the reflectivity in the s-band and the broadband ultrasonic spectroscopy as new tools for the study...

Physiologia Plantarum 148: 512–521. 2013 © 2012 Scandinavian Plant Physiology Society, ISSN 0031-9317

The reflectivity in the S-band and the broadband ultrasonicspectroscopy as new tools for the study of water relationsin Vitis vinifera L.Domingo Sancho-Knapika, Jose Javier Peguero-Pinab, Hipolito Medranob, Marıa Dolores Farinasc,Tomas Gomez Alvarez-Arenasc and Eustaquio Gil-Pelegrına,∗

aUnidad de Recursos Forestales, Centro de Investigacion y Tecnologıa Agroalimentaria, Gobierno de Aragon, 50059, Zaragoza, SpainbDepartament de Biologia, Universitat de les Illes Balears, Carretera de Valldemossa, 07071, Palma de Mallorca, SpaincUltrasound for Medical and Industrial Applications (UMEDIA) research group, Centre for Physical Technologies, C.S.I.C., 28002, Madrid, Spain

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

Received 15 October 2012;revised 26 October 2012

doi:10.1111/ppl.12007

The large water requirements of Vitis vinifera L. together with an increasein temperature and drought events imply the need for irrigation in thedriest areas of its distribution range. Generous watering may reduce grapequality so irrigation should be precisely regulated through the developmentof new methods of accurate irrigation scheduling based on plant ‘stresssensing’. Two new methods, the reflectivity in the S-band and the broadbandultrasonic spectroscopy, can be used as non-invasive and reproducibletechniques for the study of plant water relations in V. vinifera. On onehand, the measurement of reflectance at frequencies around 2.4 GHz givesan excellent accuracy when the changes in the existing area (S) between tworeflectance curves are correlated with the relative water content (RWC). Onthe other hand, an improvement of the broadband ultrasonic spectroscopybased on the enlargement of the analysis frequency window provides, apartfrom the determination of the turgor loss point (TLP), additional informationabout the leaves without additional computational cost or additional leafinformation requirements. Before TLP, the frequency associated with themaximum transmittance (f/fo), the macroscopic elastic constant of the leaf inthe Z direction (c33) and, specially, the variation of the attenuation coefficientwith the frequency (n), were highly correlated with changes in RWC. Onceturgor is lost, a shift in the parameters directly related to the attenuation ofthe signal was also observed. The use of both techniques allows for a moreconvincing knowledge of the water status in V. vinifera.

Introduction

Grapevine crop is of great importance not only froman economic point of view, but also because of itsenvironmental impact because of the extension of itscultivation around the world, especially in Europe andSpain. Moreover, grapevine crop is, for many European

Abbreviations – DW, dry weight; FW, fresh weight; P–V, pressure–volume; RWC, relative water content; TLP,turgor loss point; TW, turgid weight.

and ‘new world’ areas, an integral part of culturalheritage and landscape. A considerable amount ofvineyards are located in areas under Mediterraneantype climates (Tonietto and Carbonneau 2004) whereVitis vinifera L. is able to withstand the water deficitduring the summer drought (Chaves et al. 2010).A prolonged water scarcity or fluctuating water soil

512 Physiol. Plant. 148, 2013

availability severely reduces yield thus compromisingeconomic viability of the crop. Episodes of severe soilwater deficits are becoming more frequent and areincreasingly threatening yield and affecting berry qualityin many viticulture areas (Chaves et al. 2007). Moreover,climate change is projected to exacerbate these impacts,with more frequent and extreme high temperature anddrought events in many parts of South Europe (Garcıa-Mozo et al. 2010). In consequence, the climate changerepresents a risk for the wine industry and may force ashift of production to cooler areas in parallel with theuse of new varieties better adapted to warmer and dryerconditions (Schultz and Stoll 2010). Increasing waterscarcity could lead to a more frequent use of irrigationfor affordable crop productivity as well as to importantchanges in the optimum areas for different grape varieties(Chaves et al. 2007).

As agriculture is considered the largest waterconsuming sector (accounting between 50 and 85% ofwater consumption), and given the large areas coveredby grapevine crop and the high water requirements ofgrapevines along the growing season (Netzer et al. 2005,Zhang et al. 2007), there is an increasing concern aboutthe optimization of water use in vineyards irrigation tosecure a more environmentally sustainable viticulture.On the other hand, and even though the relationship isnot always coincident, the most generalized tendency formain grapevine cultivars shows that generous wateringcan reduce the quality of the fruit, through a decrease incolor and sugar content, an imbalanced acidity andinterfering the flavonoid development (Flexas et al.2010). Provided the large dependence of berry qualityparameters on soil water availability, irrigation shouldbe precisely regulated to achieve reasonable fruit quality(Medrano et al. 2003, Keller et al. 2008, Romero et al.2010, Pou et al. 2011) through the development of newmethods of accurate irrigation scheduling and control(Jones 2004).

It has been suggested that the use of plant ‘stresssensing’ is a better way to implement adequate irrigationscheduling than only estimating the atmospheric waterdemand or the soil moisture status directly (Jones 1990,2004, 2007). Plant sensing can be achieved by the directmeasurement of leaf water potential (�) (Jones 2004),continuous changes in leaf turgor with leaf-clamps(Zimmermann et al. 2008) or indirect measurement ofleaf water content (Sancho-Knapik et al. 2010, 2011a).The functional response of the plant in relation to itswater status can be also predicted by monitoring differentplant physiological responses (Cifre et al. 2005), such asstomatal conductance (Medrano et al. 2002, Vilagrosaet al. 2003) or leaf temperature (Grant et al. 2006,Suarez et al. 2009). Alternative methods for plant stress

sensing based on the response of the plant material toa certain stimulus have also been proposed, becausethe physical properties of plant tissues have been foundto vary according to the degree of hydration. In thisway, Carter (1991) and Carter and McCain (1993) foundthat a decrease in leaf water content was generallyassociated with an increase in reflectance throughoutthe 400–2500 nm wavelength range spectrum. Sincethen, several authors employed different techniquesconcerning infrared frequencies (Penuelas et al. 1993,Sims and Gamon 2003, Seelig et al. 2009, Wu et al.2009).

Some other techniques have been proposed for theestimation of plant water status. In this sense, lowerelectromagnetic frequency ranges have also been tested(Jordens et al. 2009). Martınez et al. (1995) describeda time domain reflectometry method to estimate leafdisk water status by measuring reflectivity on theX-band (7–12 GHz) with a non-portable laboratoryequipment. More recently, Menzel et al. (2009) proposeda non-invasive technique for measuring the changesin the dielectric properties of a whole plant whenintroduced in a microwave cavity resonator. In spiteof the good results obtained by Martınez et al. (1995)and Menzel et al. (2009), the complexity of theexperimental setup and the low portability preventedthe applicability of such methods for the developmentof practical tools to characterize plant water statusunder field conditions so far. More recently, Sancho-Knapik et al. (2011a) used a microwave digital cordlesstelephony patch antenna to measure the reflectivity ata frequency of 1730 MHz (L-band). The method yieldedan accurate estimation of relative water content (RWC)in Populus euramericana leaves with a technologicallysimple device that constitutes a solid support for thedevelopment of a portable tool for determining plantwater status under field conditions. However, thedimension of the antenna (80 mm in diameter) and thefrequency range may limit the usefulness of this antenna.The use of another range of frequencies, especially inthe free and widely used S-band, and the reduction ofthe antenna dimension are two requirements for a wideruse of this technology.

Another emergent technique, the broad-band ultra-sonic spectroscopy technique (Gomez Alvarez-Arenaset al. 2009, Sancho-Knapik et al. 2010, 2011b, 2012),has been proven as a non-destructive, non-invasive,non-contact and reproducible method for the dynamicdetermination of leaf water status. It is based on theexcitation of thickness resonances on the leaves andon the analysis of the spectral response in the vicinityof the first order thickness resonance. This approachhas the advantage to make it possible to consider the

Physiol. Plant. 148, 2013 513

leaf as a homogeneous and isotropic layer with effec-tive properties. The merit of this simplified approachis that computational time to extract leaf parametersfrom the analysis of the first thickness resonance canbe reduced so much so that it can be operated on areal-time and an in-situ basis. In addition, the obtainedeffective properties have revealed to be a faithfully repro-duction of the actual value of leaf parameters – likethickness or density – that can be measured by alter-native methods (Gomez Alvarez-Arenas et al. 2009)and to exhibit a straightforward relation with otherleaf properties of interest, specifically, changes in thestandardized frequency (f/fo) at the maximum transmit-tance (leaf resonant condition). This parameter has beenrevealed as a good indicator of the RWC of leaves withcontrasting structural features (Gomez Alvarez-Arenaset al. 2009). Other parameters such as the quality-factor(Q-factor) (Sancho-Knapik et al. 2011b) or the macro-scopic elastic constant of the leaf (c33) (Sancho-Knapiket al. 2012) have also been well related to RWC, butothers like the attenuation of the wave (α) or the vari-ation of the attenuation coefficient with the frequency(n) had never been evaluated before. In particular, thefaithful determination of this later parameter requiresthe analysis of the leaf ultrasonic response over anenlarged frequency window that is not limited to thevicinity of the first thickness resonance. As explained inGomez Alvarez-Arenas et al. (2009) and Alvarez-Arenaset al. (2009), the enlargement of the frequency windowimplies the necessity to consider the spatial anisotropyin the acoustic properties of the leaves. However, recentstudies in V. vinifera leaves (Farinas et al. 2012) havedemonstrated that the spatial anisotropy in the normaldirection of these leaves is considerably lower than inother species, so that these leaves can be used as anoptimum test bench to apply this extended frequencystudy while keeping the same theoretical modeling ofthe leaf based on a one layer approach and deter-mination of effective properties. New data can so beobtained (like n), but as the mathematical model forthe leaf remains the one-effective-layer one, none of theinconvenience derived from the necessity to consideran anisotropic multilayered model are encountered. Inthis way, the main objectives of this work were (1)to analyze the usefulness of a commercially availableantenna with morphological and resonant properties thatfits the requirements suggested above and (2) to obtainthe values of f/fo, Q-factor, c33, α and n in V. viniferaleaves in order to start setting up promising tools for thedetection of grapevine water status that would allow afine regulation of the irrigation calendar in field growinggrapevines, improving the water use efficiency and thegrape quality.

Materials and methods

Plant material and experimental conditions

Measurements were carried out on mature leaves ofV. vinifera cv. Grenache. In the early morning duringsummer, branches were collected from the sun exposedside of the plants, placed in plastic bags and carriedto the laboratory. Once there, leaf petioles were re-cut under water to avoid embolism and kept immersed(avoiding the wetting of leaves) for 24 h at 4◦C until fullleaf rehydration. It was considered that over-rehydrationdid not take place because dark spots or dark areas werenot observed, corresponding the dark green to regionswere the leaf air space was completely infiltrated withwater (Nardini et al. 2001). After rehydration, one setof 15 leaves was destined for ultrasound measurements(Gomez Alvarez-Arenas et al. 2009) and other set of 15leaves was used for microwave measurements (Sancho-Knapik et al. 2011a). Leaves were weighed and measuredat constant time intervals (approximately 30 min) toachieve different levels of RWC, starting at full saturation(turgid weight, TW). Leaf dry weight (DW) was estimatedafter keeping the plant material in a stove (24 h, 60◦C).The RWC was then calculated following the expression:RWC = (FW − DW)/(TW − DW), being FW the samplefresh weight at any moment.

Pressure–volume analysis

P–V relationships were determined following the free-transpiration method described in previous studies(Corcuera et al. 2002, Brodribb and Holbrook 2003).The water relations parameters analyzed were the leafwater potential at the turgor loss point (�TLP, −MPa),the maximum bulk modulus of elasticity (εmax, −MPa)and the RWC at the TLP (RWCTLP). The third orderpolynomial relationship found between RWC and �

(R2adj = 0.98, P < 0.0001) was used for the estimation of

� in each one of the leaves where ultrasonic parameterswere obtained.

The reflectivity in the S-band

The set up for microwave measurements consistedof a microwave Cable and Antenna Analyzer (ZVH4,100 kHz to 3.6 GHz, ROHDE & SCHWARZ, Munchen,Germany) that fed a signal to a dual band internalprinted circuit board (PCB) antenna (Acara 2.4,EAD, Buckingham, UK) (frequency range 2.4 and5.2 GHz, groundplane independent, linear polarizationand 66 × 16 × 0.8 mm). After calibration, and with nomaterial under test being present at the microwaveantenna, the impedance of the antenna is perfectly


matched to the main circuitry. Under this condition,no electrical wave reflections occur within the circuitry.The presence of water at the vicinity of the antenna,however, alters the antenna’s impedance. The resultingmismatch of impedances is produced by inserting a thintissue (the leaf) with a different dielectric constant fromthe one the antenna was designed for (air). The valueof the sample dielectric constant is highly influencedby its water content (Martınez et al. 1995), so thecorresponding reflectivity coefficient will be a directfunction of the amount of water being present at or nearthe antenna (Sancho-Knapik et al. 2011a).

With the antenna device, we measured on each leafand at different RWC levels the relationship between themicrowave frequency (from 2.25 to 2.55 GHz) and themagnitude (dB). After all the measurements were done,we analyzed which range of frequencies had a bettercorrelation with the values of RWC. For this range, wecalculated the existing area (S) between the curve fullhydrated (RWC = 1) and another single curve (RWC = i)following the next equation:

Si =x=a∑

x=b

(MgRWC=1 (x) − MgRWC=i (x)

)(1)

where a and b are the start and the end values of thefrequency range and Mg is the magnitude (dB). Once Swas obtained, it was directly correlated with RWC.

The broadband ultrasonic spectroscopy technique

The broadband ultrasonic spectroscopy techniqueis well described and schematically depicted inGomez Alvarez-Arenas (2003a) and Sancho-Knapiket al. (2010, 2011b). Briefly, the experimental set-upconsists of two pairs of specially designed air-coupledpiezoelectric transducers working at a frequency rangeof 0.3–1.2 MHz, and with a radiating diameter areaof 20 mm (Gomez Alvarez-Arenas 2003a, 2004). Thesetransducers are positioned facing each other at a distanceof 2 cm. A high voltage (100–400 V) square semicycle(duration of 0.67 μs) is applied with a Panametrics5077 pulser-receiver (Olympus, Center Valley, PA) tothe transmitter transducer that converts this electricalsignal into an ultrasonic pulse and launches it intothe air. The receiver transducer collects this signaland converts it into an electrical one, then it isamplified (up to 59 dB) and filtered (low-pass filter at10 MHz). Eventually, an oscilloscope (Tektronix 5052TDS, Tektronix Inc., Beaverton, OR) digitizes it, averagesa number of waveforms to reduce the high frequencynoise (typically up to 100 waveforms), performs the fastFourier transform and transfers the data to a computer

for storage and further calculations. The experimentalprocedure of the ultrasonic technique is as follows.First, transmission from a transmitter is directly measuredinto the receiver, providing a calibration of the system.Then a leaf is held for a few seconds between thetransducers at normal incidence. When the ultrasoundsimpact normally on the leaf surface, part of the energyis transmitted through the leaf, and reaches the backsurface. Then part of the energy is transmitted throughthe interface (air) and received at the receiver transducer.The major modification of the approach here proposed isto consider a frequency band that not only considers thevicinity of the first thickness resonance, but a frequencywindow as large as possible considering restrictions inthe frequency band of the ultrasonic sensors. For thispurpose, measuring frequency window was set from 0.3to 1.2 MHz.

The parameters directly measured were the magnitudeand the phase of the transmission coefficient in thefrequency domain (Sancho-Knapik et al. 2012). Then, inreal time, the values of the resonant frequency (f), qualityfactor (Q), attenuation coefficient (α), macroscopiceffective elastic constant in the leaf thickness direction(c33) and the variation of the attenuation coefficient withthe frequency (n) of the leaf were calculated followingthe procedure described in Sancho-Knapik et al. (2012),being afterwards related to RWC. Each single valueof f was divided by the maximum value obtained atRWC = 1.00 (fo) for each leaf, calculating, in this way,the standardized frequency (f/fo) associated with themaximum transmittance at the peak curve (Sancho-Knapik et al. 2011b). The Q factor was defined as theratio between the resonance frequency and the widthof the resonance peak measured at 3 dB below themaximum value (Gomez Alvarez-Arenas 2003b). Theinverse of the quality factor (1/Q) was employed, because1/Q is directly related to the attenuation coefficient (α).An increase in 1/Q or in α reflects either an increase inthe irregularity of the acoustic pathway (increase in thenumber of scatterers: cavitation) or an increase of therelative influence on energy conversion mechanisms (i.e.variation of heat generation by alteration of the influenceof the friction between different tissues or by differentcomponents of the same tissue). The macroscopiceffective elastic constant of the leaf, c33, relates thecompressional deformation in the thickness directionwith the stress applied in the same direction; it isgiven in MPa (Landau and Lifshitz 1959). To describethe variation of the attenuation coefficient with thefrequency, a power law is considered, see Eqn 1, wherethe parameter n is used as a measure of this variation:

α = α0fn (2)


This power law (see Eqn 2) has been used in previousworks and by other researchers to describe the variationof the attenuation with the frequency in many differentmaterials (like organic tissues and porous materials) andover large frequency ranges (Szabo 1994, 2000, GomezAlvarez-Arenas et al. 2002, 2010). The parameter nvaries normally from 0 to 4 and its value is determinedby the physical mechanism that produces the attenuationof the wave. For example, for an attenuation originatedby the presence of scatterers in the Rayleigh zone,n = 4, while for ideal linear viscoelastic losses, n = 2.In addition, for many porous materials, where theattenuation is mainly determined by the friction betweenthe fluid and the solid phases, variation of the attenuationwith the frequency respond to an empirical law givenby Eqn 2 with n = 1. In this sense, the determination ofthe variation of the n-factor with RWC in V. viniferaleaves can provide significant information about themicrostructural (histologic) changes that are produced inthe leaves by the loss of water.

Statistical analysis

On one hand, the relationship between RWC and S wasadjusted to a linear regression. On the other hand, therelationships between RWC and f/fo, Q-factor, c33 andα were adjusted to a four parameter logistic curve foreach leaf studied (Sancho-Knapik et al. 2010, 2011b).This function was selected because the inflexion pointis directly inferred from the equation and becausedescribes the evolution between two ‘equilibrium states’,before and after the turgor loss point. Finally, therelationship between RWC and n and the relationshipbetween � and f/fo were adjusted to a linear segmentedmodel. A Student’s t-test was used to compare the valuesof RWC at the TLP (RWCTLP) obtained from the P–Vcurves and the values of RWC at the inflexion pointfrom the f/fo, c33, 1/Q and α curves (Sancho-Knapiket al. 2010, 2011b) and the join point from the n curve.Moreover, a Student’s t-test was used to compare thevalues of � at the TLP obtained from the P–V curvesand the join point from the relationships between � andthe parameters derived from ultrasonic measurements.All statistical analyses were performed with the programSAS version 8.0 (SAS, Cary, NC, USA).

Results

Fig. 1A shows the relationship between the microwavefrequency (GHz) and the magnitude (dB) for one leafat different RWC values for all the frequency rangemeasured (from 2.25 to 2.55 GHz). Each curve representsthe measurement for a given water content. An overall

A

B

Fig. 1. Relationship between frequency (GHz) and magnitude (dB) forone leaf at different RWC values. (A) Shows all the frequency rangemeasured and (B) shows the frequency range use to calculate S in Eqn 1.

Fig. 2. Relationship between the RWC and the surface between twocurves (S) obtained with Eqn 1.

shift in the reflectance coefficient expressed in dB(Magnitude) was consistently observed as the leaf lostwater. The range of frequencies selected to calculateS was from 2.30 to 2.32 GHz (Fig. 1B). A very highcorrelation was found between RWC and S (Fig. 2) whenadjusted to a linear model (R2

adj = 0.995, P < 0.0001).In Fig. 3A the mean values of f/fo obtained

from ultrasonic measurements are represented againstdifferent levels of RWC. The relationship between RWCand f/fo was adjusted to a four parameter logistic curve(R2

adj = 0.99, P < 0.0001), which is characterized by theexistence of an inflexion point, corresponding to the TLP(Sancho-Knapik et al. 2011b). The mean value of theTLP for V. vinifera leaves estimated by the standardizedfrequency corresponded to a RWC of 0.86 ± 0.01,which was not statistically different at P < 0.05 fromthat estimated from the P–V curves (Table 1). InFig. 3B the mean values of f/fo obtained from ultrasonicmeasurements are represented against different levelsof � (−MPa). The relationship between � and f/fo


A

B

Fig. 3. Relationships between the standardized frequency (f/fo) and theRWC (A) and the water potential (�, −MPa) (B) for Vitis vinifera cv.Grenache leaves.

Table 1. Parameters derived from the P–V curves for Vitis vinifera cv.Grenache: water potential at the turgor loss point (�TLP, −MPa), relativewater content at the turgor loss point (RWCTLP) and the maximum bulkmodulus of elasticity (εmax, −MPa). Data are expressed as mean ± SE of10 leaves.

P–V parameters V . vinifera cv. Grenache

�TLP (−MPa) 1.99 ± 0.03RWCTLP 0.85 ± 0.01εmax (−MPa) 11.91 ± 0.28

was adjusted to a linear segmented model (R2adj = 0.92,

P < 0.0001), which is characterized by the existenceof a join point at a � value of −1.92 ± 0.04 MPa. Thisvalue was not statistically different at P < 0.05 from �TLP

estimated from the P–V curves (Table 1).Fig. 4 shows the relationships between RWC and

the mean values of macroscopic elastic constant of theleaf in the Z direction (c33), the inverse of the qualityfactor of the leaf first thickness resonance (1/Q), theattenuation of the leaf first thickness resonance (α) andthe mean values of the variation of the attenuationcoefficient with the frequency (n). The relationshipsbetween RWC and c33, 1/Q and α were also adjustedto a four parameter logistic curve (R2

adj = 0.960, 0.988

and 0.988, respectively; P < 0.0001). The mean valueof the TLP for V. vinifera leaves estimated by c33 wasnot statistically different at P < 0.05 from that estimatedfrom the P–V curves (Table 1), whereas the TLP derivedfrom 1/Q and α measurements were slightly lower. Therelationship between RWC and n was adjusted to a linearsegmented model (R2

adj = 0.966, P < 0.0001), which ischaracterized by the existence of a join point at a RWCvalue of 0.80 ± 0.02.

Discussion

The large water requirements of V. vinifera duringthe growing season (Netzer et al. 2005, Zhang et al.2007) implies the need for irrigation in the driest areasof its distribution range to avoid constraints on thephotosynthetic activity due to stomatal closure (Flexaset al. 1998, Chaves et al. 2010). The stomatal behaviorin grapevine has been proposed as characteristic of adrought-avoiding, isohydric plant (Zufferey et al. 2011).Thus, the reduction in transpiration regulates the xylemtension, limiting the risks of a hydraulic failure bycavitation (Cochard et al. 2002, Brodribb et al. 2003,Choat et al. 2010). However, more recently, Zuffereyet al. (2011) have proposed that stomatal closure inV. vinifera cannot regulate by itself the risk of drought-induced cavitation, proposing that the diurnal cyclesof cavitation and refilling in the petioles act as a‘hydraulic fuse’ that exacerbate the ability for reachingrisky water potential in the whole water pathway. Thehigh vulnerability found for leaf petioles (estimatedthrough the water potential at which plants lose 50%of hydraulic conductivity, PLC50 < −1.0 MPa) reportedin this article indicates the extreme sensitivity of theleaves in this plant species to small changes in leaf waterpotential, as long as the existence of this ‘hydraulic fuse’preserves the leaf from further consequences in termsof whole plant hydraulics but reduces the capability forkeeping certain levels of photosynthesis. The triggeringof this ‘hydraulic fuse’ in this species occurs when thewater potential is well above the TLP measured here(approximately −2 MPa, Table 1) and those reported byAlsina et al. (2007) for several grapevine cultivars. Thisfact implies that drastic changes in the leaf physiologicalperformance of V. vinifera would happen in a verynarrow water potential range.

Among the different methods that have been usedfor the estimation of the leaf water status, the use ofthe reflectance in the infrared region of the spectrum,especially in the so-called ‘water bands’ (Carter 1991,Penuelas et al. 1993, Seelig et al. 2009), faces theproblem of the strong influence of the leaf thicknesschanges at positive turgor pressures (Sancho-Knapik


A B

C D

Fig. 4. Relationship between the RWC and the macroscopic effective elastic constant (c33) (A), the inverse of Q-factor (1/Q) (B), the attenuation ofthe leaf first thickness resonance (α, m−1) (C) and the variation of the attenuation coefficient with the frequency (n) (D) for Vitis vinifera cv. Grenacheleaves. Data are expressed as mean ± SE of 15 leaves.

et al. 2011a). Effectively, these authors confirmed thatthe leaf experiences a marked decrease in thicknessuntil the turgor is lost, which counteracts the changesin reflectance values due to leaf water losses (Seeliget al. 2009). On the other hand, the ‘pressure-probe’ developed by Zimmermann et al. (2008), whichmeasures changes in turgor pressure in intact leaves,and the estimation of reflectivity at the L-band with amicrowave antenna (Sancho-Knapik et al. 2011a) canalso serve as a tool for the detection of changes in theleaf water content above the turgor loss point. However,both methods imply a close contact between the deviceand the leaf surface, which in a long time recording canderive in leaf bleaching and other problems.

The measure of the reflectance at a frequency closeto 2.4 GHz gives an estimation of the changes in thedielectric property of the leaves as the water contentdecreases (Martinez et al. 1995). The good correlationsfound between the parameter S (see Eqn 1) and theRWC in V. vinifera suggest a promising use of thistechnique in this species, especially in combination withthe physiological information given by the ultrasonicmethod (Sancho-Knapik et al. 2011b). The new antennatested in this article improves the benefits of the one used

by Sancho-Knapik et al. (2011a), both in dimensions,commercial availability and frequency range.

The air-coupled broadband ultrasonic spectroscopyallows the determination of leaf water status in anon-contact and non-invasive way, which constitutes anoteworthy progress with respect to the other techniquesabove mentioned. Among the different variables thatcan be registered, the frequency associated with themaximum transmittance (f/fo) is an optimum parameterfor the estimation of plant water status above the TLPin V. vinifera (Fig. 3A). Moreover, the changes foundin the macroscopic elastic constant of the leaf in the Zdirection (c33), which is known to be dependent on thefrequency (Sancho-Knapik et al. 2011b), were highlycorrelated with changes in RWC, particularly before theTLP (Fig. 4A). Finally, the variation of the attenuationcoefficient with the frequency (n) strongly decreasedwith the RWC at positive turgor pressures (Fig. 4D). Thedetermination of this parameter can only be achievedby considering the extension of the frequency windowanalysis proposed in this work. This extension canquestion the applicability of the one-layer effectivemedium acoustic model used to extract the propertiesof the leaf from the measured spectra. As a matter of


fact, this model can only be done if the anisotropybetween the different leaf layers in the thicknessdirection can be considered negligible. Availableevidences suggest that grapevine leaves can be soconsidered. Election of V. vinifera leaves is critical inthis point because of the low attenuation in these leavesas compared with other species. For example, whileattenuation in V. vinifera leaves is about 800 Np m−1

at 0.6 MHz, attenuation coefficient (extrapolated atthe same frequency) for other species is 1200 Np m−1

(Epipremnum aureum), 1600–1800 Np m−1 (Prunuslaurocerasus), 800–1500 Np m−1 (Ligustrum lucidum),2000–3000 Np m−1 (Platanus hispanica) and1300–1400 Np m−1 (Populus × euramericana) (GomezAlvarez-Arenas et al. 2009, Farinas et al. 2012).Thanks to this relatively lower attenuation coefficient ingrapevine leaves, measuring frequency window can beset to the 0.3–1.2 MHz range, which is large enough toget a faithful estimation of the parameter n. In addition,the variation found in n may suggest that the mainsource of ultrasonic attenuation varies from scatteringby small scatterers (n close to 4) to viscoelastic losses(n close to 2). Further investigations are required tocorrelate these variations of the dominant mechanismof ultrasonic losses with histological variations inthe leaves. Although these three variables accuratelydefined the RWC of this species above the turgorloss point, n was the parameter that showed a greatpercentage of variation of the dynamic range duringthis phase (ca. 62%), when compared with f/fo, (ca.45%) and c33 (ca. 50%). In this sense, it is worthwhileto note that while f/fo, and c33 are determined by theelasticity of the leaf, 1/Q, α and n are determined bythe various mechanisms that produce the attenuation ofthe ultrasonic wave: presence of scatterers and energyconversion phenomena. Therefore it is possible toobserve a different evolution of these parameters whenthe leaf RWC or water potential varies depending onhow this variation may affect the leaf microstructure andhow these changes affect the different factors involvedin setting up the ultrasonic response of the leaf.

Otherwise, the air-coupled broadband ultrasonicspectroscopy can be used for the determination of theTLP in V. vinifera. Effectively, no significant differenceswere found between the RWCTLP derived from analy-sis of P–V isotherms and those obtained by calculatingthe inflexion point in the RWC vs f/fo and c33 rela-tionships, respectively, which was previously reportedby Sancho-Knapik et al. (2012) in L. lucidum, Pop-ulus × euramericana and Platanus × hispanica and P.laurocerasus. The accumulation of evidences suggeststhat the use of ultrasonic measurements would consti-tute a valuable tool for assessing the turgor loss in plant

species. Recently, Bartlett et al. (2012) have revisitedthe ecological importance of the TLP in terms of waterpotential as a major physiological determinant of plantwater stress response. In case this parameter, rather thanthe RWC at zero turgor, is preferable the use of the rela-tionship between � and f/fo can also be used (Fig. 3B).Once TLP is overpassed, the different ultrasonic vari-ables are also well informative about the changes in thewater status of V. vinifera leaves. In this phase, a shift inthe parameters directly related to the attenuation of thesignal (α, 1/Q) was observed (Fig. 4B, C). However, 1/Qand n are not optimum to quantitatively monitor plantwater status once turgor is lost (when RWC is below 0.85)because of the large scattering in the signal. For this RWCrange, c33 and α could constitute a better way for a moreaccurate estimation of water status in grapevine leaves.A substantial improvement in the accuracy of RWC esti-mation below TLP can be achieved by a simultaneoususe of both ultrasounds and microwave parameters.

In conclusion, this study has proven that the twotechniques here presented can be suitable for theestimation of the leaf water status in V. vinifera throughthe different phases experienced by the leaves in thedehydration process. The measurement of reflectanceat frequencies close to 2.4 GHz gives an excellentaccuracy when the changes in S are correlated withthe value of RWC. The information given by the air-coupled broad band ultrasonic spectroscopy does notlie on one single variable, yielding information fromdifferent and non-redundant parameters, which allowsfor a more convincing knowledge of the water status inthis plant species. The combined use of both techniquesand the development of single and portable device forusing these methods under field conditions constitute achallenge that deserves further efforts.

Acknowledgements – This study was partially supported bythe projects AGL2010-21153-C02-02 (Ministerio de Cien-cia e Innovacion, Spain) and DPI2011-22438 (Ministerio deEconomıa y Competitividad, Spain). Financial support fromGobierno de Aragon (A54 research group) is also acknowl-edged. Work of J. J. P.-P. is supported by a ‘‘Juan de laCierva’’-MICIIN post-doctoral contract.

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