WUEc of the WS treatment was maintained, albeit to a lesser extent (p < 0.05), during the rewatering, marking a 9.1% increase in comparison to WW (Fig. 6).

4. Discussion The overall trial results indicate that when grafted with Sangiovese grapevines, the newly selected rootstock M4 can improve

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Fig. 5. Seasonal trends of daily mean whole-canopy transpiration (Tc ) per unit leaf area (LA) measured in well-watered and water-stressed Sangiovese grapevines grafted on SO4 (䊉, ) and M4 (, ). Duration of the water deficit period (50% of vine transpiration) is shown by the horizontal bar. Vertical bars indicate standard error (SE, n = 3). Arrow indicates date of re-watering.

tolerance to water deficit and WUE as compared to the traditional SO4 stock. One such indicator is the seasonal variation of pd : M4 significantly affected the rate of supplied-water uptake since these vines showed less negative pd than SO4 at any date during the water stress. The seasonal soil water content trends (Fig. 3) were overall quite similar to those of pd albeit within-treatment variability increased at lower levels of soil moisture. This behavior is not too much surprising since once the guides are inserted into the soil it is assumed that validity of the reading extends to a cylinder having guide length as its height and distance between guides as its diameter. Therefore, one single reading might not catch patchiness of humidity in the pot volume profile especially if we consider that the automated supply system delivers fairly small volumes (about 300 mL) several times a day depending on canopy transpiration rate. Then, it is likewise unlikely that differences in soil water contents themselves might have been the primary driver of the different gas exchange response of the two stocks. In fact, volumetric soil water content between the two treatments only differed upon the first date of measurements during stress (DOY 189), whilst on the remaining dates the same significance was not found due to overwhelming within treatment variability. Although differences of pd between the two WS treatments did not affect seasonal NCER/LA rates, it seems quite likely that the root system of the two rootstock genotypes reacts differently to an abrupt water shortage. In a recent paper, Corso et al., 2015 have shown that ungrafted M4 stocks subjected to a water deficit have higher reactive oxygen species (ROS) detoxification ability than the drought susceptible 101-14 rootstock and suggest that this could result in root lateral growth to be maintained resulting, in turn,

Fig. 6. Seasonal trends of daily mean canopy water-use efficiency (WUE) calculated as the ratio of NCER to Tc for well-watered and water-stressed Sangiovese grapevines grafted on SO4 (䊉, ) and M4 (, ). Duration of the water deficit period (50% of vine transpiration) is shown by the horizontal bar. Vertical bars indicate SE (n = 3). Arrow indicates date of re-watering.

in higher water uptake capacity from the soil. Such responses also suggest that rootstock type can modify scion response to water stress conventionally described as iso- or anisohydric (Tardieu and Simmonneau, 1998). Conversely, the effects we found in this study on whole-canopy gas exchange (i.e., net photosynthesis and transpiration) seem to contradict earlier findings from Meggio et al. (2014) and Corso et al. (2015) who reported an increase in leaf function of M4 plants at the most severe levels of water deficit in the soil. Part of such discrepancy might indeed reflect the fact that these authors worked with small ungrafted, non fruiting vines grown under semicontrolled conditions, whereas our test vines were quite close to the standard of mature vines in the field. Indeed, when we calculated the seasonal mean of NCER/LA over the whole water deficit period (25 days) for the WS treatments they were almost identical (3.22 ␮mol m−2 s−1 vs. 3.28 ␮mol m−2 s−1 for SO4 and M4, respectively), while Tc /LA of WS-M4 was reduced by 15% as compared to WS-SO4 (0.604 mmol m2 s−1 in M4 against 0.695 mmol m2 s−1 in SO4). Thus, plotting daily values of Tc /LA vs. NCER/LA (Fig. 7) revealed that better drought tolerance in M4 manifests as ability to achieve the same NCER/LA values of SO4 at a more conservative water loss rate, although such gap seems to narrow when very low Tc /LA are approached. The above statement shifts attention to WUE and, more specifically, on the effects a given rootstock can exert on the water-use efficiency of the grafted scion (Virgona et al., 2003; Soar et al., 2006; Meggio et al., 2014). To our knowledge, no data are yet available for physiological WUE assessed on a whole-canopy basis. The most comprehensive study in this connection was conducted over two quite contrasting seasons on cv. Shiraz grafted to seven scion-

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Fig. 7. Linear relationship between Tc /LA and NCER/LA for the WS treatments. Data points (SO4, , M4 ) are mean daily values from beginning (DOY 184) to end of stress (DOY 208). Equation for WS-M4 was: y = 5.4622x − 0.0222, R2 = 0.72, p < 0.01; equation for WS-SO4 was: y = 4.3634x + 0.1832, R2 = 0.52, p < 0.05. Intercepts were different at p < 0.05, whereas slopes were ns.

stock combinations varying for scion-induced vigor and drought tolerance (Soar et al., 2006). Water-use efficiency, putatively evaluated as the slope of the linear regression of the gs vs. A, resulted in no significant differences for 2003 and overall mild differences in 2002. In their paper, Meggio et al. (2014) claim that M4 had higher WUEi at gs lower than 100 mmol m−2 s−1 , although intrinsic WUE data are not presented. In our study, evidence is provided that while in SO4 variation of (WUEws /WUEww ) × 100 was negligible throughout the three stages of the study (i.e., 99.5%, 99.8% and 93.4% for pre-stress, stress and rewatering, respectively), the same parameter calculated for M4 jumped from the 98.0% calculated pre-stress to the 122.1% computed during the stress and still settled at 114.6% during rewatering. Thus, it is quite nicely shown that Sangiovese vines grafted on M4 acquire ability to maintain the same assimilation rates of SO4 at lower water loss and such feature is partially held upon rewatering. Conversely, SO4’s behavior as per WUE under water stress resembles quite closely the pattern ˜ (2015) in own-rooted recently described by Tarara and Pérez-Pena Cabernet Sauvignon grapevines in Washington State (US) and subjected to different regulated deficit irrigation (RDI) approaches; whole canopy integrated WUE did not differ among water supply regimes because daily cumulative values of NCER per canopy and Tc changed proportionally. Linking seasonal variation of canopy water use efficiency (WUEc ) to vine performance was the second goal of our work. Such comparison is quite troublesome in the grapevine due to either methodological issues (i.e., whole-canopy gas assessment should be at hand) and to the fact that grape composition also responds to a series of factors not necessarily closely related to water use efficiency. A recent study conducted by Merli et al. (2015) showed for Sangiovese vines grafted on SO4 and subjected to a dry down cycle (i.e., water supply progressively reduced from 100% to 30% of WW controls) that WUEc started to decline in WS vines when water supply was lowered below 50% and, at a similar yield level, grape composition was also impaired in terms of lower must soluble solids at harvest and unchanged color and phenolic concentration in spite of smaller berries and higher relative skin growth. In this study, we found that unchanged WUEc assessed in SO4-grafted vines during stress matches overall mild grape compositional differences between WW-SO4 and WS-SO4 with the exception of anthocyanins which were markedly dampened by water stress in SO4. On the other hand, it is apparent that WS-M4 vines were overall “more ripen” that the WW-SO4, although the increase in anthocyanins did not reach significance. Technically speaking, it is quite remarkable that in front of a

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quite similar yield reduction per vine due to the water limitation (about 0.9–1.0 kg), the SO4 grafted vines did not show any, albeit expected, improvement in grape composition which, conversely, was clearly seen in M4. This latter pattern can be quite easily explained with the improved source-sink balance (Table 1) of the WS-M4 treatment combination confirming that, according to Kliewer and Dokoozlian 2005, sugar accumulation is a quite trustable indicator of the supply/demand function. Interestingly, the fractional WS/WS increase in carbon/fruit mass (+18%) approximates the corresponding increase in ◦ Brix much better that the LA/F, which increased by 38% vs. a ◦ Brix gain of 9%. Besides that the LA/F vs. ◦ Brix relationship often reported in literature saturates around 1.5 m2 /kg (Kliewer and Dokoozlian, 2005), it should be noted that “total” leaf area might significantly deviate from “functional” leaf area especially when a stress factor is imposed. A quite challenging effect to be explained is why WS-SO4 vines despite having a slightly lower, yet not significant, must sugar concentration than their respective WW control concurrently showed a drastic drop in berry pigmentation (Table 3). This seems a clear case of decoupling between sugar and anthocyanins accumulation that, according to recent papers (Sadras and Moran, 2012; Poni et al., 2013; Palliotti et al., 2014b) might be affected by different factors. More specifically, Sadras and Moran (2012) have shown that while high temperatures (≥35 ◦ C) decouples the sugar:anthocyanins ration by delaying the onset of color accumulation, water deficit established shortly before veraison could partially restore this ratio. In our trail, both of these factors acted simultaneously since high temperatures were registered in both WS treatments (Fig. 2) and no differences in overheating were seen among the two rootstocks. Thus, the largely different color accumulation between the two WS treatments should look for different explanations. Indeed, Mori et al. (2007) showed that, in red varieties such as Cabernet Sauvignon, raising temperature from 25 to 35 ◦ C might decrease total anthocyanins by more than 50%. However, in their paper Mori et al. also showed that the low anthocyanins content was due more to accelerated degradation of already formed color rather than inhibition of mRNA transcription of the anthocyanin biosynthetic genes. Such a response shifts attention to differential gene expression that the stress treatment might have induced in the two rootstocks genotype. Corso et al. (2015) showed that, upon stress, M4 roots and leaves showed a higher induction of resveratrol and flavonoid biosynthetic genes, respectively. Although in our study we did not perform any genomic and transcriptomic analysis, it should be worth investigating if differential gene expression also extends to functions related to biosynthesis or degradation of already synthesized anthocyanins.

5. Conclusions Our water deficit study carried out on Sangiovese grapevines grafted on SO4 and M4 stocks by halving vine water supply from pre-veraison until about three weeks after veraison clearly indicated that whole canopy water use efficiency increased during water stress in WS-M4 and part of this increment was also maintained during the rewatering phase. Analysis of whole-canopy net CO2 gas exchange and transpiration revealed that higher WUEc in M4 grafted vines was due to ability to maintain under stress photosynthesis rates similar to SO4 while reducing transpiration. Higher WUEc also matched with better wine composition at harvest for vines grafted on M4 as compared to the SO4 stock. Being water stress severity and air temperature measured at the chambers’ outlet similar in both WS treatments, it is assumed that the wide differences observed in anthocyanin accumulation between the WS treatments links to possible different gene expression under drought leading to a mechanism which might interfere with

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synthesis and/or degradation of anthocyanins under high temperature conditions. Finally, it has to be acknowledged that preliminary indication for higher M4 tolerance to drought found in this oneseason pot study will need to be fortified with longer term assessment under field conditions. Among the factors that a single season pot study does not allow to address exhaustively are carryover effects on next season performance and additional variation due to the fact that, under incipient water stress, grapevine roots differing in their genetic pedigree might have a different attitude at exploring deeper and more humid soil layers. Acknowledgment Authors wish to thank Dr. David Verzoni for careful revision of the English style. References Bauerle, T.L., Richards, J.H., Smart, D.R., Eissenstat, D.M., 2008a. Importance of internal hydraulic redistribution for prolonging lifespan of roots in dry soil. Plant Cell Environ. 31, 177–186. Bauerle, T.L., Smart, D.R., Bauerle, W.L., Stockert, C., Eissenstat, D.M., 2008b. Root foraging in response to heterogeneous soil moisture in two grapevines that differ in potential growth rate. New Phyt. 179, 857–866. Chaves, M.M., Zarrouk, O., Francisco, R., Costa, J.M., Santos, T., Regalado, A.P., Rodrigues, M.L., Lopes, C.M., 2010. Grapevine under deficit irrigation: hints from physiological and molecular data. Ann. Bot. 105, 661–676. Corso, M., Vannozzi, A., Maza, E., Vitulo, N., Meggio, F., Pitacco, A., Telatin, A., D’Angelo, M., Feltrin, E., Negri, A.S., Prinsi, B., Valle, G., Ramina, A., Bouzayen, M., Bonghi, C., Lucchin, M., 2015. Comprehensive transcript profiling of two grapevine rootstock genotypes contrasting in drought susceptibility links the phenylpropanoid pathway to enhanced tolerance. J. Exp. Bot. 66, 5739–5752. Düring, H., 1994. Photosynthesis of ungrafted and grafted grapevines: effects of rootstock genotype and plant age. Am. J. Enol. Vitic. 45, 297–299. Gambetta, G.A., Manuck, C.M., Drucker, S.T., Shaghasi, T., Fort, K., Matthews, M.A., Walker, M.A., McElrone, A.J., 2012. The relationship between root hydraulics and scion vigour across Vitis rootstocks: what role do root aquaporins play? J. Exp. Bot. 63, 6445–6455. Granett, J., Walker, A.M., Kocsis, L., Omer, A.D., 2001. Biology and management of grape Phylloxera. Ann. Rev. Entomol. 46, 387–412. Iland, P.G., et al., 1988. Leaf removal effects of fruit composition. In: Smart, R.E. (Ed.), Proceedings of the Second International Cool Climate Viticulture and Oenology Symposium. Auckland, New Zealand, pp. pp. 137–138. ISTAT, 2015. Istituto Nazionale di Statistica. http://www.istat.it/it/agricoltura-ezootecnia. Kliewer, W.M., Dokoozlian, N.K., 2005. Lear area/crop weight ratios of grapevines: influence of fruit composition and wine quality. Am. J. Enol. Vitic. 56, 170–181. Lipka, Z., Tanner, H., 1974. Une nouvelle méthode de dosage rapide de l’acide tartrique dans les moÛts les vins at autres boissons (selon Rebelein). Revue Suisse de Agric. Vitic. Arboric. 6, 5–10. Long, S.P., Hallgren, J.E., 1985. Measurement of CO2 assimilation by plants in the field and the laboratory. In: Coombs, J., Hall, D.D., Long, S.P., Scurlock, J.M.O. (Eds.), Techniques in Bio-Productivity and Photosynthesis. Pergamon Press, Oxford, UK, pp. 62–93. Lovisolo, C., Perrone, I., Carra, A., Ferrandino, A., Flexas, J., Medrano, H., Schubert, A., 2010. Drought-induced changes in development and function of grapevine (Vitis spp.) organs and in their hydraulic and non-hydraulic interactions at the whole-plant level: a physiological and molecular update. Funct. Plant Biol. 37, 98–116. Meggio, F., Prinsi, B., Negri, A.S., Di Lorenzo, G., Lucchini, G., Pitacco, A., Failla, O., Scienza, A., Cocucci, M., Espen, L., 2014. Biochemical and physiological responses of two grapevine rootstock genotypes to drought and salt treatments. Aust. J. Grape Wine Res. 20, 310–323. Merli, M.C., Gatti, M., Galbignani, M., Bernizzoni, F., Magnanini, E., Poni, S., 2015. Water use efficiency in Sangiovese grapes (Vitis vinifera L.) subjected to water stress before veraison: different levels of assessment lead to different conclusions. Funct. Plant Biol. 42, 198–208.

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