Relationships between stomatal behavior, xylem vulnerability to cavitation and leaf water relations in two cultivars of Vitis vinifera

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Relationships between stomatal behavior, xylem vulnerability to cavitation

and leaf water relations in two cultivars of Vitis vinifera

Sergio Tombesia,*, Andrea Nardinib, Daniela Farinellia and Alberto Palliottia

aDepartment of Agricultural and Environmental Sciences, University of Perugia, Borgo XX giugno

74, 06121 Perugia, Italy bDepartment of Life Sciences, University of Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy

*Corresponding author, e-mail: [email protected]

Current understanding of physiological mechanisms governing stomatal behavior under water stress

conditions is still incomplete and controversial. It has been proposed that coordination of stomatal

kinetics with xylem vulnerability to cavitation (VC) leads to different levels of isohydry/anisohydry in

different plant species/cultivars. In this study, this hypothesis is tested in Vitis vinifera cultivars

displaying contrasting stomatal behavior under drought stress. The cv Montepulciano (MP, near-

isohydric) and Sangiovese (SG, anisohydric) were compared in terms of stomatal response to leaf and

stem water potential, as eventually correlated to different petiole hydraulic conductivity (kpetiole) and

VC, as well as to leaf water relations parameters. MP leaves showed almost complete stomatal closure

at higher leaf and stem water potentials than SG leaves. Moreover, MP petioles had higher maximum

kpetiole and were more vulnerable to cavitation than SG. Water potential at the turgor loss point was

higher in MP than in SG. In SG, the percentage reduction of stomatal conductance (PLgs) under water

stress was almost linearly correlated with corresponding percentage loss of kpetiole (PLC), while in MP

PLgs was less influenced by PLC. Our results suggest that V. vinifera near-isohydric and anisohydric

genotypes differ in terms of xylem vulnerability to cavitation as well as in terms of kpetiole, and that the

coordination of these traits leads to their different stomatal responses under water stress conditions.

Abbreviations – gs, stomatal conductance; Kmax, maximum hydraulic conductance; kpetiole, petiole

hydraulic conductivity; MP, Montepulciano; PLC, percentage loss of hydraulic conductance; PLgs,

percentage reduction of stomatal conductance; RWC, relative water content; RWCtlp, relative water

content at turgor loss point; SG, Sangiovese; VC, vulnerability curves; ε, leaf modulus of elasticity;

πo, osmotic potential at full turgor; πtlp, turgor loss point; Ψleaf, leaf water potential.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ppl.12180


Introduction

Plant-level gas exchange rates, photosynthesis, productivity and survival, ultimately depend on leaf-

level stomatal regulation of carbon gain versus water loss (Jones 1998). Under water stress conditions,

the onset of partial or total stomatal closure prevents excessive tissue dehydration and protects the

plant hydraulic system from runaway cavitation and catastrophic hydraulic failure that might occur

because of uncontrolled decline of leaf and stem water potential (Tyree and Sperry 1988). The ability

of different species to maintain a positive carbon balance under drought stress conditions is largely a

consequence of their different stomatal response to declining leaf water potentials (Chaves et al.

2010). On the basis of stomatal kinetics under water stress conditions, plants are generally classified

in two categories: isohydric species, which prevent water potential drop by means of early stomatal

closure, and anisohydric species, which maximize photosynthetic gain by keeping stomata open

despite larger water potential drops (Tardieu and Simonneau 1998). As an example, Vitis vinifera

comprises cultivars classified as near-isohydric, while other cultivars are known to be largely

anisohydric (Schultz 2003, Medrano et al. 2003, Poni et al. 2007, Palliotti et al. 2009, Pou et al. 2012),

although the classification into one or the other category is somehow difficult and uncertain because

of different experimental conditions and techniques adopted in different studies (Chavez et al. 2010).

The mechanism(s) responsible for stomatal closure under water stress are still controversial and

debated (Comstock 2002). Some studies have suggested that chemical signals are the main factor

driving stomatal closure, and further hypothesized that differential species-specific sensitivities to

[ABA] changes are at the basis of isohydric vs anisohydric behavior (Davies and Zang 1991, Tardieau

and Simonneau 1998, Lovisolo et al. 2002). On the other hand, stomatal closure has been reported to

be correlated to changes in turgor pressure of both epidermal and mesophyll cells (Cowan 1977,

Syvertsen 1982, Ache et al. 2010). Turgor loss is a critical outcome of water stress, and

osmoregulation leading to lower turgor loss point (Ψtlp) is generally regarded as an important strategy

adopted by plants to improve drought tolerance (Bartlett et al. 2012). In fact, plant species that

maintain positive turgor pressure at very low leaf water potential are also known to keep high stomatal

conductance, gas exchange and growth rates at relatively lower soil water potential than species

characterized by less negative Ψtlp values (Abrams and Kubiske 1990, Sack et al. 2003, Baltzer et al.

2008, Mitchell et al. 2008, Blackman et al. 2010).

Stomatal conductance to water vapor has been reported to be coordinated with liquid phase

hydraulic conductance of the soil-to-leaf pathway (Saliendra et al. 1995, Comstock 2002, Meinzer

2002), suggesting a possible feedback link between stomatal regulation and hydraulic signals (Nardini

and Salleo 2000, Franks 2004). Stomatal control of transpiration is known to be involved in xylem

cavitation avoidance mechanisms (Jones and Sutherland 1991, Tyree and Sperry 1989). Salleo et al.

(2000) have reported that the onset of partial stomatal closure in transpiring Laurel plants occurs when

stem water potential approaches the cavitation threshold, as revealed by concurrent production of


ultrasound acoustic emissions at the stem and leaf level (Tyree and Dixon 1983).

Xylem hydraulic efficiency and vulnerability to cavitation-induced embolism are correlated to

several anatomical traits, including xylem conduit dimensions and arrangement, as well as to pit

membrane features (Sperry and Saliendra 1994, Lo Gullo et al. 1995, Choat et al. 2008, Lens et al.

2011). Previous studies have reported differences in terms of stem hydraulic conductance among

different genotypes of V. vinifera. In particular, Schultz (2003) has suggested that higher stem specific

conductivity was at the basis of the isohydric behavior of the cultivar Grenache when compared to the

anisohydric Syrah. Much less is known about the eventual relationships between xylem vulnerability

to cavitation and the relative level of isohydry of different grape cultivars, although it has been

suggested that contrasting hydraulic vulnerability to cavitation of different genotypes might also be an

important determinant of their different stomatal responses to declining water potential (Schultz

2003). Alsina et al. (2007) have reported large variability of vulnerability to cavitation across eight

different grape cultivars, with values of leaf water potential inducing 50% loss of hydraulic

conductance (Ψ50) ranging from –1 MPa in cv Parellada to about –3 MPa in cv Sauvignon Blanc.

However, xylem vulnerability was not correlated to drought tolerance mechanisms at the leaf level as

estimated on the basis of water relation parameters derived on water potential isotherms, in contrast

with data gathered from inter-specific comparisons (Vilagrosa et al. 2010). Overall, the above studies

did not provide clear-cut evidence about eventual correlations between vulnerability to cavitation and

stomatal regulation in grape.

In the present study, two grapevine cultivars were investigated for their stomatal and hydraulic

responses to drought stress. In particular, we selected two genotypes known for their contrasting

stomatal behavior i.e. the cv Sangiovese which is anisohydric, and the cv Montepulciano which

displays a near-isohydric behavior (Palliotti et al. 2009). The two cultivars are known to differ from

each other also in terms of xylem anatomy and other functional properties linked to photosynthetic

efficiency and energy dissipation potential like chlorophyll and carotenoids contents (Palliotti et al.

2008, 2011). Our aim was to identify the eventual relationships and mechanistic link between stomatal

behavior and xylem vulnerability to cavitation, as well as eventual coordination of the relative level of

isohydry/anisohydry to water relation parameters correlated to drought resistance.

Materials and methods

Plant material

This study was conducted during 2012 on 7-year-old potted V. vinifera vines of cultivar Sangiovese

(clone VCR30) and cultivar Montepulciano (clone R7), both grafted onto 1103 Paulsen rootstock and

grown in an outdoor area close to the Faculty of Agriculture of the University of Perugia (Region of

Umbria, central Italy, 42°58’N, 12°24’E, elevation 405 m a.s.l.). Pots (60 liters volume) were filled

with loam soil having field capacity and permanent wilting point water contents of 30.2 and 16.7%

(v/v), respectively. At the end of February, each vine was pruned to retain four spurs with two buds


each. All shoots were oriented upright using suitable stakes. Ten vines per cultivar were used and

initially maintained at ~90% of maximum soil water availability. Daily water supply per pot was

determined by monitoring the soil water content with a Diviner 2000 capacitance probe (Sentek

Environ Tech., Sentek Environment Technologies, Stepney, South Australia), using access tubes

located in three pots per cultivar. Measurements were performed at 100 mm, 170 mm and 250 mm of

depth from the soil surface in the pot. Water was added in the required amounts every day at 8 pm.

Additional experiments were performed in 2013 on the same vines used in 2012. Vines were kept

irrigated to field capacity until 7 July, when drought was imposed by suspending irrigation and

closing pots into plastic bags. The drought treatment was continued until complete leaf abscission.

Stomatal conductance vs water potential

In 2012, stomatal conductance (gs) measurements were carried out on adult, non senescent, primary

leaves inserted between the 5th and the 10th node from the cane base. Measurements were performed

under saturating light conditions (PPFD > 1200 µmol photons m–2 s–1) using an open gas exchange

system (ADC-System, LCA-3, Hoddesdon, UK) equipped with a Parkinson leaf chamber.

Measurements were repeated during three consecutive sunny days (17, 18 and 19 July 2012) between

08.00 and 10.00 a.m. Two canes per vine, sampled from five different vines per cultivar, were

harvested at 08.00 by cutting the stem under water. Canes were transported to the laboratory and

allowed to dehydrate in the light. At different time intervals, leaves were measured for gs and

immediately detached to record their water potential using a pressure chamber (Soilmoisture Corp,

Santa Barbara, CA, USA).

In 2013, stomatal conductance (gs) measurements were carried out on adult, non senescent,

primary leaves grown between the 5th and the 10th node from the cane base in order to assess if

measurements of gs carried out on detached shoots were eventually biased by shoot cutting procedure.

Measurements were carried out between 12.00 a.m. and 1.00 p.m. from 8 July up to 16 July on 5 vines

per cultivar using the same equipment described above. Leaves were measured for gs and immediately

detached and wrapped in plastic film to record their water potential (Ψleaf) using a pressure chamber

(Soilmoisture Corp, Santa Barbara, CA, USA). Stem xylem water potential (Ψstem) was measured on

mature leaves that had been wrapped in plastic film and aluminum foil prior to the measurements

(McCutchen and Shackel 1992).

Xylem anatomy

In August 2012, three petioles per vine were sampled from five vines per cultivar. Petioles were

sampled from leaves grown between the 5th and the 10th node from the cane base. The basal portion

(~20–30 mm) of petioles was then sampled and immediately placed in ice and refrigerated until

sectioned. Samples were embedded in an agar solution (0.2%) and cross sections (four per sample) 20

µm thick were obtained using a microtome (2700 Frigocut, Reichert-Jung, Nossloch, Germany).


Sections were stained with Iodine green to increase the contrast. Photographs of the cross-sections

were taken with a Leica ICCA camera (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany)

coupled to a Leica DMLB light microscope (Leica Microsystems Wetzlar GmbH). Images were

acquired with Leica IM1000 software (Leica Microsystems Digital Imaging, Cambridge, UK). Two

photographs were taken from each cross-section at 200× magnification. Measurements of vessel

diameters were taken using SIGMASCAN PRO 5.0 software (Systat Software Inc., San Jose, CA, USA)

(Palliotti et al. 2011). Mean diameter distributions of vessels were then reported in frequency classes

of 5 µm expressed as percentage of the total number of vessels.

The theoretical hydraulic conductance (kh) (kg m MPa–1 s–1) was calculated according to the

modified Hagen-Poiseuille law as reported by Tombesi et al. (2010):

128

where d is the radius of the vessel (m), obtained from the mean of the largest and smallest radii, ρ is

the fluid density (assumed to be 1000 kg×m3 or equal to that of water at 20°C) and η is the viscosity

(assumed to be 1×10–9 MPa s–1, or equal to that of water at 20°C).

Vulnerability curves

Vulnerability curves were constructed using the double-ended pressure sleeve method described by

Cochard et al. (1992) and Ennajeh et al. (2011). A custom built apparatus was used, consisting of a T

steel fitting (50 mm long) that was used as sleeve, and two compression fittings at the two ends of the

sleeve. Rubber corks were used to seal the sleeve. Internal distance between corks when the sleeve

was sealed was ~ 4 mm. Measurements were performed in August 2012 on samples collected between

8.00 and 13.00. Minimum length of petioles used in this study was 11 cm i.e. substantially longer than

the longest xylem vessel in petioles of V. vinifera (Chatelet et al. 2011). Hydraulic conductance of

petioles was measured by connecting one sample end to a vertical plastic tubing filled with a filtered

(0.2 µm) 20 mM KCl solution. The pressure head was maintained at 6 kPa. Water flow through the

petioles was measured continuously while the air pressure inside the chamber was gradually

increased. Flow was measured gravimetrically by collecting the fluid from the distal end in pre-

weighted sponge pieces fitted in plastic tubes. Flow readings were taken over 1 min time intervals.

At the beginning of each experiment, the petioles were flushed at P = 0.2 MPa for 30 min, to

remove any eventual native embolism. After flushing samples, maximum hydraulic conductance

(Kmax) was measured. Then, the pressure in the chamber was progressively increased to 0.3, 0.5, 0.8,

1.0, 1.3, 1.5, 1.8, 2.0, 2.3 and 2.5 MPa. Sample hydraulic conductance (K) was measured at steady

state (10 min after the desired pressure value was reached) at each pressure step. Percentage loss of

hydraulic conductance (PLC) at each pressure value was calculated as:


PLC = 100 × (Kmax–K)/Kmax

The leaf specific petiole hydraulic conductivity (kpetiole) was finally estimated according to Zufferey et

al. (2011) as:

kpetiole = (Kmax×L)/Aleaf

where L is petiole length and Aleaf is leaf blade surface area.

Relationship between percent loss of gs (PLgs) and PLC was obtained by interpolating gs value

from the regressions between gs and Ψstem for Ψstem values at which PLC values were measured. PLgs

representing as percentage of the maximum gs of the curve.

Pressure-volume curves

Leaf water potential isotherms, commonly known as pressure-volume (P-V) curves, were measured in

August 2012 on ten mature leaves per cultivar (two leaves from each of five different vines). Leaves

were harvested at 06.00 a.m. and petioles were kept immersed in deionized water for 2 h to allow full

rehydration. Leaves with Ψleaf < –0.05 MPa after rehydration were immediately discarded. Each leaf

was weighted using an analytic balance (OHAUS PA64C, OHAUS Corp., USA) and water potential

was immediately measured by a pressure chamber. Leaves were progressively dehydrated on the

bench and successive measurements of leaf weight and water potential were taken. Turgor loss point

(πtlp) and relative water content (RWC) at turgor loss point (RWCtlp) of each leaf was calculated as the

intersection point of the two phase linear equation representing the relationship between leaf RWC

and Ψleaf (Brodribb and Holbrook 2003). Leaf modulus of elasticity (ε) and the osmotic potential at

full rehydration (πo) were respectively calculated as the slope of the pressure potential curve between

100–RWC=0 and πtlp and the y-intercept of the solute potential curve (see Bartlett et al. 2012).

Differences between genotypes were assessed using the Student’s t-test (P<0.05). The

significance of regressions was tested using Pearson Product Moment Correlation. Test of

homogeneity of regression slope was used to assess if the slope of correlations was similar, an

ANCOVA was performed in order to test whether the null hypothesis was satisfied.

Results

While predawn water potential was similar in Sangiovese and Montepulciano, stem water potential at

midday was more negative in Sangiovese than in Montepulciano (Fig 1A). A similar pattern was

observed for leaf water potential (Ψleaf), which in both cultivars reached more negative values than

Ψstem (Fig 1B). In both genotypes, gs decreased as a function of declining Ψleaf and Ψstem, both when

measured on desiccating detached shoots or on whole drought-stressed plants (Fig. 2). The

relationship was significantly fitted by a sigmoid regression in all cases and the water potential


inducing stomatal closure was independent on the method used. The two fitted curves were

significantly different depending on the genotype (test of homogeneity of regression slope P=0.0025,

P=0.005 and P=0.036 for relationships in fig 2A, 2B and 2C, respectively). However, stomatal

conductance measured on leaves from intact plants was larger than that measured on detached shoots.

Sangiovese had lower maximum gs than Montepulciano. Sangiovese also showed a very progressive

(almost linear) gs response to decreasing water potential and underwent complete stomatal closure at

Ψ = –2.0 MPa (Fig 2A, B and C). Montepulciano leaves displayed a sharp decrease of gs at Ψleaf < –

0.8 MPa and Ψstem < –0.5, and reached full stomatal closure at Ψleaf = –1.4 MPa and Ψstem = –1.0 MPa,

already.

Both πtlp and πo were significantly lower in Sangiovese than in Montepulciano (Table 1). On the

other hand, RWCtlp and ε were not significantly different in the two genotypes. Overall, the

relationship between RWC and Ψleaf was similar for the two cultivars in the water potential range

between 0 and –1.5 MPa. Below this threshold value, however, Sangiovese leaves retained higher

RWC than Montepulciano (Fig. 3) as Ψleaf continued to decline.

The two cultivars also displayed differential vulnerability to xylem embolism. Petiole PLC was

significant larger in Montepulciano in the P range from –0.5 to –1.3 MPa, with respect to Sangiovese

(Fig 4). Water potential values inducing 20% (P20) and 50% (P50) loss of hydraulic conductivity were

significantly lower in cv Sangiovese than in cv Montepulciano, while both cultivars reached 80% loss

of hydraulic conductivity at similar xylem pressures (Table 2). Leaf specific petiole hydraulic

conductivity (kpetiole) was approximately 40% higher in Montepulciano than in Sangiovese, averaging

about 7.1 and 5.0 mmol m–1 s–1 MPa–1, respectively (Table 2).

In both cultivars, PLgs was significantly correlated to PLC in a sigmoid fashion (Fig. 5).

However, cv Sangiovese had a higher gs sensitivity to PLC, as the PLgs to PLC ratio was close to 1

over the PLC range between 10 and 60%. On the other hand, the cv Montepulciano showed almost no

stomatal adjustment down to PLC of about 20%. After this PLC level, gs started to decline sharply and

PLgs approached 100% at PLC = 40%.

In Sangiovese petioles, xylem tissue had significantly more vessels in the 5–10 µm diameter

class when compared to Montepulciano, whereas this latter cultivar had significantly more vessels in

10–15 µm, 15–20 µm and 25–30 µm diameter classes than the former (Fig. 6). In Sangiovese, a larger

fraction of hydraulic conductance was apparently supported by smaller xylem conduits when

compared to Montepulciano (Fig. 7 and Table 2). About 50% of the theoretical xylem hydraulic

conductance was supported by xylem conduits smaller than 18 and 21 µm in Sangiovese and

Montepulciano, respectively (Table 2).

In both cultivars, PLgs was larger than PLC, but had reached similar values at Ψ = –2.3 MPa and

–2.0 in Montepulciano and Sangiovese, respectively (Fig. 8).

Discussion


Montepulciano and Sangiovese vines displayed different levels of anisohydry. In both cultivars, Ψstem

declined during the central hours of the day but the decline was more marked in Sangiovese. This

different behavior can be attributed to different stomatal kinetics that resembled those previously

reported by Schultz (2003) for Grenache and Syrah cultivars, and were interpreted as evidence for

different levels of iso/anisohydry. Our results show that under water stress, Sangiovese reduces

stomatal conductance more gradually than Montepulciano and that, at relatively low water potentials

(< –1.5 MPa), it maintains higher gas exchange rates than Montepulciano. These results are in

accordance with previous data obtained on the same genotypes under field conditions (Palliotti et al.

2009). Furthermore, these results contribute to explain the different behavior observed in these same

cultivars under irrigation and after water stress acclimation (Palliotti et al. 2014). In our study, gs was

correlated both with Ψleaf and Ψstem. Stomatal closure was observed approximately at the same Ψleaf

and Ψstem values in both lab and field experiments, although the two curves differed in the central

region. In the field experiment carried out in 2013, gs values were higher than those recorded in the

experiment carried out in 2012 on excised sample, and this difference was probably due to the

severing of canes. However, the consistency of water potential values triggering stomatal closure in

the two experiments suggest that bench shoot dehydration could be a useful method for fast screening

of stomatal behavior across different grape genotypes.

Differently from other studies carried out on grapevine cultivars with different levels of

anisohydry (Tramontini et al. 2013, Hochberg et al. 2013), in our experiments the near-isohydric

cultivar Montepulciano had larger gs than Sangiovese. This is in accordance with the larger hydraulic

conductance of Montepulciano canes (Palliotti et al. 2011) and petioles in comparison with those of

Sangiovese. These results also differ from those reported by Beis and Patakas (2010) which did not

found any significant difference in plant hydraulic conductance in two grapevine cultivars with

different stomatal sensitivity to water stress. According to Schultz (2003), contrasting stomatal

behaviors among grapevine cultivars could be explained in terms of their different hydraulic

architecture. Our additional hypothesis was that differential vulnerability to cavitation might be at the

basis of contrasting stomatal kinetics under water stress conditions. In fact, xylem cavitation is a

hydraulic signal triggering stomatal closure (Salleo et al. 2000, Nardini et al. 2001). In turn, stomatal

regulation of transpiration limits stem and leaf water potential drop (Domec and Johnson 2012). The

regulation of water potential prevents further cavitation and embolism in the leaf and in the stem

(Tyree and Sperry 1988). Indeed, vulnerability curves of petioles, that could be considered as 'fuses' in

the hydraulic circuit (Zufferey et al. 2011), revealed that the near-isohydric cultivar Montepulciano

was more vulnerable to cavitation than the anisohydric one (Sangiovese), in agreement with Zufferey

et al. (2011) and Domec and Johnson (2012). Hence, our results apparently support the hypothesis of

coordination of stomatal closure with xylem vulnerability to cavitation, although only in Sangiovese

PLC was almost linearly correlated with PLgs, while the same relationship was weaker in

Montepulciano.


Different stomatal behaviors as related to water potential drop and consequent triggering of

cavitation events can be also interpreted as a functional consequence of contrasting hydraulic anatomy

in different cultivars (Chouzouri and Schultz 2005). In fact, xylem vulnerability to cavitation and

embolism is dependent on xylem anatomy. As an example, mean cavitation pressure was correlated

with inter-vessel pit structure in different Acer species (Lens et al. 2011) and Cai and Tyree (2010)

demonstrated that in aspen, xylem vessels with large diameter suffered 50% PLC at xylem pressures

less negative than those necessary to embolize smaller conduits. As previously reported by some of us

(Palliotti et al. 2011), Sangiovese petioles had smaller xylem conduits than Montepulciano, in

accordance with relatively higher resistance to xylem embolism of the former cultivar with respect to

the latter. Moreover, in Sangiovese a larger fraction of total xylem hydraulic conductance was

apparently supported by smaller vessels.

Alternatively or in addition to cavitation-associated hydraulic signals, bulk leaf water status can

influence stomatal closure, which has been reported to be correlated or coordinated to the turgor loss

point (Cowan 1977). In the present study, πtlp was more negative in Sangiovese (anisohydric) than in

Montepulciano (near-isohydric) genotype, thus suggesting a possible coordination of this parameter

with stomatal behavior in response to bulk leaf water potential changes. In contrast with Beis and

Patakas (2010), in the present study we observed differences also in terms of osmotic potential (πo)

between the two cultivars, but no difference in terms of ε and RWCtlp. These results support the

hypothesis that also in Vitis vinifera genotypes, eventual differences in terms of πtlp are mainly driven

by osmotic adjustment and not by modifications of mechanical properties of cell walls, in agreement

with previous reports on several different species (Lenz et al. 2006, Baltzer et al. 2008, Mitchel et al.

2008, Bartlett et al. 2012). In both Sangiovese and Montepulciano, πtlp was reached when almost 90%

of stomatal conductance reduction or complete stomatal closure had occurred, already. Such results

are consistent with previous studies showing that initiation of stomatal closure is largely uncoupled

from bulk leaf water status (Mott and Franks 2001, Brodribb et al. 2003). On the other hand, in both

cultivars near-complete stomatal closure occurred at Ψleaf close to πtlp, similarly to observations

reported by Brodribb et al. (2003) in several different species and with data reported for walnut and

loblolly pine by Cochard et al. (2002) and Domec et al. (2009). Our data suggest that although πtlp is

not the main factor triggering partial stomatal closure, it is indeed correlated to the occurrence of

complete halt of leaf-level gas exchange.

Stomatal behavior in Montepulciano was apparently addressed at keeping high gas exchange

rates at moderately negative water potentials (Ψstem > –0.5), commonly reached under well-watered

conditions (fig 1). Moreover, Montepulciano petioles were more vulnerable to cavitation than

Sangiovese. If the stomatal kinetics were exclusively dominated by hydraulic signals, genotypes with

more vulnerable hydraulic apparatus would be expected to suffer gas exchange reduction even for

relatively small drops of leaf/stem water potential, as commonly experienced under non-stressful

conditions during the middle part of the day when irradiance and temperature are at their peaks.


However, Montepulciano vines under well-watered conditions were able to transpire at faster rates

than Sangiovese (Palliotti et al. 2011). Under these conditions Ψleaf normally drops down to –0.8 MPa

(Zufferey et al. 2011) and Ψstem drops to –0.6 MPa, and, according to our data, PLC of Montepulciano

would be expected to be significantly higher than that of Sangiovese. According to the hypothesis of

complete coordination of stomatal behavior with xylem vulnerability to cavitation, stomatal

conductance should be lower in Montepulciano than in Sangiovese under these conditions. Our data

show an opposite behavior, suggesting that stomatal kinetics are not coordinated with xylem

vulnerability in the same way in all cultivars and at any level of water stress. This might arise as a

consequence of the fact that stomatal conductance is more closely related to actual values of kpetiole

rather than to relative loss of hydraulic conductivity. In fact, in accordance with previously reported

data on vine canes (Palliotti et al. 2011), Montepulciano had higher kpetiole than Sangiovese. As a

result, even at higher PLC in the former than in the latter cultivar, the actual water transport efficiency

per unit leaf surface area in the two cultivars might be similar. As an example, at Ψstem = –0.8 MPa, gs

was similar in the two cultivars, despite the fact that PLC of Montepulciano was higher (39%) than

that of Sangiovese (26%). However, taking into account the maximum kpetiole measured for the two

cultivars, at this water potential level it can be calculated that the actual kpetiole would still be slightly

higher in Montepulciano than in Sangiovese (4.6 vs 3.6 mmol m–1 s–1 MPa–1, respectively). This

would translate in similar water transport rates per unit leaf surface area and, hence, similar leaf

hydration and stomatal opening. However, in both cultivars stomatal closure occurred at Ψstem close to

those at which 50% of PLC occurs (P50). These results support the coordination of stomatal closure

with xylem vulnerability and the validity of P50 as an indicator of xylem hydraulic safety (Meinzer et

al. 2009, Choat et al. 2012).

In conclusion, different stomatal behaviors of Montepulciano and Sangiovese grapevines,

respectively near-isohydric and anisohydric, properly correlate to different levels of xylem

vulnerability to water stress-induced cavitation, provided actual values of stem/petiole hydraulic

conductivity are taken into account. Previous studies have analyzed many grapevine cultivars for their

contrasting stomatal behavior, and in several cases the same cultivar was classified as isohydric or

anisohydric by different studies (Chaves et al. 2010, Lovisolo et al. 2010). Experimental conditions

can be claimed as possible causes for such contradictory results. Our results point out that, depending

on the water potential range at which the experiment is carried out, the same cultivar can behave in

relatively opposite ways. Previous studies have proposed that cultivar-specific differences in stomatal

control in response to water stress arise as a result of differences in the ABA signaling and perception

machinery (Soar et al. 2006, Perrone et al. 2012a) and/or as a consequence of different patterns of

aquaporins expression and/or activation (Vandeleur et al.2009, Perrone et al.2012b, Pou et al 2013).

Although our data point to a mechanistic linkage between stomatal behavior and vulnerability to

cavitation, the possibility that other phenomena integrated hydraulic signals and triggered or mediated

stomatal closure cannot be ruled out.


Author contributions – S.T., A.N. and A.P. conceived and planned the study. S.T. carried out the

experiment, analyzed the data and wrote the first draft of the manuscript. A.P. planted and managed

the vines that were used in the study, and reviewed the manuscript. A.N. revised and edited the

manuscript. A.P. and D.F. helped in the analysis of the data, revised and edited the manuscript, and

obtained funds to support the project.

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Figure legends

Fig. 1. Stem water potential (Ψstem) (A) and leaf water potential (Ψleaf) (B) pattern on 8/7/2013 in

Sangiovese and Montepulciano grapevine. Data were collected from five vines per each cultivar.


Fig. 2. Relationship between leaf water potential (Ψleaf) and stomatal conductance (gs) in primary

leaves of detached shoots of Sangiovese and Montepulciano grapevine in 2012 (A). Data were

collected from five vines per each cultivar. Data were fitted to a sigmoid curve (Sangiovese R2=0.81,

P<0.001; Montepulciano R2=0.89, P<0.001). Relationship between leaf water potential (Ψleaf) (B),

stem water potential (Ψstem) (C) and stomatal conductance (gs) in Sangiovese and Montepulciano

grapevine in 2013. Data were collected from five vines per each cultivar. Data were fitted to a sigmoid

curve [(B) Sangiovese R2=0.86, P<0.001; Montepulciano R2=0.83, P<0.001; (C) Sangiovese R2=0.86,

P<0.001; Montepulciano R2=0.89, P<0.001].

Fig. 3. Relationship between leaf relative water content (RWC) and leaf water potential (Ψleaf) in

leaves of Sangiovese and Montepulciano grapevines. Data were obtained from 10 leaves per cultivar.


Fig. 4. Relationship between percent loss of hydraulic conductance (PLC) and stem water potential

(Ψstem) (MPa) in petioles of Sangiovese and Montepulciano grapevines. Each point is the mean of five

vines (n=5) ± SE. Data of each cultivar were fitted in a sigmoidal curve (Sangiovese R2=0.98,

P<0.001; Montepulciano R2=0.96, P<0.001).

Fig. 5. Relationship between percentage loss of leaf stomatal conductance (PLgs) and percentage loss

of petiole hydraulic conductance (PLC) in Sangiovese and Montepulciano grapevines. PLgs was

calculated on the basis of the regression curves shown in Fig 4. Data of each cultivar were fitted in a:

sigmoidal curve (Sangiovese R2=0.98, P<0.001; Montepulciano R2=0.99, P<0.001).


Fig. 6. Number of xylem vessels (% of total) per vessel size class (µm) in petioles of Sangiovese and

Montepulciano grapevines. Bars are mean values of five vines (n=5) per cultivar ± SE.

Fig. 7. Relationship between cumulated theoretical hydraulic conductance (% of total) and vessel

diameter (µm) in petioles of primary leaves in Sangiovese and Montepulciano grapevines.


Fig. 8. Relationship between percentage loss of leaf stomatal conductance (PLgs), percent loss of

petiole hydraulic conductance (PLC) and leaf water potential (Ψleaf) in Montepulciano (A) and

Sangiovese (B) grapevine. Solid vertical lines represent the leaf water potential at turgor loss point

(πtlp) in Montepulciano (A) and Sangiovese (B) leaves.


Table 1. Leaf water potential at turgor loss point (πtlp) (MPa), modulus of elasticity (ε) (MPa), relative

water content at turgor loss point (RWCtlp) (%), and osmotic potential at full turgor (πo) (MPa) of

leaves of Sangiovese and Montepulciano grapevines. Values are means of leaves sampled from five

vines (n=5) ± SE. For each parameter significant differences are indicated with * (P<0.05).

Sangiovese Montepulciano t-test

πtlp –1.67 ± 0.09 –1.36 ± 0.08 *

πo –1.38 ± 0.04 –1.16 ± 0.08 *

RWCtlp 95.50 ± 0.81 96.55 ± 0.39 ns

ε 7.82 ± 0.75 7.96 ± 0.61 ns


Table 2. Maximum diameter of xylem vessels (µm) accounting for 20, 50 and 80% (D20, D50, D80

respectively) of total theoretical hydraulic conductance (as shown in Fig. 7); xylem pressures (MPa)

inducing percentage loss of conductance (PLC) of 20, 50 and 80% (P20, P50, P80) in petioles and leaf

specific petiole hydraulic conductivity (kpetiole) in Sangiovese and Montepulciano grapevines. Values

are means of leaves from five vines (n=5) ± SE. For each parameter significant differences are

indicated with * (P<0.05).

Sangiovese Montepulciano t-test

D20 (µm) 14.63 ± 0.23 16.27 ± 0.19 *

D50 (µm) 18.59 ± 0.31 20.93 ± 0.42 *

D80 (µm) 22.33 ± 0.33 26.23 ± 0.53 *

P20 (MPa) –0.67 ± 0.09 –0.49 ± 0.05 *

P50 (MPa) –1.25 ± 0.07 –1.08 ± 0.05 *

P80 (MPa) –1.78 ± 0.08 –1.79 ± 0.05 ns

kpetiole (mmol m–1 s–1 MPa–1) 5.04 ± 0.46 7.13 ± 1.19 *

Documents

Relationships between stomatal behavior, xylem vulnerability to cavitation and leaf water relations in two cultivars of Vitis vinifera