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Shoot hydraulic characteristics, plant water status and stomatal response in olive trees under different soil water conditions

J. M. Torres-Ruiz & A. Diaz-Espejo & A. Morales-Sillero & M. J. Martín-Palomo & S. Mayr & B. Beikircher & J. E. Fernández

Received: 8 March 2013 /Accepted: 13 May 2013 /Published online: 4 June 2013

Abstract Aims To evaluate the impact of the amount and distri- bution of soil water on xylem anatomy and xylem hydraulics of current-year shoots, plant water status and stomatal conductance of mature ‘Manzanilla’ ol- ive trees. Methods Measurements of water potential, stomatal conductance, hydraulic conductivity, vulnerability to embolism, vessel diameter distribution and vessel den- sity were made in trees under full irrigation with non- limiting soil water conditions, localized irrigation, and rain-fed conditions. Results All trees showed lower stomatal conductance values in the afternoon than in the morning. The irri- gated trees showed water potential values around −1.4 and −1.6 MPa whereas the rain-fed trees reached lower

values. All trees showed similar specific hydraulic con- ductivity (Ks) and loss of conductivity values during the morning. In the afternoon, Ks of rain-fed trees tended to be lower than of irrigated trees. No differences in vul- nerability to embolism, vessel-diameter distribution and vessel density were observed between treatments. Conclusions A tight control of stomatal conductance was observed in olive which allowed irrigated trees to avoid critical water potential values and keep them in a safe range to avoid embolism. The applied water treat- ments did not influence the xylem anatomy and vul- nerability to embolism of current-year shoots of ma- ture olive trees.

Keywords Cavitation . Olive . Irrigation .

Vulnerability to drought-induced embolism . Water stress . Xylem anatomy

Introduction

According to the cohesion theory (Dixon and Joly 1894; Askenasy 1895) water ascends plants in a metastable state. The driving force is generated by the negative pressure at the evaporating surfaces of the leaf. The tension is transmitted through a continuous water col- umn from leaves to roots and lowers their water poten- tial below the potential of the surrounding soil. This causes water uptake from the soil and its upward move- ment to the aerial part of the plant. The decrease in water potential as the soil gets dry may cause cavitation of

Plant Soil (2013) 373:77–87 DOI 10.1007/s11104-013-1774-1

Responsible Editor: Rafael S. Oliveira.

J. M. Torres-Ruiz (*) : A. Diaz-Espejo : J. E. Fernández Irrigation and Crop Ecophysiology Group, Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS, CSIC), Avenida Reina Mercedes, n.º 10, 41012 Sevilla, Spain e-mail: [email protected]

A. Morales-Sillero : M. J. Martín-Palomo Departamento de Ciencias Agroforestales, ETSIA, University of Sevilla, Carretera de Utrera, km 1, 41013 Seville, Spain

S. Mayr: B. Beikircher Department of Botany, University of Innsbruck, Sternwartestr. 15, 6020 Innsbruck, Austria

# European Union 2013

conduits and the resulting embolism to loss of hydraulic conductivity (K) (Tyree and Zimmerman 2002). Stoma- tal closure is one of the most effective mechanisms to avoid critical water potential values (Choat et al. 2012). Accordingly, also olive trees minimize water losses under high water demand conditions by stomata regula- tion (Fernández et al. 1997; Tognetti et al. 2009; Boughalleb and Hajlaoui 2011). Stomatal closure is known to be especially advantageous in environ- ments with wide fluctuations both in evaporative demand and soil moisture (Franks et al. 2007), as those in which the olive tree is widely grown (Fernández and Moreno 1999).

The relationships between stomatal conductance (gs), leaf water potential (Ψl), K and environmental variables are complex. Feedback mechanisms between these variables (Chaves et al. 2003; Lovisolo et al. 2010) and differences between cultivars (Winkel and Rambal 1990; Fernandez et al. 2008) have been reported. In addition, irregular distribution of water in the rootzone may trigger root-to-shoot signals in- ducing stomatal closure (Dry and Loveys 1999; Dodd 2005). This is caused by some irrigation systems, as localized irrigation, in which a fraction only of the rootzone is wetted by irrigation. In olive, Fernández et al. (2003) reported restricted transpiration in trees under localized irrigation, but they could not unravel whether the stomatal closure was induced by a chem- ical signal involving abscisic acid (ABA) generated in the roots remaining in the drying soil or by a hydraulic signals, as e.g. the drop of hydraulic conductance, caused by soil drying. Bacelar et al. (2007) analyzed the effect of the soil water regime on gas exchange and xylem hydraulic properties of olive cultivars. They reported that water stress caused a marked decline of gs and an increase in xylem vessel density in all cultivars. Some of those cultivars also showed a re- duction in vessel diameter. They worked, however, with 1-year-old plants in pots and their experiments did not include localized irrigation. Tognetti et al. (2009) reported that the loss of hydraulic conductance is an important signal for the stomatal control of tran- spiration in olive trees under drying soil conditions. A better understanding of how soil water regime and plant water status influence stomata regulation and hydraulics of olive cultivars is important for a efficient manage- ment of water used in irrigated orchards. The interest in this area of research is considerable given that 2.3 out of the 10.5 Mha of olive surface are irrigated, mostly with

localized irrigation systems (International Olive Coun- cil, www.internationaloliveoil.org; Pastor 2005).

We evaluated the effect of different soil water re- gimes on xylem anatomy, xylem hydraulics and gas exchange in current-year shoots of mature ‘Manzanil- la’ olive trees. Measurements of xylem anatomical parameters, Ψl, gs, specific hydraulic conductivity (Ks) and percentage loss of conductivity (PLC) and analysis of the vulnerability to embolism were made in 41-year-old ‘Manzanilla’ olive trees along a full irri- gation season. The trees were under three different soil water regimes: rain-fed conditions, localized irrigation in which part of the rootzone remained in drying soil, and full irrigation that kept the whole rootzone under non-limiting soil water conditions. We hypothesized that differences in soil water conditions imposed by the mentioned water treatments limit plant hydraulics and lead to stomatal regulations as well as to acclima- tion in xylem anatomical traits in olive trees.

Materials and methods

Experimental site and water treatments

This study was carried out at ‘La Hampa’ experimental farm, 15 km from Seville, southwest Spain (37º 17′ N, 6º 3′ W, 30 m a.s.l.). Climate in the area is Mediterranean with a wet, mild season from October to April and a hot, dry season from May to September. Trees in the orchard were 41-year-old ‘Manzanilla de Sevilla’ (from now on ‘Manzanilla’) olive trees at 7 m×5 m spacing (286 trees ha−1). The soil of the orchard is a sandy loam (Xerochrept) of 1.6–2.0 m depth. The texture is quite homogeneous, both vertically and horizontally (Moreno et al. 1988), with mean values of 14.8 % clay, 7.0 % silt, 4.7 % fine sand and 73.5 % coarse sand. Laboratory measurements showed a volumetric soil water content (θv) of 0.33 and 0.09 m

3 m−3 for a soil matric potential of −0.01 and −1.5 MPa respectively. Field measurements showed θv=0.22 m

3 m−3 at field capacity conditions. Experiments were made during the irrigation season

of 2009, from May 6, day of year (DOY) 126, to October 2 (DOY 275). Three adjacent 0.2 ha plots were used in the project, each under a different water treat- ment: 1) a rain-fed treatment (R), in which rainfall was the only source of water supply until a recovery irriga- tion was applied from September 8 (DOY 251) to the end of the season. This consisted on supplying daily

78 Plant Soil (2013) 373:77–87

three times more water than to the LI trees (described below), in a circle of ca. 2 m radius around the trees; 2) Localized irrigation (LI), in which trees were irrigated daily throughout the irrigation season with enough wa- ter to replace 100 % of the crop evapotranspiration (ETc). The irrigation system consisted of a lateral pipe per tree row with five 3 L h−1 drippers per tree, 1 m apart. This system leaves part of the roots under soil drying conditions during the irrigation season. The irri- gation dose was calculated with the crop coefficient approach (Allen et al. 1998), with coefficients adjusted for the orchard conditions by Fernández et al. (2006); and 3) Full irrigation (FI), in which trees were irrigated with a 0.4 m×0.4 m grid of pipes with a 2 L hour−1

dripper in every node. The grid covered a surface of 8 m×6 m, with the tree in the middle, enough to keep non-limiting soil water conditions in the whole rootzone throughout the irrigation season. The FI trees were irrigated every other day, to avoid hypoxia.

Soil water and weather measurements

Soil water profiles were measured every 7–10 days during the experimental period. We used a PR2-Profile probe (Delta-T Devices Ltd, Cambridge, UK) with three access tubes per tree in three trees per treatment, at 0.50 m, 1.50 m and 2.25 m from the trunk to cover possible heterogeneities in soil water distribution. In each access tube θv values were measured at 0.1, 0.2, 0.3, 0.4, 0.6 and 1.0 m depth. These values were used to calculate the relative extractable water (REW) according to Granier (1987). Simultaneous measurements of root distribution and soil water content in the orchard down to 2.4 m depth made by Fernández et al. (1991) showed that the explored top meter of soil was enough for a reliable average value of θv in the rootzone of the experimental trees.

Main weather variables were recorded every 30 min with a weather station (Campbell Scientific Ltd., Leices- tershire, UK) under standard conditions, located at ca. 50 m from the orchard. These data were used to calcu- late the FAO56 Penman-Monteith potential evapotrans- piration, ETo (Allen et al. 1998).

Leaf water potential and stomatal conductance

Values of Ψl at predawn, in the morning (9–10 GMT) and in the afternoon (14–15 GMT) were measured on June 23 (DOY 175), July 28 (DOY 209), August 25

(DOY 237) and October 1 (DOY 274). We sampled two fully expanded leaves of the current year per tree in three trees per treatment. Measurements were made with a Scholander-type pressure chamber (Soilmoisture Equip- ment Corp., Santa Barbara, California, USA), following recommendations by Turner (1988) and Koide et al. (1989). We measured gs at the same time as Ψl both in the morning and in the afternoon. We used a LI-6400 portable photosynthesis system (Licor Inc., USA) and sampled the same type and number of leaves as for Ψl.

Hydraulic conductivity and native embolism

Five branches per treatment of 1.2 m in length with at least one 0.4 m long current-year shoot, respectively, were cut under water (to avoid air entering into the vessels), wrapped in plastic bags with wet paper towel inside (to prevent transpiration) and transported to the laboratory. To avoid any possible influence of the sampling position on the results, all branches were sampled from similarly-oriented parts of the canopy, at ca. 1.5 m above ground. The sampling was made at the same time, in the same trees and from the same part of the canopy as the gs and Ψl measurements. Once in the laboratory, we submerged one current- year shoot of each branch in a container with perfusion solution (see below). From each shoot we sampled one ca. 30 mm long segment under water, removed its bark and trimmed sample ends with a razor blade. All the sampled segments were collected from the mid-part of the shoots. The native K of each segment was then determined with a XYL’EM® apparatus (Bronkhorst, Montigny-les-Cormeilles, France) as:

K ¼ F ΔP

L ð1Þ

where F mass flow rate of a solution through the segment, ΔP is the applied pressure gradient driving the flow and L is the sample length. We used a filtered (0.22 μm) 50 mM KCl perfusion solution made with distilled water, and applied a pressure gradient of 3 kPa until a steady-state native K was attained. Previous experiments in olive showed that this water head is below the threshold at which embolized vessel opened at both ends are flushed and artificially contribute to K (data not shown). Flushing was obtained after initial K measurements by perfusion at 150 kPa for 20 min. The pressure was then lowered again down to 3 kPa to

Plant Soil (2013) 373:77–87 79

determine maximum K. Percent loss of conductivity (PLC) (i.e. native embolism) was calculated as:

PLC ¼ 100 � 1−native K . maximum K

� � ð2Þ

The Ks was calculated by dividing native K by the mean cross-sectional area of the sample.

Vulnerability curves

On November 2 (DOY 306), twenty 1.5 m long branches with 3–4 current-year shoots, respectively were sampled under water from 3 to 5 representative trees of each treatment, and transported to the laboratory as described above. The branches were used to determine vulnerabil- ity to drought-induced embolism by the bench-top dry- ing technique (Tyree and Dixon 1986; Sperry and Tyree 1988). During dehydration, xylem water potential (Ψx) was measured in intervals until the desired values down to −10 MPa were reached. Branches then were stored for

1.5 h in a plastic bag with a wet paper towel inside to allow the equilibrium between Ψl and Ψx. Measurements of Ψx were made in two leaves per branch and after- wards, one segment of ca. 30 mm length per current-year shoot was sampled, following the procedure described above for native K measurements and PLC determina- tions. We generated one vulnerability curve per treatment by plotting PLC versus Ψx. For fitting the vulnerability curves obtained from each treatment we used a Weibull function (Neufeld et al. 1992) with an additional inde- pendent factor to consider the levels of embolism mea- sured at Ψx≃0:

PLC ¼ 100−y0ð Þ− 100−y0ð Þe− x bð Þc þ y0 ð3Þ

being x the Ψx, b the Ψx for a PLC of 63 %, c a dimensionless parameter controlling the shape of the curve and y0 the PLC at Ψx=0 MPa. These points were fitted to Eq. 3 using Excel’s solver function. The Ψx

IA (

m m

)

0

5

10

15

20

P (

m m

)

0

2

4

6

8

10

FI LI R

E T

( m

m )

0

2

4

6

8 ETo ETc

DOY 2009

120 140 160 180 200 220 240 260 280

R E

W

0,0

0,5

1,0

FI LI R

May June July August September October

a

b

c

Fig. 1 Time courses of (a) reference (ETo) and crop evapotranspiration (ETc), b collected precipitation (P) and irrigation amounts (IA) supplied to trees of each treatment, and (c) relative extractable water (REW) for each treatment. DOY day of year. Arrows indicate sampling dates for variables shown in Figs. 2, 3 and 4

80 Plant Soil (2013) 373:77–87

values corresponding to 50 % loss of hydraulic conduc- tivity (P50, MPa) due to embolism formation and its 95 % confidence intervals were calculated from each vulnerability curve.

Anatomical measurements

On November 2 (DOY 306), current-year shoots of similar characteristics than those used for K measure- ments were collected from four different representa- tive trees per treatment. One 10 mm segment was sampled from each shoot, dehydrated in an acetone series and embedded in SPURR resin. Sections were cut with an ultramicrotome, stained with toluidine blue

(1 %) for 3 min, rinsed in water and photographed with a digital camera attached to a light microscope (Olympus BX61). Images from each section were divided into four parts of similar area. Vessel density (number of vessels in a given area) was determined in two of these parts using Adobe Photoshop CS3 soft- ware (Adobe Systems Incorporated, USA). The vessel diameter was calculated for each vessel from its sur- face area, previously determined with the mentioned software, assuming a circularity of 1. The diameter distribution per treatment was determined after classi- fying the vessels into bin diameters (diameter size classes of 2 μm width). The resulting values were expressed as percentage of vessel number from total in each class. Some 300 to 500 vessels were measured per section.

Statistical analysis

Data sets were tested for normality with the Kolmogorov- Smirnov test and homogeneity of variances was deter- mined by Brown & Forsythe test. Differences in Ψl, gs, K, PLC, vessel density and percentage of vessel in each bin diameter between water treatments were evaluated by a one-way analysis of variance (ANOVA). When the dif- ferences were significant, a multiple comparison of means (post hoc Tukey honest significant difference test) was carried out. Statistical comparisons were considered significant at p<0.05. P50 values calculated from the vulnerability curves obtained for trees of each water treatment were considered significantly different when their 95 % confidence intervals did not overlap.

Repeated measures analyses of variance (ANOVA) over time were carried out to test differences in Ψl, gs, K and PLC between FI and LI trees during the season. All analyses were performed by using STATISTICA software (StatSoft, Inc., USA) and Sigmaplot (SPSS Inc., USA).

Results

Weather conditions and soil moisture

Except for peak values of ETo recorded at the end of May, the time course of the atmospheric demand dur- ing the experimental period was as usual in the area, with high values in the middle of the summer and decreasing values from the end of August (Fig. 1a).

P re

d a w

n

l (M

P a )

-6

-5

-4

-3

-2

-1

0

FI LI R

M o rn

in g

l (M

P a )

-6

-5

-4

-3

-2

-1

160 180 200 220 240 260 280

A ft

e rn

o o

n

l (M

P a

)

-6

-5

-4

-3

-2

-1

DOY 2009

June July August September

a a b

a a

b

a a

b

n.s.

a a

b

a

a

b

a

a

b

a b c

a a

b

a

a

b

a

a

b

n.s

a

b

c

Fig. 2 Time courses of leaf water potential (Ψl) measured at (a) predawn, b at 9.00–10.00 GMT (morning Ψl), and (c) at 14.00– 15.00 GMT (afternoon Ψl). Data points are average of six values; vertical bars represent ± the standard error. Different letters indicate statistically significant difference (p<0.05) be- tween treatments. n.s. no significant difference. The dashed line indicates the beginning of the recovery irrigation applied to the R trees. DOY day of year

Plant Soil (2013) 373:77–87 81

The irrigation amounts applied to the LI trees (Fig. 1b) were enough to maintain REW values close to field capacity throughout the irrigation season (Fig. 1c). In the FI plot, REW values showed conditions close to saturation. Total amounts of water supplied to LI and FI trees were 3361.8 m3 ha−1 (88 % ETc) and 16121 m3 ha−1 (420 % ETc), respectively. The high irrigation supplies in the FI treatment were justified by the need of ensuring non-limiting soil water conditions in the whole rootzone. REW values in the R plot de- creased during the irrigation season, until the recovery irrigation applied from DOY251 to DOY275, when 1482.3 m3 ha−1 of water were supplied. This, together with the first rainfall events after the summer dry period (Fig. 1b) increased REW values to ca. 0.9 (Fig. 1c).

Plant water status and stomatal conductance

Similar values of Ψl at predawn were recorded in the LI and FI trees during the dry season (Fig. 2a). In

contrast, R trees showed decreasing Ψl values at predawn throughout the summer, in agreement with the gradual decrease in REW (Fig. 1c). In the LI and FI trees, Ψl values were close to −1.5 MPa both in the morning and the afternoon throughout the dry season (Figs. 2b and c), indicating no increase in water stress during the season. Although slightly, the Ψl was

M o rn

in g

g s

(m o

l m -2

s -1

)

0,00

0,05

0,10

0,15

0,20

0,25

0,30

FI LI R

DOY 2009 160 180 200 220 240 260 280

A ft

e rn

o o

n

g s

(m o

l m -2

s -1

)

0,00

0,05

0,10

0,15

0,20

0,25

a

b

c

a

ab

b

a

a

b

a a

b

a a

b

a

ab

b

a

a

b

a

a

b

June July August September

a

b

Fig. 3 Time courses of sto- matal conductance (gs) mea- sured at (a) ca. 9.00–10.00 GMT and (b) 14.00–15.00 GMT. Data points are aver- age of six values; vertical bars represent ± the standard error. Different letters indi- cate statistically significant difference (p<0.05). The dashed line indicates the be- ginning of the recovery irri- gation applied to the R trees. DOY day of year

Table 1 Values of the vapour pressure deficit of the air (Da) recorded in the morning (9–10 GMT) and in the afternoon (14– 15 GMT) of the days when main physiological variables were measured

DOY Da (kPa)

Morning Afternoon

174 1.47 3.73

209 2.10 4.66

234 1.85 3.42

274 0.30 1.65

82 Plant Soil (2013) 373:77–87

consistently lower in LI trees than in the FI trees during the whole season (Table 3) but not on a day-by-day basis (Fig. 2). In the R trees, Ψl values during the dry period were significantly lower than in LI and FI trees. Simi- larly to predawn, Ψl decreased in the R trees both in the morning and in the afternoon throughout the season as the soil water was depleted. The most negative values were recorded at the end of August (morning Ψl=−4.2± 0.4 MPa; afternoon Ψl=−4.8±0.4 MPa), when lowest REW were recorded (Fig. 1c). The water supplied by the recovery irrigation and the rainfall events in autumn (Fig. 1b) caused Ψl of R trees to raise to values similar to LI and FI trees (Fig. 2).

In LI and FI trees, gs values in the morning were over 0.2 mol m−2 s−1 during the entire dry season (i.e. until the first rainfall events), while R trees showed much lower gs values (Fig. 3a). In all trees, gs in the afternoon was lower than in the morning during the drought season but similar at the end of the study period (Fig. 3). Both in the morning and in the after- noon, the LI trees used to show lower gs values than the FI trees although differences were not always significant day by day. But, when the entire season is considered, the gs was consistently lower in LI than in

FI (Table 3). Marked differences in Da between morn- ing and afternoon were recorded during the whole season (Table 1).

Hydraulic and anatomical characteristics

No pronounced differences or trends were observed between treatments in Ks or PLC of morning samples (Fig. 4a, b). In the afternoon, Ks in the R trees tended to be lower than in the irrigated trees with significant differences at DOY 209. In the FI and LI trees, no significant differences or trends in Ks or PLC were found, either day by day or during the season (Fig. 4; Table 3). Corresponding to the course in Ks, PLC of R trees peaked at DOY 209 (ca. 60 %), while PLC values were similar on all other sampling days (Fig. 4c, d).

Trees from all treatments showed a similar vulnera- bility to embolism (Fig. 5a) and no difference between P50 (Table 2). Figure 5a shows that native PLC at Ψx close to 0 MPa was 15–30 % in all treatments. Accord- ingly, anatomical analysis revealed similar vessel diam- eter distributions in all treatments (Fig. 6). Vessel den- sity tended to be highest in R trees, followed by LI and FI trees, but differences were not significant (Table 2).

DOY 2009

160 180 200 220 240 260

P L

C

0

20

40

60

80

160 180 200 220 240 260 280

Afternoon

n.s.

n.s.

n.s.n.s. n.s.

n.s.

n.s. n.s.

n.s.

n.s. n.s. n.s.

a

b d

June July August September June July August September

n.s. n.s.

n.s. a

a

bc

Morning

K s

(k g

m -1

s -1

M P

a -1

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 FI LI R

Fig. 4 Average values of specific hydraulic conductivity (Ks) and percentage loss of hydraulic conductivity (PLC) at 9.00– 10.00 GMT (i.e. morning) (a, b respectively) and at 14.00– 15.00 GMT (c, d respectively) (i.e. afternoon). Data points are average of three to five values; vertical bars represent±the

standard error. Different letters indicate statistically significant difference (p<0.05); n.s. no significant difference. The dashed lines show the beginning of the recovery irrigation applied to the R trees. DOY day of year

Plant Soil (2013) 373:77–87 83

Discussion

Stomatal control of transpiration and embolism formation

Results of the present study suggest a tight control of Ψl in olive (Fig. 3a, b). Despite of the two-fold increase in Da between morning and afternoon (Table 1), Ψl was maintained fairly constant and around 1.4–1.6 MPa, which allowed FI and LI trees to avoid critical Ψl values. Critical thresholds in olive have been reported to be around −1.5 MPa (Sofo et al. 2008). Differences in gs between morning and afternoon were proportional to

those observed in Da (Fig. 4), assuring a good balance between the increase in water demand and supply. This behaviour is typical for olive, a species well adapted to drought, with stomatal closure from early in the morning as an efficient strategy to reduce water losses and thus decreases in water potential (Fernández et al. 1997; Cuevas et al 2010). Despite restrictive stomatal closure recorded also in the R trees, these trees reached Ψl values more than two-fold lower than irrigated trees (Figs. 2 and 3).

Interestingly, similar PLC values (Fig. 4) were ob- served in all treatments although only R trees were exposed to low Ψl during the study. Since vulnerability curves also reflected some native PLC, a recomputed vulnerability curve based on data of all treatments was constructed (Fig. 5b). This curve enabled to compute the air entry pressure (Pe), indicating the threshold xylem

0

20

40

60

80

100

R LI FI

Pe

x (MPa) -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0

P L C

0

20

40

60

80

Recomputed data Recomputed vulnerability curve

Pe

b

a

Fig. 5 a Xylem vulnerability to embolism curves of current- year shoots of FI (○), LI (white triangle) and R (white square) trees, represented as the percentage loss of conductivity (PLC), as a function of decreasing xylem water potential (Ψx). Data points are the average of five to seven samples; vertical bars represent ± the standard error. The dashed grey line indicates 50 % loss of hydraulic conductivity; b All data represented in Fig. 5a were recomputed considering PLC=0 at Ψx=0, and the resulting vulnerability curve fitted and plotted. The black dashed line represents the tangent through the midpoint of the vulnerability curve and its x-intercept represents the air entry pressure (Pe) following Meinzer et al. (2009)

Table 2 Values both of the xylem tension inducing 50 % loss of hydraulic conductivity (P50) and their 95 % confidence interval (95 % CI). Also shown are the mean vessel densities (n=4) found in current-year shoots from trees of each treatment. P50 values which 95 % CI do not overlap are considered significantly differ- ent. No significant differences were found on vessel density be- tween treatments (p<0.05); SE = standard error

Treatment P50 (MPa) Vessel density (vessels mm−2)

R −4.35 (−3.67;−4.97) 618±63 SE LI −4.02 (−3.50;−4.52) 555±69 SE FI −4.26 (−3.59;−4.91) 499±75 SE

Diameter classes ( m) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36

D is

tr ib

u tio

n (

% )

0

2

4

6

8

10

12

14

16

18

20

FI LI R

Fig. 6 Xylem vessel diameter distribution measured in four cross-sections per treatment from current-year shoots. From 300 to 500 vessels were measured per section. Vertical bars represent ± the standard error. No significant differences were found between treatments

84 Plant Soil (2013) 373:77–87

pressure at which loss of conductivity begins to increase rapidly (Meinzer et al. 2009). In our case, Pe was around −1.3 MPa, demonstrating that water potentials observed in irrigated trees (i.e. FI and LI trees) were too high for any relevant induction of embolism. We thus conclude that their native embolisms were formed prior to our first measurements, maybe in an early stage of xylogenesis. During the study period, irrigated trees were obviously able to keep their water potential in a safe range and to avoid further embolism. It is well known that under non- extreme conditions, gs is regulated to keep Ψl within a safety margin avoiding the development of embolism (Brodribb and Holbrook 2003; Cochard et al. 2002; Meinzer et al. 2009; Choat et al. 2012). In contrast, R trees were not able to balance water deficits: observed minimum Ψl of ca. −4.8 MPa and maximum PLC of ca. 60 % (Figs. 2 and 3) correspond relatively well with the vulnerability curve. The full recovery of Ψl and gs in the R trees observed on DOY 274, after the water supplies of September (Fig. 1b), agrees with the well known capacity of the olive tree to recover quickly from water stress after the summer dry period (Lavee and Wodner 1991; Moriana et al. 2007).

Heterogeneity in soil water distribution within the rootzone of the LI trees can promote changes in the hydraulic conductance of the soil–leaf pathway, which may indirectly drive changes in gs and transpiration (Sperry et al. 2002). This, therefore, would explain the fact that the gs values recorded in trees with localized irrigation systems (i.e. LI trees) tended to be lower than in FI trees, which had access to water homogenously distributed in the soil (Fig. 3; Table 3). Indeed, an additional chemical root-to-shoot signalling mechanism triggered in the LI trees by roots remaining under soil drying conditions could also have influenced the sto- matal behaviour (Dodd et al. 2008), although this mechanism is still to be proven in olive. From a similar study carried out on the same trees and under the same treatments, Morales-Sillero et al. (2013)

recently reported that the reductions in gs observed in LI trees also reduced the net photosynthesis rate with regard the FI trees. They also reported that localized irrigation improved oil quality but reduced fruit yield as compared to an irrigation system able to wet the whole rootzone. Although they did not eval- uate water productivity in the orchard, they suggested that it would be higher in orchards with localized irrigation than in those with irrigation systems that wet the whole rootzone.

Acclimation in xylem anatomy and function

The applied water treatments did not influence the vulnerability to embolism (Fig. 5a, Table 2), although a significant fraction of the new wood in shoots was formed under the influence of the water treatments. Intraespecific differences in xylem resistance induced by soil moisture conditions have been reported for some species (Choat et al. 2007; Beikircher and Mayr 2009; Fichot et al. 2010), while other studies found no effects (Maherali et al. 2002; Cornwell et al. 2007). Lacking differences in hydraulic safety in studied ol- ive trees corresponded well with similarity in vessel- diameter distribution and vessel density across water treatments. Only the number of vessels per mm −2

tended to increase in trees with lower irrigation doses (Table 2). Contrasting results on the effect of the water regime on the xylem characteristics in olive have been previously reported for different olives varieties (Bacelar et al. 2007; Lopez-Bernal et al. 2010). These discrepancies could be due, apart from differences between cultivars, to the different age of the studied material, since histological characteristics may differ between young and adult olive trees (Lopez-Bernal et al. 2010). In each case, our results, reject our hypoth- esis about the influence of soil water conditions on the xylem anatomy and vulnerability to embolism of current-year shoots.

Table 3 Significances (p) of the differences in leaf water po- tential (Ψl), stomatal conductance (gs), specific hydraulic con- ductivity (Ks) and percentage loss of hydraulic conductivity

(PLC) during the season between full irrigated (FI) and localized irrigated (LI) trees calculated by using repeated measures (ANOVA) over time tests

Ψl gs Ks PLC

Predawn Morning Afternoon Morning Afternoon Morning Afternoon Morning Afternoon

p 0.025* 0.006* 0.019* 0.003* 0.000* 0.741 0.128 0.445 0.053

*Differences were considered significant when p<0.05

Plant Soil (2013) 373:77–87 85

Conclusions

Our results showed a tight control of gs in olive which allowed irrigated trees to avoid critical Ψl values and keep them in a safe range to avoid embolism. Marked stomatal closure was also observed in the R trees, but this did not impede Ψl values being lower in those trees than in the FI and LI trees. Values of Ks tended to be lower in the R than in the irrigated trees. All treatments, however, showed similar PLC values. The applied water treatments did not affect the vulner- ability to embolism, vessel-diameter distribution and vessel density of current-year shoots of mature ‘Man- zanilla’ olive trees.

Acknowledgments This project was funding both by the Spanish Ministry of Science and Innovation (research project AGL2006-04666/AGR) and the Austrian Science Fund (FWF) (project No. P20852-B16). We thank Antonio Montero for helping us in a great number of measurements. J.M. Torres- Ruiz held a doctoral fellowship from the Spanish Ministry of Science and Innovation (BES-2007-17149, MCINN).

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