Partial root zone drying exerts different physiological ...

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Functional Plant Biology http://dx.doi.org/10.1071/FP13276

Partial root zone drying exerts different physiological responses on field-grown grapevine (Vitis vinifera cv. Monastrell) in comparison to regulated deficit irrigation Pascual Romero A,E, Juan Gabriel Pérez-Pérez B, Francisco M. del Amor C, Adrián Martinez-Cutillas A, Ian C. Dodd D and Pablo Botía B A

Departamento de Viticultura, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, c/ Mayor s/n, 30150, La Alberca, Murcia, Spain. B Departamento de Citricultura, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, c/ Mayor s/n, 30150, La Alberca, Murcia, Spain. C Departamento de Calidad y Seguridad Alimentaria, Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, c/ Mayor s/n, 30150, La Alberca, Murcia, Spain. D The Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK. E Corresponding author. Email: [email protected]

Abstract. Regulated deficit irrigation (RDI) and partial root zone irrigation (PRI) were compared for 4 years at two irrigation volumes (110 mm year–1 (1) and 78 mm year–1 (2)) in field-grown grafted Monastrell grapevines (Vitis vitifera L.) to distinguish the effects of deficit irrigation from specific PRI effects. PRI-1 and RDI-1 vines received ~30% of the crop evapotranspiration (ETc) from budburst to fruit set, 13–15% from fruit set to veraison and 20% from veraison to harvest. RDI-2 and PRI-2 vines received around 20% of ETc from budburst to fruit set, no irrigation from fruit set to veraison, and recovery (21–24% ETc) thereafter. Compared with RDI-1, PRI-1 increased irrigation depth and total soil water (qv) availability in the root zone, and stimulated greater fine root growth and water uptake. Increased soil volume exploration supported greater canopy water use, vegetative development, biomass accumulation and internal water storage capacity. PRI-1 vines had higher stomatal conductance, lower leaf-level water use efficiency and increased leaf xylem sap concentration ([X-ABA]leaf) following reirrigation. Compared with RDI-2, PRI-2 decreased total qv availability, fine root growth and water uptake, gas exchange, leaf water status, [X-ABA]leaf, biomass accumulation and storage capacity. Xylem ABA decreased with total qv availability in PRI-2, probably from limited sap flow when qv in drying soil was low (20%). For this rootstock–scion combination, high irrigation volumes applied to the wet part of the roots (qv > 30%) are critical for increasing root-to-shoot ABA signalling and growth, and improving performance under semiarid conditions. Additional keywords: abscisic acid, plant biomass, root and shoot growth regulation, water stress physiology, water uptake, water use efficiency. Received 19 September 2013, accepted 6 February 2014, published online 3 April 2014

Introduction Partial root zone irrigation (PRI) is an irrigation technique that was conceived essentially as a field adaptation of laboratory splitroot experiments using partial root zone drying and was used initially in vines with the aim of restricting vine water use and vegetative growth without incurring leaf water deficit (Dry et al. 1996; Dry and Loveys 1999; Stoll et al. 2000; Kriedemann and Goodwin 2003). PRI was developed to impose soil moisture heterogeneity in the root zone by using irrigation to alternately wet and dry different parts of the plant root system. In theory, the drying of roots triggers chemical signals (an increase in ABA, or changes in other hormones and xylem sap pH) that are transported to the shoots via the xylem, altering shoot Journal compilation CSIRO 2014

physiology (Stoll et al. 2000; Dodd et al. 2006). The effect traditionally associated with PRI was that increased xylem ABA concentration ([X-ABA]) would reduce vegetative growth and plant water use more than fruit growth, thereby maintaining yield and increasing water use efficiency (WUE) (Stoll et al. 2000). In addition, the irrigated roots supply sufficient water to the shoots to maintain the shoot water status and prevent plant water deficit (Dry et al. 2000a, 2000b). Many studies have focussed on the impact of PRI on production of the stress hormone ABA and how to increase the intensity of ABA signalling (Stoll et al. 2000; Dodd et al. 2006; Liu et al. 2006, 2008; Dodd et al. 2008a, 2008b, 2010, 2011; Ahmadi et al. 2010b; Martín-Vertedor and Dodd 2011; www.publish.csiro.au/journals/fpb

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Einhorn et al. 2012; Romero et al. 2012; Puértolas et al. 2013), because this hormone controls many of the adaptive responses that plants have evolved to conserve water, such as stomatal closure, reduced canopy area and increased root biomass (Wilkinson and Hartung 2009). However, PRI experiments have not always detected differences in ABA signalling compared with conventional deficit irrigation (Marsal et al. 2008; Rodrigues et al. 2008; Savic et al. 2009; Ahmadi et al. 2010b; Wang et al. 2012b; Pérez-Pérez et al. 2012). Although the PRI technique was initially developed to exploit chemical root-to shoot signalling, many recent studies comparing PRI and conventional deficit irrigation techniques (which have applied the same amount of water) in different crops and experimental (controlled or field) conditions have reported a wide range of PRI-specific responses, some of which were independent of root-to-shoot ABA signalling (Pérez-Pérez et al. 2012). These included better soil aeration, and enhanced soil enzymatic activities and activity of soil microorganisms (Wang et al. 2008; Li et al. 2010); higher mineralisation of soil organic C and N (Sun et al. 2013); higher soil N availability, N use efficiency, and improved plant N uptake, nutrition and distribution (Wang et al. 2013); decreased soil evaporative losses and a slight increase in irrigation efficiency (Marsal et al. 2008); decreased total vine water use and sap flow rates at high evaporative demands (Rodrigues et al. 2008; Collins et al. 2010); greater root biomass in controlled environments (Mingo et al. 2004; Shao et al. 2008, 2011; Kaman et al. 2011; Affi et al. 2012; Wang et al. 2012c); changes in root water uptake patterns, root growth and distribution in the field (Gu et al. 2004; Abrisqueta et al. 2008; Collins et al. 2010; Pérez-Pérez et al. 2012; Romero et al. 2012); increased root–shoot hydraulic conductivity and root water uptake in the wet root zone, producing a ‘compensatory effect’ (Ahmadi et al. 2011; Hu et al. 2011; McLean et al. 2011; Romero et al. 2012; Yang et al. 2013); decreased bundle-sheath cell leakage of CO2 and enhanced photosynthetic capacity in controlled split-root systems (Tahi et al. 2007; Wang et al. 2012a); smaller guard cells and lower stomata density, via modulating stomatal morphology under a high N rate (Yan et al. 2012); higher root nutrient uptake capacity and higher xylem ionic concentrations (Wang et al. 2012b); higher photosynthesis levels and plant water use in field conditions (Romero et al. 2012); and improved leaf-level WUE (Ahmadi et al. 2010b; Egea et al. 2011). Nevertheless, plant responses to PRI vary enormously, especially in field conditions. McLean et al. (2011) indicated that species appear to lie on a continuum of PRI response, from those responding primarily at the leaf level to those responding mainly via root-level compensation. At present, there is still considerable controversy regarding the effects of PRI in wine grapes (Vitis vinifera L.) and other crops (Dodd 2009; Sadras 2009). Some researchers reported no improvement in water relations, plant performance, water use, crop yield or fruit quality compared with conventional drip irrigation at the same irrigation amounts, especially in field conditions (dos Santos et al. 2003; de Souza et al. 2003, 2005a, 2005b; Bravdo et al. 2004; Gu et al. 2004; Intrigliolo and Castel 2009; Myburgh 2011). Multiple reasons have been suggested for the varied and inconsistent effects of PRI, including different soil types

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(Kriedemann and Goodwin 2003; Sepaskhah and Ahmadi 2010) varieties, rootstocks, environmental and experimental conditions (de Souza et al. 2005a, 2005b; Chaves et al. 2007; Rodrigues et al. 2008; Chaves et al. 2010), different intensities and modulation of the chemical signal (Li et al. 2011a), different distribution of the soil water content (Dodd et al. 2008a, 2008b), different species adaptations to soil moisture heterogeneity (McLean et al. 2011), root hydraulic redistribution (Smart et al. 2005) and methodological problems in applying PRI and unsuitable irrigation management (De la Hera et al. 2007). In addition, the benefits of PRI relative to deficit irrigation may also depend on (1) genotypic variation in root distribution and the proportion of root biomass exposed to drying soil (MartínVertedor and Dodd 2011; Kaman et al. 2011), and (2) the soil water content maintained in the wet root zone (Hutton and Loveys 2011; Romero et al. 2012; Romero and Martínez-Cutillas 2012; Wang et al. 2012b; Einhorn et al. 2012) determined by irrigation management (irrigation frequencies and volumes). All these new findings provide a further stimulus to understand (and perhaps optimise) PRI in wine grapes under field conditions by integrating physiological and agronomical research. This work compared two different irrigation techniques (conventional regulated deficit irrigation (RDI) and PRI) at the same irrigation volumes (the moderate and low amounts of water usually used by growers to irrigate wine grapes in southeast Spain) with the aim of distinguishing the effects of deficit irrigation per se from any specific PRI effects (placement of water). Differences between RDI and PRI in root growth and distribution, root water uptake patterns, sap [X-ABA], soil–plant water relations, trunk diameter fluctuations, vine vigour and plant biomass accumulation were studied in field-grown Monastrell wine grapes, in a 4-year experiment in a semiarid environment. Materials and methods Field conditions, plant materials and irrigation treatments This research was carried out in a 1-ha vineyard at the Centro Integrado de Formación y Experiencias Agrarias (CIFEA) experimental station in Jumilla, Murcia (Spain, 3820 N, 1580 W, 395 m above sea level). The soil was a 60-cm-deep fine clay (48% clay, 30% silt and 22% sand; field capacity 35%). The irrigation water, from a well, had an electrical conductivity of 1.6 dS m–1. The climate is Mediterranean semiarid, with hot, dry summers, scanty annual rainfall (less than 300 mm year–1) and a total annual reference evapotranspiration (ETo) of around 1200 mm (Table 1). The grapevines were 13-year-old Vitis vinifera L. cv. Monastrell (syn. Mourvedre), a red wine variety, grafted onto V. vinifera cv. 1103 Paulsen rootstock. The training system was a bilateral cordon trellised to a three-wire vertical system. The vine rows ran north-north-west to south-south-east, and the planting density was 2.5 m between rows and 1.25 m between vines (3200 vines ha–1). Six two-bud spurs (12 nodes) were left after pruning; in May, nonproductive green shoots were removed from each vine in the same manner for all treatments, according to the growers’ practice in the area. During 4 consecutive years (2009–12), a moderate RDI strategy was applied under conventional drip irrigation

Comparative physiological effects of PRI and RDI


(RDI-1) and under PRI (PRI-1). In PRI-1 and RDI-1, the vines received ~30% of the crop evapotranspiration (ETc) from budburst to fruit set, 13–15% of the ETc from fruit set to veraison and 20% of the ETc from veraison to harvest (Table 2). From 2009 to 2011, a more severe RDI strategy was also applied under conventional drip irrigation (RDI-2) and under PRI (PRI-2). In RDI-2 and PRI-2, the vines received around ~20% of the ETc from budburst to fruit set. From fruit set to veraison, no irrigation was applied. From veraison to harvest, an irrigation recovery was established (similar to RDI1 and PRI-1). In contrast, in 2012, PRI-2 and RDI-2 vines received less water from budburst to fruit set (10% ETc) and were watered from fruit set to veraison at 18–20% ETc (Table 2). Then, from véraison to harvest, an irrigation recovery was established (similar to RDI-1 and PRI-1). A recovery of irrigation at 40% ETc was applied to all treatments from harvest to leaf fall (the end of October) every year. The average annual amount of water applied by most Monastrell grape growers in modern irrigated vineyards in the Jumilla

C

area ranges from 150 mm year–1 to 200 mm year–1 (GarcíaGarcía 2010), normally distributed across the whole cycle. In our study, the average annual amount of irrigation water applied in RDI-1 and PRI-1 was 109 mm and 110 mm, respectively; in RDI-2 and PRI-2, it was 79 mm and 77 mm, respectively (for the period 2009–2011). These deficit irrigation strategies with moderate and low amounts of water were initially designed to control excessive vegetative development during the early season, to reduce berry size and yield (by using moderate or severe water deficits before verasion) and to stimulate berry accumulation of sugar, anthocyanins and other phenolic compounds (by using a moderate water deficit after verasion) in order to improve berry quality for premium red wine production. Crop evapotranspiration (ETc = ETo Kc) was estimated using varying crop coefficients (Kc) based on those proposed by the Food and Agriculture Organisation, adjusted for the Mediterranean area and reference evapotranspiration (ETo) values (Table 1). The ETo was calculated weekly from the

Table 1. Crop coefficients (Kc) used, monthly rainfall, reference crop evapotranspiration (ETo) and atmospheric vapour pressure deficit (VPD) at the experimental site in 2009, 2010, 2011 and 2012 Month Kc January February March April May June July August September October November December

0.35 0.45 0.52 0.76 0.70 0.60 0.45

Total

Rainfall (mm) 16.6 1.7 45.4 19.1 7.3 1.6 0.1 35.9 27.5 5.7 5 57.9 224

2009 ETo (mm)

VPD (kPa)

Rainfall (mm)

42.8 50.3 85.5 110.1 152.7 188.1 195.3 112.4 74.8 73.5 58.1 38.0

0.42 0.52 0.75 0.83 1.32 2.10 2.40 1.94 1.18 1.14 0.88 0.49

20.9 20.6 36.3 15.1 22.3 39.8 0.1 33.4 42 18.7 41.5 14.4

1182

1.17

305

2010 ETo (mm)

VPD (kPa)

Rainfall (mm)

38.0 46.9 74.4 97.4 145.9 158.6 189.2 154.3 109.4 73.0 50.0 31.7

0.37 0.46 0.55 0.73 1.17 1.47 1.98 1.78 1.35 1.01 0.63 0.41

1.9 6.2 23 48.8 10.7 3.5 2.9 3.1 13.9 1.7 46.0 0.2

0.99

167.1

1169

2011 ETo (mm)

VPD (kPa)

Rainfall (mm)

35.2 63.0 73.8 96.8 140.1 166.5 188.2 174.4 117.3 78.9 41.1 34.1

0.45 0.75 0.56 1.09 1.20 1.64 2.02 2.27 1.56 1.14 0.51 0.57

7.0 2.4 55.7 23.3 5.3 1.3 4.3 3.4 46.9 54.4 81.2 3.2

1209

1.15

288

2012 ETo (mm)

VPD (kPa)

46.4 70.3 94.8 121.9 167.8 188.2 195.6 179.8 118.2 72.6 36.8 37.8

0.58 0.71 0.86 1.02 1.63 2.43 2.17 2.71 1.67 0.96 0.39 0.55

1330

1.31

Table 2. Deficit irrigation treatments applied during the experimental period (2009–12), percentage of crop evapotranspiration (ETc) used, water amounts applied in each phenological period and mean total annual applied water for each treatment PRI-1, partial root zone irrigation with 110 mm year–1; PRI-2, partial root zone irrigation with 78 mm year–1; RDI-1, regulated deficit irrigation with 110 mm year–1; RDI-2, regulated deficit irrigation with 78 mm year–1 Treatment

RDI-1 PRI-1 RDI-2 PRI-2 RDI-2 PRI-2 A

Year(s)

2009–12 2009–12 2009–11 2009–11 2012 2012

% ETc

Water applied (mm)

Budburst– fruit set (April– May)

Fruit set– veraison (early June to the end of July

Veraison– harvest (end July to midSeptember)

After harvest (midSeptember to October)

Budburst– fruit set (April– May)

Fruit set– veraison (early June to the end of July

Veraison– harvest (end of July to midSeptember)

After harvest (midSeptember to October)

Mean total annual water applied (mm year–1) (%ETc)A

26 29 19 19 10 10

15 13 0 0 18 20

20 20 24 21 21 21

40 42 42 44 37 42

28 31 20 21 11 11

30 26 0 0 36 40

35 35 42 38 37 38

16 17 17 18 15 17

109 (21%) 110 (21%) 79 (15%) 77 (15%) 99 (19%) 106 (20%)

Average of % of ETc applied every year.

D


mean values of the preceding 10–11 years, using the Penman–Monteith–Food and Agriculture Organisation method (Allen et al. 1998) and the daily climatic data collected in the meteorological station (datalogger model CR10X; Campbell Scientific, Logan, UT, USA) located at the experimental vineyard agrometeorological network belonging to the Servicio de Información Agraria de Murcia (SIAM-IMIDA). The experimental design consisted of four replicates per treatment in a completely randomised block. Each replicate contained 164 vines. Border vines in each row were excluded from the study to eliminate potential edge effects. Irrigation was applied 3–5 times per week, depending on the phenological period, and was controlled automatically. The amount of water applied in each treatment was measured with flow meters (model M170; Elster Iberconta, Basque Country, Spain). Water was applied by one pressure-compensated emitter per plant (4 L h–1) with one drip-irrigation line per row for the conventional drip irrigation in RDI and on a double line per row for the PRI. All treatments received the same annual amount of fertiliser (30 kg N, 20 kg P, 30 kg K and 16 kg Mg per ha, and 1.6 g Fe chelate per vine), which was supplied through the irrigation system from April to June. In the PRI layout, the two pipelines were joined on both sides of the trunk and placed underneath each vine row. In each pipeline in the PRI treatments, there were alternate zones with and without emitters to create dry and wet root zones within each vine row. In the PRI treatments, water was supplied to only one side of the root system at a time, alternating every 14–16 days. In the RDI treatments, irrigation water was supplied simultaneously to the entire root system. Each year, the PRI treatments were applied throughout the growing season (early April to the end of October). To apply the same amount of water in PRI and RDI, the irrigation times were doubled in the PRI-1 and PRI-2 treatments compared with RDI-1 and RDI-2, respectively. Root growth analysis Minirhizotron tubes were installed in the winter of 2010, 15 cm perpendicularly from the emitter (located 75 cm from the trunk) in one representative vine per plot, in three out of the four replicate plots of each treatment (one on each side for the PRI treatments). The tubes were made of transparent Plexiglas (Plexiglas, Lousville, KY, USA) and were 1 m long, with inside and outside diameters of 64 mm and 69.7 mm, respectively; each tube was sealed to prevent light leaks. The tubes were installed vertically. The total length of each buried tube was 60 cm. Although soil depth probably varied within the vineyard, the tubes could not be installed deeper than 60 cm because of a very compact and hard soil layer below this depth. The part of the tube protruding from the soil surface was covered with a black plastic sheet to prevent light from entering the tube. On 5 August 2011, after more than 3 years of maintaining the irrigation treatments, images of roots every 20 cm from the soil surface were captured. Each image (21.59 cm 19.56 cm) was captured using a CI-600 root growth scanning system (CID Bioscience Inc., Camas, WA, USA), which consisted of a rotating, linear scan head and a laptop computer. Linear and nondistorted scanned colour images of the same profile captured by the system were grouped by specific software to overlap

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panoramas (Hugin ver. 2010.2.0.3bd31edd2dca, built by Aron Helser; http://hugin.sourceforge.net/). The resulting image was cropped to obtain a normalised image size (19 cm 60 cm) in all treatments, neglecting areas with distortions produced during the scanning process. Then, the image were analysed with WinRHIZO Tron MF software (Regent, Quebec City, QC, Canada), a manual root measurement program that allows image analysis from the minirhizotron underground scanner system, which does not always offer a good contrast between roots and their background. With this program, roots were traced manually, obtaining the position, length and initial and final diameter for each root segment. For each depth (0–25 cm and 25–50 cm) and for each selected diameter class (2 mm), these data were used to calculate, the length, area and volume of roots, considering each segment as a cylinder with the diameter averaged between the initial and final diameter of the segment considered. In this study, the length, area and volume of roots were expressed as the total of each of these parameters per unit of volume of soil sampled. Density parameters such as: root length density and root volume density were calculated, whose numerical values were expressed by the generic equation (Eqn 1) based on the expression proposed by Johnson et al. (2001) in similar studies: DXR ¼

X ; A DF

ð1Þ

where DXR is the root density considered for parameter X, X is the parameter such as the length (cm) or volume (cm3) of the roots calculated under observation with the program WinRHIZO Tron MF, A is the area of observation and DF is the depth of field of the scanner within the minirhizotron (0.65 cm). To study the development of the entire root system in more detail, between four and nine representative vines per treatment were completely excavated from the soil at the end of the experiment (December 2012). Two trenches in the soil (4–5 m length 0.5 m width 1.5 m depth) were dug on both sides of a row of 3–4 vines (in two rows per treatment and in different plots) at 50 cm from the vine trunk. To maximise root extraction while minimising damage, vine rows were washed with pressurised water to remove some of the surface soil and the trenches were completely filled with water and left for 3 or 4 days. Then vines were taken from the soil as carefully as possible with the aim of keeping the root system intact. The root system of each vine was separated from aboveground material and immediately washed in situ to remove the soil before the roots were labelled, photographed and weighed. The aboveground accumulated biomass (trunk and cordons) of each vine was also weighed. In the laboratory, photographs of the root system of each vine were corrected using Photoshop ver. CS6 (Adobe Systems Software, Ireland). From these images, estimates of the total length and volume of the roots of each vine were calculated using the program WinRHIZO Tron MF, according to the procedure described above. Soil water content, vine water status, gas exchange and ABA signalling The volumetric soil water content (qv) was measured 2–3 times per week during the experiment with a Diviner 2000 portable soil moisture probe (Sentek Pty Ltd, Stepney, SA, Australia).


Polyvinyl chloride access tubes were installed to a depth of 60–70 cm and readings were taken close to the vines, 10–15 cm from the drip head and oriented perpendicularly to the drip lines, at depths of 10–60 cm (maximum depth) for four replicates per treatment (one per plot). The scaled frequency (SF) values were converted to qv using a capacitance probe calibration equation (qv = 47.38 SF3.12, r2 = 0.93) for clay soil (of similar texture to our vineyard soil), as proposed previously (Rose et al. 2001). Root water uptake rate (Dqv Dt–1) was estimated at each depth as the change in qv (with time) between two irrigation events. In 2009, qv was also monitored using a C-Probe FDR capacitance probes (C-Probe Corporation, Agrilink, Adelaide, SA, Australia) with wireless radiotelemetry (Adcon Telemetry, Klosterneuburg, Austria) and internet-based graphing software. Polyvinyl chloride access tubes were installed for each probe in one (RDI) or both (PRI) parts of the root zone, in one representative vine per treatment, 10–15 cm from the drip head and oriented perpendicularly to the drip lines, with sensors at depths of 10, 30 and 60 cm. Readings of qv were taken every 15 min. Each year, the stem water potential (Ys) was determined weekly from the beginning of vegetative growth until leaf fall. Eight healthy, fully exposed and expanded mature leaves from the main shoots in the middle and upper part of the vine canopy were taken per treatment (two leaves per plot). The leaves were enclosed in aluminium foil and covered with plastic at least 2 h before the midday measurement. The Ys was measured at noon (1200–1300 hours) using a pressure chamber (Model 600; PMS Instrument Co., Albany, OR, USA). During some important periods (from fruit set to veraison and from veraison to harvest), Ys was measured more frequently (twice a week: at the beginning and end of the week, and before and after an irrigation event). Gas exchange was measured between 0900 hours and 1030 hours every 7–14 days from May to October in 2009, 2010 and 2012, and also at midday (1200–1300 hours) in 2009 on selected clear days. Measurements were made on healthy, fully expanded mature leaves exposed to the sun (one leaf on each of 8 or 12 vines per treatment) and from main shoots located on the exterior canopy. Leaf photosynthesis rates (A) at early morning and at midday (Amd), stomatal conductance (gs) at early morning and at midday (gsmd) and the transpiration rate (E) at early morning and at midday (Emd) were measured with a portable photosynthesis measurement system (LI-6400, Li-Cor, Lincoln, NE, USA) equipped with a broadleaf chamber (6.0 cm2). During measurements, the leaf chamber temperature was maintained in an optimum range between 25C and 32C, the leaf-to-air vapour pressure deficit at 2.05 kPa and the relative humidity at 30–50%. Molar air flow rate inside the leaf chamber was 400 mmol mol–1. All measurements were taken at a reference CO2 concentration similar to ambient (380 mmol mol–1) and at a saturating PPFD of 1500 mmol m–2 s–1. This was done by using a red–blue light source (6400–02B LED; Li-Cor) attached to the leaf chamber. On specific days in 2009, 2 days after starting a new PRI cycle (26 August), in the middle of the PRI cycle (2 September) and at the end of the PRI cycle (8 September), xylem sap from the leaves (four leaves per treatment) was collected in the early


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morning (0800–0930 hours) and at midday (1200–1330 hours) by applying an overpressure between 0.3 MPa and 0.5 MPa (using N) for 1–4 min and then lightly touching the cut petiole with a glass capillary tube. Leaf water potential (Yl) was measured in the same leaves and gs was measured in similar leaves as close as possible to the leaves taken for xylem sap collection. The sap was immediately transferred to an Eppendorf tube, frozen in liquid nitrogen and then stored at –20C before ABA measurement by radioimmunoassay (Quarrie et al. 1988), using the monoclonal antibody AFRC MAC 252. Isotope composition At the end of July in 2009 and 2010 dry, powdered leaf samples were packed in tin capsules and analysed by isotope ratio mass spectrometry (Continuous Flow Isotope Ratio Mass Spectrometer, Micromass Isoprime, EuroVector SpA, Milan, Italy), according to del Amor and Cuadra-Crespo (2011). The N content was expressed as a percentage of dry matter. The 13 C : 12C ratios were expressed in d notation determined by Eqn 2 (Farquhar et al. 1989): 13 13 C C 12 Cstandard 12 C sample 13 ; ð2Þ d Cð‰Þ ¼ 13 C 1000 12 C standard where ‘sample’ refers to plant material and ‘standard’ refers to Vienna Pee Dee Belemnite calcium carbonate. The same d notation was used for the 15N : 14N ratio expression (d 15N) but with ‘standard’ referring to the air. Isotope secondary standards of known 13C : 12C and 15N : 14N ratios were used for calibration (United States Geological Survey (USGS)-24 and urea, respectively). Trunk growth and trunk diameter fluctuations Trunk diameter fluctuations were continuously measured in 2009 with Plantsens dendrometers (Verdtech SA, Huelva, Spain) on two representative vines per treatment. Vines with similar trunk size were chosen. The dead outer tissues of the bark were removed before installation, allowing the contact point of the sensor to rest directly on the living tissues of the bark (Fernández and Cuevas 2010). A sensor was fixed to the main trunk of each vine with a metal frame of Invar, located ~30 cm from the ground. Before installation, the sensors were calibrated individually with a voltmeter (Verdtech SA). The typical output was 0–2.45 V, representing 0 mm and 6100 mm, respectively. The trunk diameter measurement resolution was 5 mm. Measurements were recorded automatically every 15 min with a data logger (Addits, Adcon Telemetry), wireless radiotelemetry (Adcon Telemetry) and internet-based graphing software (Verde Smart Corporation-Verdtech Nuevo Campo S.A, Madrid, Spain; http://www.verdtech.es). For each vine, the stem cycle was divided into distinct phases according to Deslauriers et al. (2003, 2007). Phase 1 was a contraction phase, the period between the morning stem radius maximum and the afternoon minimum. The maximum diurnal trunk shrinkage (MDTS) was calculated as the difference between these two values. Phase 2 was an expansion phase, the total period from the stem radius minimum to the next morning’s maximum. Both overnight or afternoon recovery, and stem increment

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were considered during the expansion phase (Deslauriers et al. 2007). The ‘recovery phase’ was defined as the portion of the cycle during which the stem radius increased from the afternoon minimum until it reached the value of the maximum of the previous morning; the ‘stem increment phase’ was defined as the period from the time when the stem radius exceeded the previous morning’s maximum until the subsequent maximum, during which the stem radius continued to increase until the beginning of the shrinkage phase of the next diurnal cycle. The difference between the expansion maximum and the onset of the stem radius increment phase represented the positive stem radius change estimate. When the previous cycle maximum was not reached, a negative stem radius change was calculated. The duration (in h) of each phase was calculated. The differences in the daily maximum trunk diameter values were also calculated. Furthermore, in 2009, the trunk circumference of 16 vines per treatment (four per plot) was measured with a tape measure from the end of March to the end of May (the active vegetative

growth period). Two trunk measurements per vine were taken 20–30 cm above the soil surface, at a previously marked location on the trunk. The transverse cross-sectional areas were calculated according to Mitchell and Chalmers (1982) and the absolute trunk growth rates during this active vegetative growth period were estimated. Vine vigour and leaf area Total main shoot length and lateral shoot length were measured with a tape measure for one main shoot (east-oriented) per vine and for a previously marked lateral shoot on the main shoot. Both measurements were made in 12–16 vines per treatment (three to four vines per plot, depending on the year) and measured weekly from early April to mid-July. The number of main shoots per vine was measured each year at the end of June for 13 vines per plot. The number of lateral shoots per main shoot was also measured every year, on the same main shoot utilised for shoot length measurements. During the winters of this 4-year

Table 3. Mean values of volumetric soil water content (qv, in %) in the highest fine root density zones (0–30 cm) and in the deeper soil layers (40–60 cm), and the cumulative soil water content available (%) in the entire measurable soil profile (stock 0–60 cm) for each treatment in three representative periods of 2009 PRI-1, partial root zone irrigation with 110 mm year–1; RDI-1, regulated deficit irrigation with 110 mm year–1; PRI-2, partial root zone irrigation with 78 mm year–1; RDI-2, regulated deficit irrigation with 78 mm year–1; qv-wet, qv on the irrigated side of partial root zone irrigation (PRI) treatments; qv-dry, qv on the dry side of PRI treatment; *, P < 0.05; **, P < 0.01; ***, P < 0.001. For each column values followed by different letters are significant different according to Duncan’s multiple range test at the 95% confidence level Budburst–fruit set (April–early June) qv qv qv (0–30 cm) (40–60 cm) (0–60 cm) PRI-1 (qv-wet) PRI-1 (qv-dry) PRI-1 (average qv) RDI-1 PRI-2 (qv-wet) PRI-2 (qv-dry) PRI-2 (average qv) RDI-2 ANOVA

30.2a 25.5b 27.8ab 30.1a 28.1ab 26.1b 27.0ab 27.4ab *

39.7a 39.4a 39.5a 32.7d 35.4bc 35.3cd 35.2cd 37.9ab ***

209.7ab 217.8a 202.2bc 190.9cd 190.5cd 184.2d 186.7d 195.9cd ***

Fruit set–veraison (early June to the end of July) qv qv qv (0–30 cm) (40–60 cm) (0–60 cm) 24.5a 15.6d 20.1c 22.6b 16.2d 9.5f 13.0e 11.8e ***

33.2a 32.6a 32.9a 27.0c 28.7b 29.6b 29.0b 30.4b ***

173.1a 144.4c 158.8b 150.7c 134.7d 117.4f 125.9e 126.6e ***

Veraison–harvest (end of July to mid-September) qv qv qv (0–30 cm) (40–60 cm) (0–60 cm) 28.5a 16.1c 21.9b 22.8b 26.1ab 16.0c 21.1b 22.7b ***

35.8a 32.3bc 33.6b 26.6e 32.6bc 29.6d 31.1cd 34.2ab ***

192.9a 145.2ef 166.6c 150.8de 176.3b 137.0f 156.7d 170.7bc ***

Table 4. Mean values and average of fine root length density and root volume density of distinct root diameters (2 mm) at different depths for each treatment on 5 August 2011 PRI-1, partial root zone irrigation with 110 mm year–1; RDI-1, regulated deficit irrigation with 110 mm year–1; PRI-2, partial root zone irrigation with 78 mm year–1; RDI-2, regulated deficit irrigation with 78 mm year–1; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Separation was by Duncan’s multiple range test at the 95% confidence level. For each column values followed by different letters are significant different according to Duncan’s multiple range test at the 95% confidence level Root length density (cm cm–3)

Treatment 2 mm

1–2 mm

All diameters

0–30 cm

30–50 cm

Average (0–50 cm)

0–30 cm

30–50 cm

Average (0–50 cm)

0–30 cm

30–50 cm

Average (0–50 cm)

Average (0–50 cm)

0.49 0.53 0.75 0.95 ns

1.24ab 0.95a 0.94a 1.69b *

0.79a 0.70a 0.82a 1.25b **

0.027a 0.008a 0.036a 0.077b **

0.199bc 0.096a 0.157ab 0.254c *

0.096b 0.043a 0.084b 0.148c **

0.0042ab 0.0000a 0.0003a 0.0103b *

0.0163a 0.0057a 0.0034a 0.0336b **

0.0090a 0.0023a 0.0016a 0.0196b **

0.0036b 0.0021a 0.0029ab 0.0064c ***



study, the pruning weight was also measured from 52 vines per treatment (including the same vines in which shoot measurements were taken). The shoot number was also recorded and the shoot FW was calculated. A representative sample of the winter pruning (main and lateral shoots) was taken, and the FW and DW (dried at 65C to a constant weight) were calculated. The dry biomass and water content of the shoots were estimated. The total leaf area per vine, estimated every year at the end of June (the maximum vegetative growth period) using a nondestructive method, showed a significant relationship with the main shoot length. In the previous years, 2007 and 2008, the length and total leaf area of randomly selected main shoots (80 shoots) were measured destructively twice (on 15 May and 25 June) using a tape measure and a leaf area meter (LI-3000, Li-Cor), respectively. A significant polynomial regression equation related main shoot length (L) to main shoot total leaf area, where Arealeaf = –4045 + 96 L – 0.1993 L2 (r = 0.94, P < 0.0001). Four representative main shoots per vine from 16 vines per treatment were chosen for the main shoot length and leaf area measurements. Leaf area per vine was estimated by multiplying the average shoot leaf area by the number of shoots on the vine.

PRI-1

G

Fresh and dry matter accumulation In July 2009, two representative main shoots were taken in eight vines per treatment. All leaves from the main shoots (including those from the lateral shoots) were collected, and the FW and DW (dried at 65C to a constant weight) were determined. The FW and DW of the main and lateral shoots (without leaves) were also measured and shoot water content was calculated. The number of main shoots per vine was also counted. The FW, DW and water content of the leaves, main shoots and lateral shoots per vine were estimated. In addition, on 7 September 2009 (harvest day), two bunches of grape berries from five vines per treatment were taken, and the FW, DW and water content were obtained. The total yield of these vines was also calculated. Thus the FW, DW and water content of the fruits per vine were estimated. Statistical analysis The data were analysed using 1- and 2- way ANOVA procedures to discriminate irrigation volume and placement effects, and the means were separated by Duncan’s multiple range test, using Statgraphics Plus ver. 2.0 and ver. 5.1 software (Statistical Graphics Corp., Herndon, VA, USA). Linear regressions were fitted using SigmaPlot 2000 (Systat, Richmond, CA, USA).

RDI-1

PRI-2

RDI-2

FW (kg vine–1) Root system:

Trunk and cordons: Total:

0.94b

1.17ab

1.26a

ns

3.06

2.55

3.21

ns

4.00

3.72

4.47

1.33a*

3.41

4.74

RL (cm vine–1):

3725b*

1979a

3078b

2785ab

3

1851b***

732a

1390b

1782b

–1

RV (cm vine ):

Fig. 1. Images of representative grapevines from each treatment showing root system development, trunk and cordons after 4 years of applying irrigation treatments (December 2012), and the parameters derived from analysis of the images. PRI-1, partial root zone irrigation with 110 mm year–1; RDI-, regulated deficit irrigation with 110 mm year–1; PRI-2, partial root zone irrigation with 78 mm year–1; RDI-2, regulated deficit irrigation with 78 mm year–1; RL, total root length; RV, total root volume; ns, not significant; *, P < 0.05; ***, P < 0.001. Separation was by Duncan’s multiple range test at the 95% confidence level. Each mean value is the average of four vines in PRI-1 and RDI-1, and nine vines in PRI-2 and RDI-2.


Results Seasonal patterns of soil water content Significant spatial and temporal variation in qv occurred throughout most of the growing season in the PRI treatments, showing a heterogeneous distribution of soil moisture, and pronounced wetting and drying cycles in the upper soil profiles (0–30 cm); qv at depth (40–60 cm) was relatively constant over time (Table 3). PRI maintained wet (qv-wet) and dried (qv-dry) zones in the root system in the different phenological stages, but more severe water stress periods (from fruit set to harvest) magnified the differences between qv-wet and qv-dry. During the early season (from budburst to fruit set), the qv in the entire soil profile was maintained close to field capacity in all treatments, 30% higher on average compared with the other periods (Table 3). From budburst to fruit set, the qv in the irrigated root zone of PRI-1 vines was similar to that of treatment RDI-1 in the upper soil layer (0–30 cm), but was significantly higher in the deeper part of the soil profile (40–60 cm) (Table 3), indicating deeper water percolation through the soil under PRI-1 vines. In the early season and during the fruit set–veraison and veraison–harvest periods, although the average qv (in dry and wet parts) of the upper soil layer (0–30 cm) of PRI-1 vines was generally similar to or even lower than for the RDI-1 vines, it was significantly higher when considering the deeper soil layers (the average qv at 40–60 cm deep), and the cumulative soil water content in the entire measurable soil profile (qv at 0–60 cm), indicating higher total soil water availability in the entire root zone for PRI-1 vines than for RDI-1 vines during the season. In contrast, in these same periods, the average qv (dry and wet) in the upper and deeper parts of the soil and in the entire soil profile (qv at 0–60 cm) of the PRI-2 vines was similar to or even significantly lower than in RDI-2 vines, indicative of lower total soil water availability in the entire root zone (Table 3). Root growth, distribution and water uptake The higher qv in the deeper soil layers led to most of the fine roots in all irrigation treatments being located in the deeper zone (25–50 cm depth), the greater proportion of the roots being less than 1 mm in diameter (Table 4). The total volume of the roots per unit of soil volume was around 41% higher in PRI-1 than in RDI-1 vines, due to significantly higher root length density for roots with a diameter between 1 and 2 mm, especially at depth (25–50 cm) (Table 4). Furthermore, the average root volume density in the 0–50 cm soil profile, including all diameters, was also significantly higher for PRI-1 vines than for RDI-1 vines. In contrast, the PRI-2 vines showed significantly lower fine root length and volume density than RDI-2 vines throughout the entire soil profile (Table 4). Furthermore, after 4 years of the irrigation treatments, the PRI-1 vines showed greater root system development and a greater volume of soil explored by the roots, with significantly higher total root FW, length and volume than RDI-1 vines (Fig. 1). However, the PRI-2 and RDI-2 vines did not show significant differences in root system development. Total fresh biomass accumulation (below- and aboveground: roots, trunk and cordons) was 0.74 kg greater in PRI-1 vines than in RDI-1 vines, and 0.75 kg greater in RDI-2 compared with PRI-2

P. Romero et al.

vines, but no significant differences were found in this parameter due to the high variability (Fig. 1). During the early season period (no water stress, irrigated), the total root water uptake in the wet root zone (0–60 cm) (Dqv Dt–1) was significantly higher in PRI-1 vines than in RDI-1 (Fig. 2a). Thus the total Dqv Dt–1 (adding the two root zones) in the entire soil profile (0–60 cm) was higher in PRI-1 than in RDI-1 and the rest of the treatments in this period. However, there were no significant differences in Dqv Dt–1 in the dry and wet root zones between RDI-2 and PRI-2. In contrast, during the water deficit before verasion, Dqv Dt–1, in the wet root zone was similar in PRI-1 and RDI-1, but it was significantly lower in the PRI-1 dry root zone compared with RDI-1 vines (Fig. 2b). Thus, there were no significant differences in total Dqv Dt–1 between PRI-1 and RDI-1, although the values were significantly higher than for RDI-2 and PRI-2 (Fig. 2b). Furthermore, the PRI-2 and RDI-2 vines (not irrigated in this period) showed a similar and very-low total Dqv Dt–1 in the entire soil profile.

(a) 16

Early season period (Budburst-Fruit set)

Total v t–1: 14 (two root zones)

Total root water uptake v t–1 (0–60 cm) (mm day–1)

H

a***

12 10

ns

8

dry

b

b

6

dry wet

b

wet

4 2 0 16 14

a**

b

ab

wet

wet

wet

(b) Total v t–1: (two root zones)

10

a Pre-veraison period (Fruit set -veraison)

a***

12 b dry

b wet

*** a*** wet

8 6 b

4

a*** wet

2 0 PRI-1

b

a wet

RDI-1

b dry b dry

b dry

PRI-2

RDI-2

b dry

Fig. 2. Total root water uptake rate (Dqv Dt–1) in the wet and dry root zones of grapevine in the measurable soil profile (0–60 cm) for each treatment during different periods in 2009. (a) Early season: budburst to fruit set; (b) period before verasion (fruit set to veraison). Each bar represents the mean of four vines per treatment, with s.e. not indicated for clarity. For each root zone (dry and wet), different letters within different bars indicate significant differences among irrigation treatments. Different letters on the top of the bars represent significant differences in the total Dqv Dt–1 (dry + wet root zones) among irrigation treatments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Separation was by Duncan’s multiple range test at the 95% confidence level.



I

Table 5. Trunk growth parameters derived from dendrometer measurements for each treatment during two active vegetative growth periods in 2009. Trunk cross-sectional area absolute growth rate (AGR) for each treatment was measured during the budburst–fruit set period in 2009 PRI-1, partial root zone irrigation with 110 mm year–1; RDI-1, regulated deficit irrigation with 110 mm year–1; PRI-2 partial root zone irrigation with 78 mm year–1; RDI-2, regulated deficit irrigation with 78 mm year–1; MDTS, maximum diurnal trunk shrinkage; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. Separation was by Duncan’s multiple range test at the 95% confidence level. For each column values followed by different letters are significant different according to Duncan’s multiple range test at the 95% confidence level 30 April–2 June 2009 (active vegetative growth period) (budburst–fruit set)

PRI-1 RDI-1 PRI-2 RDI-2 ANOVA Irrigation placement Irrigation volume Volume placement

Contraction phase

Expansion phase

Trunk radial growth phase

Contraction rate (mm h–1)

Expansion rate (mm h–1)

Growth rateA (mm day–1)

Rehydration rate (mm h–1)

Trunk radial growth rate (mm h–1)

9.44a 7.19b 8.19a 6.69b * * ns ns

8.16a 6.64b 7.79a 5.29c * ** ns ns

74.70a 52.49b 49.29b 51.70b * ns * *

10.70a 6.84b 5.87b 6.52b * ns * *

11.95a 8.38ab 6.66b 4.78b ** * ** ns

26 March–27 May 2009

1–16 June 2009 (fruit set–veraison)

Trunk cross-sectional area growthB

Contraction phase

Expansion phase

MDTS (mm)

AGR (mm2 day–1)

Contraction rate (mm h–1)

Expansion rate (mm h–1)

89.38a 69.05b 61.93b 51.20b *** * *** ns

7.02a 5.50b 5.37b 5.36b * ns * ns

9.56a 8.66a 7.27b 9.34a * ns ns *

7.67 7.23 6.81 6.30 ns ns ns ns

Expansion phase (13 May–2 June)

A

Calculated by data of dendrometers. Calculated by data of trunk circumference treatment measured with a tape measure.

B

Table 6. Statistical analysis of mean values of midday stem water potential (Ys) and gas exchange parameters for each treatment and phenological period in 2009 A, leaf photosynthesis rate; Amr, leaf photosynthesis rate at early morning; Amd, leaf photosynthesis rate at midday; gs, stomatal conductance; gsmr, stomatal conductance at early morning; gsmd, stomatal conductance at midday; E, transpiration rate; Emr, transpiration rate at early morning; Emd, transpiration rate at midday. Units: Ys, MPa; A, mmol m–2 s–1; E, mmol m–2 s–1; gs, mol m–2 s–1; A/E, mmol mmol–1; A/gs, mmol mol–1; PRI-1, partial root zone irrigation with 110 mm year–1; RDI-1, regulated deficit irrigation with 110 mm year–1; PRI-2, partial root zone irrigation with 78 mm year–1; RDI-2, regulated deficit irrigation with 78 mm year–1; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. For each column values followed by different letters are significant different according to Duncan’s multiple range test at the 95% confidence level Parameter

Ys

Amr

Amd

Emr

Emd

gsmr

gsmd

A/Emr

A/Emd

A/gsmr

A/gsmd

Budburst–fruit set (early April–early June) PRI-1 –0.68 12.95 RDI-1 –0.69 12.97 PRI-2 –0.70 13.24 RDI-2 –0.68 12.64 ANOVA ns ns Irrigation volume ns ns Irrigation placement ns ns Volume placement ns ns

12.14a 11.28b 10.82b 12.06a *** ns ns *

2.67 2.69 2.73 2.54 ns ns ns ns

2.72a 2.22b 2.10b 2.64a *** ns ns *

0.14 0.14 0.14 0.13 ns ns ns ns

0.14a 0.11b 0.10b 0.13a *** ns ns *

5.01 4.93 5.06 5.19 ns ns ns ns

4.48b 5.12a 5.17a 4.64b *** ns ns **

93.19 94.24 96.59 96.82 ns ns ns ns

85.43c 101.49a 104.75a 92.65b *** ns ns *

Before veraison (June–end of July) PRI-1 –1.20a RDI-1 –1.21a PRI-2 –1.44c RDI-2 –1.34b ANOVA ***

5.27a 5.60a 2.60b 3.23b **

2.32a 2.26a 1.84b 1.94b **

1.65a 1.60a 1.12b 1.31ab *

0.11a 0.10a 0.08b 0.09b **

0.06a 0.06a 0.04b 0.05ab *

4.17 4.16 4.23 4.17 ns

3.14a 3.49a 2.31b 2.48b ***

95.54 95.52 100.19 97.20 ns

84.07b 96.33a 65.06c 67.82c ***

5.76 5.07 4.85 5.67 ns

2.59 2.54 2.33 2.44 ns

1.85 1.65 1.61 2.06 ns

0.10 0.10 0.08 0.09 ns

0.06 0.05 0.05 0.06 ns

3.56 3.51 3.55 3.60 ns

3.15 3.08 3.01 2.83 ns

95.53 95.10 99.06 95.57 ns

93.65 95.83 99.40 96.73 ns

9.85a 9.52a 8.02b 8.39b **

After veraison (end of July to mid-September) PRD-1 –1.41a 8.96 RDI-1 –1.36ab 8.72 PRI-2 –1.30b 7.94 RDI-2 –1.34ab 8.29 ANOVA * ns


P. Romero et al.

Variations in seasonal and diurnal trunk growth There were two main peaks of trunk growth during the season (Fig. S1a, b, available as Supplementary Material to this paper), mainly during May (the active vegetative growth period) and after harvest, in September. During the first growth period, cumulative maximum trunk diameter growth was higher in PRI-1 than in RDI-1 and was similar for PRI-2 and RDI-2. PRI-1 and RDI-1 maintained positive (but very low) trunk growth during June and July, compared with the other treatments. In addition, PRI-1 vines showed significantly greater average MDTS during the whole season, relative to the rest of the treatments (Fig. S1c, d). Both PRI treatments gave a significantly higher trunk diameter contraction rate during the day than the RDI treatments (Table 5). The trunk diameter expansion rate was also significantly higher in PRI-1 and PRI-2 compared with RDI-1 and RDI-2, respectively (Table 5). The greater diurnal variations of the trunk diameter in the PRI-1 vines produced significantly higher MDTS relative to the rest of the treatments. In addition, during the stem radius increment phase, the trunk growth rate was significantly higher in PRI-1 compared with the other treatments. This was also supported by the significantly higher growth rate of the trunk cross-sectional area in PRI-1 vines (n = 16) measured during the vegetative growth period (April–May 2009) (Table 5). Furthermore, during the first part

of the fruit set–veraison period (early to mid-June), the daily contraction rate of the trunk was significantly lower in PRI-2 vines than for RDI-2 and the rest of the treatments, but the expansion rate was similar. Studying the trunk expansion phase during an active vegetative growth period in 2009 (13 May–2 June) in more detail, we separated two distinct phases: rehydration and trunk radial growth. Interestingly, the rehydration rate of the trunk during this period was significantly higher in PRI-1 vines than in the vines of the other treatments (Table 5). The trunk radial growth rate during this period was similar in PRI-1 and RDI-1, but was significantly higher than in PRI-2 and RDI-2 (Table 5). There were interactive effects of the irrigation volume (high vs low) and irrigation emplacement (PRI vs RDI) on some trunk growth parameters (Table 5). Seasonal leaf water relations In 2009, during the early season, from budburst to fruit set, the RDI-1 and PRI-1 vines maintained a similar midday Ys and early to mid-morning (0900–1030 hours) gas exchange, but the gas exchange rates measured at midday (1230–1330 hours) were significantly higher in PRI-1 vines than in RDI-1 vines (Table 6). In addition, A/Emd and A/gsmd in this period, and A/gsmd before veraison (from fruit set to veraison in 2009) were significantly higher in RDI-1 than in PRI-1 vines. In general, this

14

0.20 veraison

(a) 12

a*

ns ns

***

ab bc c

10

***

8

(b)

PRI-1 RDI-1 PRI-2 RDI-2

veraison

J

0.15

a* ***

ns

ns

ns

b

6

ns

early morning 14

(c) 12

*** a

10

Recovery period

early morning 0.00

(d )

a***

0.15

a**

*

*** ns

6

Severe water stress

veraison

Recovery period

gs (mol m–2 s–1)

0.05

Severe water stress

veraison

A (mol m–2 s–1)

4

8

ab ab

***

0.10

2


a ab b b

***

4

b b

0.10

**

**

c 0.05

ns ***

2

b b b

b b

mid-day

mid-day

0.00

0 7/20

7/27

8/3

8/10

8/17

8/24

8/31

**

c

7/20

7/27

8/3

8/10

8/17

8/24

8/31

Fig. 3. Average (a, c) photosynthesis (A) and (b, d) stomatal conductance (gs) of grapevine at (a, b) early morning and (c, d) midday during the recovery period in August 2009. Each value is the average of eight measurements per treatment. Dashed lines represent the veraison period when recovery irrigation was applied. For each date, symbols with different letters indicate significant differences among irrigation treatments. ns, not significant; *, P < 0.05, **, P < 0.01; ***, P < 0.001. Separation was by Duncan’s multiple range test at the 95% confidence level.


K

Post-veraison period (4–8 August 2009) –0.6

Mid-mornings (MPa)

–0.7

(a)

*

ns

*

*

–0.8 –0.9 –1.0 –1.1 –1.2

ns

–1.3

RDI-1 PRI-1

*

–1.4

Trunk diameter fluctuations (m)

4900 4800

MDTS (m day–1) NI I

(b)

RDI-1

4700

RDI-1 33.8* 47.3*

4600 4500 4400 4300

PRI-1 97.0 79.8

PRI-1

4200 4100 4000 3900 3800

Volumetric soil water content (v%)

difference in stomatal behaviour between PRI-1 and RDI-1 was not maintained consistently in other periods before and after veraison in 2009, 2010 and 2012, when the volume of water applied had a greater effect on seasonal leaf water relations than the irrigation system per se (Table 6 and Tables S1 and S2). In contrast, during the early season in 2009, the PRI-2 vines maintained significantly lower gas exchange rates and higher A/Emd and A/gsmd at midday than the RDI-2 vines. In this period, there were also interactive effects of the irrigation volume and irrigation emplacement on the gas exchange parameters (Table 6). In addition, the PRI-2 vines had midday Ys values before veraison in 2009, and before and after veraison in 2010 that were significantly lower (more negative) than in RDI-2 vines (Table S1). Cutting the irrigation completely before veraison (as applied to PRI-2 and RDI-2 vines) significantly reduced the gas exchange levels and gas exchange efficiency at early morning and midday, compared with PRI-1 and RDI-1 vines (Table 6). Furthermore, analysing this period without irrigation, the RDI-2 vines extracted more water from the soil, as indicated by a higher total Dqv Dt–1 (0.50 mm day–1 in RDI-2 vs 0.43 mm day–1 in PRI-2; Fig. S2a, b), and maintained better vine water status than PRI-2 vines (Table 6, Table S1 and Fig. S2). Furthermore, in 2009, although the PRI-2 and RDI-2 vines were watered with the same volume of water, during the irrigation recovery period (after veraison), the PRI-2 vines showed a slower recovery of A and gs at midday than the RDI-2 vines (Fig. 3c, d). After 20 days of irrigation recovery (14 August), the RDI-2 vines had significantly higher Amd and gsmd values than the PRI-2 vines and even the rest of the treatments. However, this delay of gas exchange recovery in PRI-2 was not observed during early morning (Fig. 3a, b). In 2012, when irrigation was applied before veraison in RDI-2 and PRI-2, there were no significant differences in vine water status or gas exchange between the RDI and PRI vines, which received the same amount of water (Table S2). In general, the watered vines (RDI-1 and PRI-1) showed better water status and gas exchange, especially before veraison, than the less watered vines (especially PRI-2, which showed the lowest gas exchange rates of all the treatments; Table S2). The evolution of seasonal midday Ys for the period 2009–2011, measured before and after night irrigation, showed greater differences in Ys between the PRI and RDI treatments when Ys was measured the day after a night-time irrigation event than when Ys was measured the day before an event (Fig. S3). In general, the PRI-1 vines had seasonal midday Ys values similar to those of the RDI-1 vines, although Ys on the day following a night irrigation event was slightly lower (but not significantly so) in PRI-1 vines. Interestingly, the PRI-2 vines had significantly lower average midday Ys than the RDI-2 vines, regardless of the timing of irrigation, although the differences in Ys were accentuated following night irrigation (Fig. S3). To explain this behaviour, we analysed the daily evolution of Ys, MDTS, qv and Dqv Dt–1 during 1 week in 2009. There were greater changes in Ys after irrigation events in RDI-1 vines than in PRI-1 vines (Fig. 4). The RDI-1 vines had lower Ys, MDTS and Dqv Dt–1 (compared with PRI-1) when no night irrigation occurred, and had higher Ys, qv and Dqv Dt–1 (combining the two root zones) when night irrigation did occur (Fig. 4). However, these differences in the diurnal patterns of Ys, trunk diameter fluctuations and Dqv Dt–1 between irrigation


(c)

greater root water uptake in PRI-1

RDI-1 PRI-1wet PRI-1dry

25

20

NI

I

 t–1 (0–30 cm) v

15

PRI-1 3.26ns 5.01* RDI-1 2.91 8.23

Lower root water uptake in RDI-1

10 2

3

4

5

6

7

8

9

August Fig. 4. Evolution of the (a) mid-morning stem water potential (Ys), (b) trunk diameter fluctuations and (c) volumetric soil water content (qv) during 1 week for grapevines under partial root zone irrigation (PRI-1) and regulated deficit irrigation (RDI-1) with 110 mm year–1 in August 2009. Dqv Dt–1 (0–30 cm) represents the average of root water uptake estimated from continuous measurements using C-Probe FDR soil capacitance probes(C-Probe Corporation) at two different depths (10 cm and 30 cm). For differences between treatments: ns, not significant; *, P < 0.05. NI no irrigation event; I, measurement was performed following an irrigation event. Separation was by Duncan’s multiple range test at the 95% confidence level

events were not observed clearly between the less watered treatments PRI-2 and RDI-2 (data not shown). Carbon isotopic discrimination, vine growth and biomass accumulation Interestingly, before veraison period in 2009, the carbon isotopic (13C : 12C) ratio (d 13C) in PRI-1 vines was significantly lower (–24.72‰) than in the other treatments (Fig. 5a, b), which

L


P. Romero et al.

showed similar carbon isotopic compositions of –23.55‰, –22.88‰ and –22.88‰ RDI-1, PRI-2 and RDI-2, respectively. The nitrogen isotopic composition (d 15N) value was higher for PRI-1, RDI-1 and RDI-2, but for PRI-2, it was significantly lower (21.6%) than for PRI-1. However, in 2010, no significant differences were found (Table S1). In addition, the PRI-1 vines had more vigour (expressed as average pruning weight, leaf area and water content in the shoots during the experimental period 2009–12) (Table 7). Significant positive correlations were found between the carbon and nitrogen isotopic composition and biomass accumulation (Fig. 5c, d). Furthermore, in 2009, the total dry matter content (especially in main shoots), fresh matter content (in main and lateral shoots and fruits) and water content (in main and lateral shoots and fruits) were also increased significantly in PRI-1 vines compared with RDI-1 vines and the rest of the treatments (Fig. 6). Interestingly, compared with RDI-2, the PRI-2 vines had lower growth and vigour, expressed as a significant reduction in the number of lateral shoots, the total leaf area (Table 7), the DW and FW (main shoots) and the total water content per vine (particularly in the main shoots and leaves; Fig. 6). ABA signalling The PRI treatments changed significantly the leaf xylem sap ABA concentration ([X-ABA]leaf) measured in the morning at the beginning, middle and end of a typical PRI cycle after veraison in

–20

–26

(a)

a

(c)

p < 0.05 b

–24

b

–22 –20 –18 –16 3.0

–23 r = 0.42*

–24

b

p < 0.05 ab a

2.0

(d )

2.5

ab 2.0

Leaf N (‰)

Leaf N (‰)

–22

–25

(b) 2.5


–21

b

Leaf C (‰)

Leaf C (‰)

2009 (Table 8). At the beginning of the PRI cycle, in the early morning (and, on average, during the whole morning), the PRI-1 vines had a [X-ABA]leaf that was significantly higher than that for RDI-1 and the other treatments, but maintained similar gs and average qv of the dry and wet parts in the entire soil profile (10–60 cm). The total average qv of the dry and wet parts in the entire soil profile (10–60 cm) was correlated significantly with [X-ABA]leaf following rewatering for the PRI-1 and RDI1 vines (Fig. 7a). In addition qv (10–30 cm) was also significantly correlated with [X-ABA]leaf at the end of a PRI cycle for PRI-2 and RDI-2 vines (Fig. 7c). Significant relationships were also found between qv in the dry side and [X-ABA]leaf for PRI-1 and PRI-2 vines (Fig. 7b, d). Furthermore, in PRI-1, the greater qv heterogeneity between the dry and wet root zones during the development of the PRI cycle did not significantly alter the ABA signalling or stomatal behaviour in the middle or at the end of the PRI cycles, compared with RDI-1 (Table 8). In contrast, at the end of the PRI cycle, in the early morning, the PRI-2 vines had the lowest [X-ABA]leaf and significantly lower gs than RDI-2 and the rest of the treatments. This effect was also associated with greater qv heterogeneity between the dry and wet root zones at the end of the PRI cycle (compared with RDI-2), and with the minimum qv reached in the dry root zone in PRI-2 (11% in the upper 0–30 cm and 20% at 0–60 cm). Similar stomatal behaviour was observed in the PRI-2 vines (compared with RDI-2 vines and the other treatments) midway through the PRI cycle in the early morning, without significant changes in [X-ABA]leaf (Table 8).

1.5 1.0

r = 0.54*

1.5 1.0 0.5

0.5 0.0

0.0

PRI-1

RDI-1

PRI-2

RDI-2

0

200 400 600 800 1000 1200 1400 1600 1800

Dry matter weight (g vine–1) Fig. 5. (a, b) Leaf C and N isotopic composition for each treatment at veraison in 2009, and (c, d) relationships between the C and N isotopic discrimination and dry matter accumulation in Monastrell grapevines in 2009. (a, c) Leaf d 13C; (b, d) Leaf d 15N. Each single point is the average of four measurements per treatment (one per plot). Linear regressions were fitted to the data. *, P < 0.05.



M

Table 7. Mean values of vine vigour parameters of Monastrell grapevines for each treatment and year, and the average during the experimental period 2009–2012 PRI-1, partial root zone irrigation with 110 mm year–1; RDI-1, regulated deficit irrigation with 110 mm year–1; PRI-2, partial root zone irrigation with 78 mm year–1; RDI-2, regulated deficit irrigation with 78 mm year–1; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Separation was by Duncan’s multiple range test at the 95% confidence level Parameter

Year

PRI-1

Final main shoot length (cm)

2009 2010 2011 2012 Mean

135a 95a 115a 77a 114a

Number of main shoots

2009 2010 2011 2012

13 12 12 11

Final lateral shoot length (cm)

2009 2010 2011 2012

17.93 6.73 26.75 6.84ab

Number of lateral shoots per main shoot

2009 2010 2012

12 10a 7a

Total leaf area (m2 per vine)

2009 2010 2011 2012 Mean

7.04a 3.89a 5.18a 2.63 5.28a

6.18ab 3.39a 4.31ab 2.25 4.60ab

Pruning weight (kg per vine)

2009 2010 2011 2012 Mean

0.73 0.52a 0.51a 0.26a 0.58a

FW of main shoot (g per shoot)

2009 2010 2011 2012 Mean

57.05 44.02a 44.45a 22.47a 48.33a

Dry biomass of shoots (g per vine)

2009 2010 2011 2012 Mean

329 285a 295a 120a 303a

Water content of the shoots (g per vine)

2009 2010 2011 2012 Mean

394 231 211a 140a 278a

Discussion Root growth, water uptake, water status, gas exchange and ABA signalling PRI-1 vines had more water available in the entire soil profile than RDI-1 vines (Table 3), especially at depth (40–60 cm), which stimulated more fine root growth, and a larger and more extensive root system that allowed greater soil exploration and water capture (Table 4, Fig. 1). The invigorating and

RDI-1

PRI-2

RDI-2

ANOVA

118b 76b 98b 65bc 97b

128ab 82ab 114ab 61c 109ab

* * * ** *

13 12 11 11

13 12 12 11

ns ns ns ns

15.90 6.06 20.75 5.91bc

17.13 6.46 24.88 5.38c

ns ns ns ***

12 10a 6b

ns * *

5.82b 2.42b 3.79b 1.93 4.00b

6.31ab 3.01ab 4.97a 2.18 5.08a

* * * ns *

0.63 0.46ab 0.43ab 0.24ab 0.51b

0.60 0.33b 0.33b 0.20bc 0.42c

0.59 0.33b 0.47a 0.17c 0.46bc

ns ** * *** ***

53.42 39.41ab 36.86ab 20.64ab 43.13ab

47.96 29.94b 30.30b 17.13bc 35.33c

49.52 29.66b 39.57ab 15.02c 39.54bc

ns * * ** **

270 255ab 265ab 113ab 263ab

257 180b 193b 94bc 210c

269 187b 264ab 79c 240bc

ns * * ** ***

339 205 166ab 124ab 237b

311 148 142b 103bc 200b

320 141 203a 90c 221b

ns ** * *** ***

127ab 87ab 104ab 73ab 104ab 13 12 11 12 17.10 6.10 22.50 7.44a 12 10ab 7ab

11 8b 6b

drought-tolerant rootstock used in this study, 1103P, can exploit available deep water more efficiently than other rootstocks (Alsina et al. 2011). Similarly, PRI increased root biomass in controlled environments (Mingo et al. 2004; Shao et al. 2008; Kaman et al. 2011) and root growth into wet soils and into deeper soil layers in field conditions, relative to conventional deficit irrigation (Dry et al. 2000a, 2000b; Gu et al. 2004; dos Santos et al. 2007; Abrisqueta et al. 2008).

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P. Romero et al.

1800

Dry matter weight (g vine–1)

(a) 1600 1400

Cumulative:

1000

b

b

b

a*

ab

b

b

800 ns

600 400

0 6000

Fresh matter weight (g vine–1)

a**

1200

ns

200

a*

(b)

a* Cumulative: a*

bc b

a

a

c

b

c

ab

b

bc

ab a

b

5000

bc c

4000

a***

b b

3000

a*

2000

a

b

b

b

b

b

*

a

1000 5000 0

a**

(c) Cumulative:

Water content (g vine–1)

Main shoots Lateral shoots Leaves Fruits

b

ab ab a

a***

4000

b 3000

b c

***

a

b b

b

2000 *

a 1000

0

a

b

*

a

**

b b

b b

PRI-1

RDI-1

PRI-2

a

a ab a RDI-2

Fig. 6. Mean values of the (a) DW, (b) FW and (c) water content of the main and lateral shoots, leaves and fruits in grapevine for each treatment in 2009. For each plant organ (main shoots, lateral shoots, leaves and fruits), different letters within the different bars indicate significant differences among irrigation treatments. Different letters on the tops of the bars indicate significant differences in total plant values. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Separation was by Duncan’s multiple range test at the 95% confidence level.

The induction of fine root growth, and increased root tip number and surface area of fine roots after rewatering in PRI may be involved (Kang and Zhang 2004; Li et al. 2011b). Increased root osmotic potential and a greater free polyamine production in PRI roots have been related to increased root growth and density (Du Toit 2005). During periods not under deficit irrigation, PRI-1 vines maintained significantly greater total root water uptake (Dqv Dt–1) in the wet root zone compared with RDI-1 vines

(Fig. 2a), allowing greater water extraction from the entire soil profile than for RDI-1 vines (Fig. 2a). However, the smaller irrigation volumes during deficit-irrigated periods (e.g. from fruit set to veraison) decreased the qv availability in the wet root zone and decreased total Dqv Dt–1 of PRI-1 plants compared with RDI-1 plants (Fig. 2b). Thus the root system of Monastrell grapevines can compensate for the decreased water availability on the non-irrigated side of the vine during PRI by increasing root water uptake on the irrigated side during periods when the entire wet root zone had a high soil water content (qv-wet >30% at 10–60 cm). The PRI-1 and RDI-1 vines suffered similar degrees of water stress (as indicated by the seasonal Ys; Table 6), although altered spatial distribution of qv, root growth and root water uptake supported greater leaf water use and photosynthesis in PRI-1 vines, especially at midday and in periods with high irrigation volumes (e.g. from budburst to fruit set). In contrast, in previous studies that maintained soil moisture near to field capacity on the irrigated side of PRI vines showed decreased leaf water use and gs, and an increased WUE, resulting in a similar or better water status (Dry and Loveys 1999; Stoll et al. 2000; Davies et al. 2002; Du Toit et al. 2003; dos Santos et al. 2003; Kang and Zhang 2004; Chaves et al. 2007, 2010). In contrast, the reduced qv availability in the wet root zone and lower total soil water available in PRI-2 vines (Table 3) did not stimulate root growth, and PRI-2 vines were unable to compensate by increasing water uptake. This was reflected in lower root water uptake and decreased water status and gas exchange (Tables 6 and 8; Table S1) during water stress periods before veraison, and in slower and incomplete gas exchange recovery after veraison in PRI-2 vines (Fig. 3). Similar leaf N and chlorophyll contents in PRI-2 and RDI-2 vines (data not shown) indicated no quantitative losses in the photosynthetic apparatus or damage to the biochemical photosynthetic machinery. However, the more prolonged exposure of PRI-2 roots to drier soil may have caused anatomical root changes such as the loss of succulent secondary roots (North and Nobel 1991), and increased resistance to water uptake and transport, thus decreasing root hydraulic conductivity (Hu et al. 2011), and whole-vine hydraulic conductance and evaporative flux in this rootstock–scion combination (Romero et al. 2012). Enhanced stomatal closure and lower Amd and gsmd in PRI-2 vines (compared with RDI-2 vines) were not related with higher [X-ABA]leaf during the drying cycles (Table 8). Instead, the RDI-2 vines had higher [X-ABA]leaf, gs and Amd levels than PRI-2, indicating no correlation between leaf water use and [X-ABA]leaf, as recently reported (Ahmadi et al. 2010b; Einhorn et al. 2012; Wang et al. 2012b). Likewise, increased [X-ABA]leaf in PRI-1 vines (compared with RDI-1 vines) was not associated with decreased gas exchange and growth. In contrast to previous reports showing a robust relationship between [X-ABA]leaf and gs (Davies et al. 2002; Loveys et al. 2004; Liu et al. 2005), other nonhydraulic signals (cytokinins and ethylene) and their interactions (rather than changes in the concentration of a single hormone or signal) may also be involved in modulating leaf gas exchange under PRI (Pérez-Pérez et al. 2012; Wilkinson et al. 2012).


Table 8.


Mean values of leaf xylem ABA concentration, [X-ABA]leaf, stomatal conductance at early morning and midday and volumetric soil water content at the beginning, middle and end of a typical 15-day partial root zone irrigation (PRI) cycle after veraison in 2009 ns, not significant, * P < 0.05; ** P < 0.01; *** P < 0.001. Separation by Duncan’s multiple range test at the 95% confidence level 26 August (beginning of the PRI cycle) Early morning Midday Average (0800 hours) (1200 hours) morning

[X-ABA]leaf (nM) PRI-1 RDI-1 PRI-2 RDI-2 ANOVA

727a 384b 488b 397b *

523 322 416 473 ns

625a 348b 452b 435b *

2 September (mid-PRI cycle) Early morning Midday Average (0800 hours) (1200 hours) morning 584 590 711 893 ns

26 August (beginning of the 2 September (mid-PRI cycle) PRI cycle) Early morning Midday Early morning Midday (0800 hours) (1200 hours) (0800 hours) (1200 hours) Stomatal conductance (mol m–2 s–1) PRI-1 0.075 RDI-1 0.078 PRI-2 0.081 RDI-2 0.085 ANOVA ns

Depth

O

– – – – –

26 August (beginning of the PRI cycle) Early morning (0900 hours) 10–30 cm 10–60 cm

Volumetric soil water content (%) PRI-1 (wet) 28a PRI-1 (dry) 21b PRI-1 (average) 25ab RDI-1 24ab PRI-2 (wet) 22ab PRI-2 (dry) 17b PRI-2 (average) 20b RDI-2 21b ANOVA *

31 26 29 25 25 24 25 26 ns

0.092a 0.091a 0.070b 0.085a *

0.153a 0.130ab 0.094ab 0.088b *

2 September (mid-PRI cycle) Early morning (0900 hours) 10–30 cm 10–60 cm 30a 15d 22bc 23bc 24b 12d 18bcd 18cd ***

34a 23bc 28b 25bc 26b 21c 24bc 24bc ***

[X-ABA]leaf generally peaked at early to mid-morning and decreased at midday (Table 8), as seen in other field studies (Ahmadi et al. 2010b). Transient increases in [X-ABA]leaf, mainly following reirrigation in PRI-1, suggests that rewatering the dry part of the root system liberated rootsynthesised ABA to the transpiration stream once these roots began to contribute proportionally to total sap flow (Dodd et al. 2006, 2008a; Topcu et al. 2007; Romero et al. 2012). However, this effect has not always been observed experimentally (Einhorn et al. 2012; Pérez-Pérez et al. 2012). During PRI, [X-ABA]leaf depends mainly on the qv in the wet root zone, as modelled by Dodd et al. (2008a, 2008b) and observed experimentally (Einhorn et al. 2012; Romero et al. 2012; Wang et al. 2012b). If ABA production and transport were completely dependent upon the water status of the dry roots, PRI-1 and PRI-2 would have had similar [X-ABA]leaf values because they had similar qv (no significant differences) in the dry root zone (Tables 3 and 8). However, the higher [X-ABA]leaf in PRI-1 (compared with PRI-2) at the beginning and end of the PRI cycle suggests that [X-ABA]leaf strongly depends on the qv in the wet root zone (Dodd et al. 2008a, 2008b). Contrary to the expected negative correlation between

460 583 534 762 ns

522 587 623 828 ns

8 September (end of the PRI cycle) Early morning Midday Average (0800 hours) (1200 hours) morning 738a 720a 452b 725a *

518 439 439 457 ns

628 580 445 591 ns

8 September (end of the PRI cycle) Early morning Midday (0800 hours) (1200 hours) 0.082a 0.078a 0.049b 0.070a *

– – – – –

8 September (end of the PRI cycle) Early morning (0900 hours) 10–30 cm 10–60 cm 27a 14de 21bc 21bc 26ab 11e 18cd 18cd ***

31a 22cd 27bc 23bcd 27ab 20d 23bcd 24bcd ***

[X-ABA]leaf and qv, [X-ABA]leaf and total qv-avg (10–60 cm) (average of the dry and wet parts) in the entire soil profile (10–60 cm) were positively correlated only following rewatering of the PRI-1 and RDI-1 vines (Fig. 7a); with no significant correlations observed in the middle or at the end of the PRI cycle or with the PRI-2 and RDI-2 treatments (data not shown). Furthermore, differences in the average qv (at 10–60 cm) do not explain observed changes in [X-ABA]leaf and gs, during a PRI cycle (a similar average qv at 10–60 cm) was observed between PRI and RDI; Table 8). Instead, accounting for the relative contribution of both sides (qv-wet vs qv-dry) during a PRI cycle seem to be important, as modelled by Dodd et al. (2008a) and observed experimentally (Hutton and Loveys 2011). Thus significant correlations between qv-dry and [X-ABA]leaf (Fig. 7b–d), suggest that (1) there is an optimal qv range for ABA synthesis and accumulation, especially following rewatering (qv-wet > 30% and qv-dry 26% at 10–60 cm, as in PRI-1 , Table 8, Fig. 7b) and (2) at the end of PRI cycle, low qv-dry (qv-dry 11% at 10–30 cm and qv-dry 20% at 10–60 cm; Table 8, Fig. 7c, d) probably limited sap flow (and ABA transport) from roots in drying soil, as in PRI-2 vines. Similarly, Wang et al. (2012b) recommended that qv in the wet root zone should be

P


P. Romero et al.

End of PRI cycle

Start of PRI cycle 1000

900

[X-ABA]leaf (nM) (early morning)

r = 0.82*

(c)

800

600 400 200

PRI-1 RDI-1

[X-ABA]leaf (nM) (average morning)

(a) 800

0

r = 0.93***

700 600 500 400

PRI-2 dry side RDI-2

300 200 10

20 22 24 26 28 30 32 34

700

900

(b) r = 0.90*

600 500 400 300

PRI-1 PRI-2

200 18 20 22 24 26 28 30 32 34

v-dry side (%) (10–60 cm)

[X-ABA]leaf (average morning)

[X-ABA]leaf (nM) (average morning)

800

15

20

25

v (%) (10–30 cm)

vavg (%) (10–60 cm)

(d)

800

r = 0.86**

700 600 500 400 PRI-1 PRI-2

300 200 6

8

10 12 14 16 18 20

v-dry side (%) (10–30 cm)

Fig. 7. (a) Relationship between the xylem sap ABA concentration ([X-ABA]leaf) at early morning and the average volumetric soil water content, qv, in the entire root zone (10–60 cm) for vines under partial root zone irrigation (PRI-1) and regulated deficit irrigation (RDI-1) with 110 mm year–1. (b) Relationship between [X-ABA]leaf and qv in the dry root zone (10–60 cm) at the beginning of a typical partial root zone irrigation (PRI) cycle in late August 2009, for PRI-1 vines and vines under PRI with 78 mm year–1 (PRI-2). (c) Relationship between [X-ABA]leaf and qv (10–30 cm) for PRI-2 vines and vines under regulated deficit irrigation with 78 mm year–1 (RDI-2). (d) Relationship between [X-ABA]leaf and qv in the dry root zone (10–30 cm) at the end of a typical PRI cycle in late August 2009. Each point is one replicate per treatment (one per plot). Linear and non-linear regressions were fitted to the data. Significance level of the relationships: *P < 0.05; **P < 0.01; ***P < 0.001.

maintained relatively high, whereas in the drying zone, it should not be very low, both conditions being crucial to maintain high soil and plant water status while sustaining ABA signalling. Vine vigour, trunk growth patterns, WUE and biomass accumulation PRI vines frequently exhibit stronger control of vegetative growth compared with those grown using conventional deficit irrigation (Loveys et al. 1999; Stoll et al. 2000; dos Santos et al. 2003, 2007; Du Toit et al. 2003; de Souza et al. 2005a, 2005b; Chaves et al. 2007). However, in many other studies that applied the same amount of water using both PRI and conventional deficit irrigation techniques, no significant differences in vegetative development occurred (Dry et al. 2000a, 2000b; Bravdo et al. 2004; Gu et al. 2004; Marsal et al. 2008; Intrigliolo and Castel 2009). Some studies even reported increased shoot growth and plant height (Cameron et al. 2008; Talluto et al. 2008), pruning weight (Dry et al. 2001), plant dry biomass (Wang et al. 2008) or leaf area (Antolín et al. 2006; De la Hera et al. 2007; Shao et al. 2011; Romero and Martínez-Cutillas 2012) with PRI. Similarly, PRI-1 vines also showed significantly greater trunk growth and pruning weight than RDI-1 vines and the other treatments

(Table 7), in accordance with the greater root water uptake and leaf CO2 assimilation. In PRI-1 vines, greater fresh and dry biomass allocation compared with the RDI-1 vines was directed not only to the roots but also to the main shoots and fruits (Fig. 6), as in other PRI studies (Shao et al. 2008, 2011; Zegbe et al. 2007). However, this contrasted with other studies where PRI did not significantly affect aboveground biomass allocation (Savic et al. 2009; Ahmadi et al. 2010a) or where PRI even increased root biomass at the expense of a biomass allocation to the stem, leaves and fruits (Mingo et al. 2004). Interestingly, the significantly greater vegetative growth and plant biomass accumulation of PRI-1 vines (Fig. 6), with similar leaf water status, and higher gas exchange and canopy water use, imply a more favourable internal vine water status than RDI-1 vines (Romero and Martínez-Cutillas 2012). This is confirmed by the greater water content of the different stem tissues (main and lateral shoots and fruits) relative to those of RDI-1 vines (Fig. 6). In addition, the lower foliar d13C (higher isotopic discrimination against 13C, Fig. 5a) indicates that PRI-1 vines maintained more open stomata than RDI-1 vines (as decreased gs is reflected in increased d 13C due to reduced discrimination by Rubisco; Farquhar et al. 1989), allowing greater carbon assimilation throughout the growing season (de Souza et al.


2003, 2005c; Wang et al. 2010). This allowed greater fractions of water and photoassimilates to be allocated to different plant organs in PRI-1, in order to sustain growth, as indicated by the significant correlation between total plant DW and d13C (Fig. 5c). In addition, although d 15N in PRI-1 was not significantly different from RDI-1, PRI-1 increased significantly d 15N (in 2009, Fig. 5b, d) and total leaf area compared with PRI-2 (Table 7), which could indicate that the larger root system and higher soil water content in PRI-1 enhanced available N in the soil and plant N accumulation (Wang et al. 2012c; Sun et al. 2013). Drying and rewetting cycles in the soil induced by PRI could also stimulate higher soil organic matter mineralisation and thus more mineral N being available for the plants (Wang et al. 2009). Daily trunk diameter fluctuations, which depend mainly on the water stored in the phloem and related tissues, cambium and living tissues of the bark (Fernández and Cuevas 2010), showed significantly higher diurnal trunk contraction and recovery or expansion rates, and consequently increased MDTS in PRI-1 vines (Table 5, Fig. S1). Similarly, a greater and faster recovery of trunk diameter during the afternoon was reported in PRI compared with RDI almond (Prunus dulcis (Mill.) D. A. Webb) trees (Egea et al. 2011). Higher MDTS reflected greater diurnal exchange of water between the xylem and phloem, indicating a greater capacity to accumulate and store water (at night) and to release it to the transpiration stream during the day in PRI-1 vines. Higher MDTS could be due to greater potential gradients between the xylem and living tissues (Fernández and Cuevas 2010), as a consequence of greater soil–plant water potential gradients and higher transpirational fluxes (Montoro et al. 2012; Ortuño et al. 2006). In tree species, greater internal water storage sustained maximum transpiration and therefore kept the stomata open for longer periods of time (Goldstein et al. 1998). Besides, utilising internal water reserves would allow the vines to be less dependent, in the short term, on soil water content (Zweifel et al. 2001). This could explain the lower variation in leaf water status between irrigation events in PRI-1 compared with RDI-1 vines (Fig. 7). In contrast, the PRI-2 vines (more water-stressed) showed greater restriction of vegetative development than the RDI-2 vines (Table 7), and no compensatory root growth and water uptake. This resulted in a less favourable shoot water supply and vine water status, a lower water storage or release capacity of the trunk under severe soil water deficit, and lower canopy water use in PRI-2 than in RDI-2 vines, despite applying the same total amount of water. Conclusions Physiological responses induced by PRI were due to both the placement of irrigation and reductions in irrigation volumes. Monastrell-1103P grapevines compensated for the decreased water availability on the non-irrigated side of the vines under PRI by increasing root growth and root water uptake on the irrigated side (compared with RDI) when high irrigation volumes were applied (qv-wet >30% at 10–60 cm). This increased qv availability in the entire root zone also stimulated greater canopy water use (more open stomata), vegetative development, plant biomass accumulation and internal water


Q

storage and release capacity compared with RDI-1 vines, despite the total amount of water applied being the same. In contrast, the smaller irrigation volumes applied in PRI-2 decreased the total qv availability, fine root growth and water uptake, and substantially reduced the gas exchange, leaf water status, [X-ABA]leaf and plant biomass accumulation compared with RDI-2 vines. The significant qv–[X-ABA]leaf relationships indicated that [X-ABA] decreased with decreased total qv availability in PRI-2 plants (especially at the end of PRI cycles), probably due to limited sap flow from roots in drying soil when qv was too low (qv-dry 20% 10–60 cm)). More investigation is required to define the thresholds and optima of qv in wet and dry root zones in wine grapes, their relationships with ABA signalling and other hormones (cytokinins and ethylene), and their implications for irrigation scheduling and berry quality in PRI. Further research will determine whether these distinct PRI-specific physiological changes alter the yield response, berry composition and wine quality. Acknowledgements This work was financed by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Subprograma Nacional de Recursos y Tecnologías Agrarias through the Project RTA2008–00037C04–04, with the collaboration of the European Social Fund (ESF) European Union-FEDER 80%. Región de Murcia PO-07-33. PR gratefully acknowledges a doctoral contract in the INIA-CCAA system supplied by INIA and cofinanced by the ESF. We thank Santiago López Miranda for support with leaf area measurements in 2009. We also thank Atanasio Molina Molina, Aniceto Turpín Bermejo, Antonio Lucas Bermudez and Cristobal Marín for their work in vineyard management; Juan Jose Sánchez Ruiz, Jose María Rodriguez de Vera-Beltrí and Francisco Martínez López for field assistance and support in laboratory analyses; and David J. Walker for assistance with manuscript preparation and correction of the English. ICD thanks the European Commission (FP7- KBBE-2009–3–245159 – SIRRIMED (Sustainable use of irrigation water in the Mediterranean region) project) for continued support of the work on partial root zone drying.

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