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Plant and SoilAn International Journal on Plant-SoilRelationships ISSN 0032-079XVolume 369Combined 1-2 Plant Soil (2013) 369:257-268DOI 10.1007/s11104-012-1568-x

Non-invasive pressure probes magneticallyclamped to leaves to monitor the waterstatus of wheat

Helen Bramley, Wilhelm Ehrenberger,Ulrich Zimmermann, JairoA. Palta, Simon Rüger & KadambotH. M. Siddique

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REGULAR ARTICLE

Non-invasive pressure probes magnetically clamped to leavesto monitor the water status of wheat

Helen Bramley & Wilhelm Ehrenberger &

Ulrich Zimmermann & Jairo A. Palta &

Simon Rüger & Kadambot H. M. Siddique

Received: 26 August 2012 /Accepted: 11 December 2012 /Published online: 29 December 2012# Springer Science+Business Media Dordrecht 2012

AbstractBackground and aims Being able to monitor the hy-dration status of a plant would be useful to breedingprograms and to providing insight into adaptation towater-limited environments, but most current methodsare destructive or laborious. We evaluated novel non-invasive pressure probes (commercial name: ZIM-

probe) for their potential in monitoring the water statusof wheat (Triticum aestivum L.) leaves.Methods The probes consist of miniature pressure sen-sors that clamp to the leaves via magnets and detectrelative changes in hydration status. Probes were clampedto leaves of six individual plants of the cultivar Wyal-katchem at the stem elongation stage and comparedagainst traditional plant water relations measurements.Results Output from the probes, called patch-pressure(Pp), correlated well with leaf water potential andtranspiration of individual plants. Variation betweenplants in the original clamp pressure exerted by themagnets and leaf individual properties led to varia-tions in the amplitude of the diurnal Pp profiles, butnot in the kinetics of the curves where Pp respondedsimultaneously in all plants to changes in the ambientenvironment (light and temperature).Conclusions Drying and rewatering cycles and analy-sis of the curve kinetics identified several methods thatcan be used to test comparisons of water status mon-itoring of wheat genotypes under water deficit.

Keywords Turgor .Water potential . Temperature .

Drought

Introduction

All land plants must take up water from the soil andtransport it to leaves to replace the water lost to theatmosphere through transpiration. Mechanisms that

Plant Soil (2013) 369:257–268DOI 10.1007/s11104-012-1568-x

Responsible Editor: John McPherson Cheeseman.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-012-1568-x) containssupplementary material, which is available to authorized users.

H. Bramley (*) :K. H. M. SiddiqueThe UWA Institute of Agriculture, M082,The University of Western Australia,35 Stirling Highway, Crawley, WA 6009, Australiae-mail: helen.bramley@uwa.edu.au

W. Ehrenberger :U. Zimmermann : S. RügerZIM Plant Technology GmbH,Neuendorfstr. 19, 16761 Hennigsdorf, Germany

W. EhrenbergerLehrstuhl für Biotechnologie, Biozentrum,Universität Würzburg,Am Hubland, 97074 Würzburg, Germany

J. A. PaltaCSIRO Plant Industry,Private Bag No. 5, Wembley, WA 6913, Australia

J. A. PaltaSchool of Plant Biology,The University of Western Australia,35 Stirling Highway, Crawley, WA 6009, Australia

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improve efficiency of water transport and minimisewater loss are therefore, advantageous in ephemeralor arid environments where water is the most limitingresource (Smith et al. 1989). Efficiency in water trans-port and transpiration efficiency (amount of carbonfixed per unit of water transpired) also influence cropproductivity and drought resistance, and where cropsare irrigated, the amount of water applied to achieveoptimal growth (Flexas et al. 2010; Galmés et al.2010; Morison et al. 2008). With the demand forincreased crop production to feed the world’s escalat-ing population and the adverse changes in climatepredicted for many cropping regions (Easterling et al.2007), there is a need to identify hydraulic traits thatbreeders can select to develop cultivars better adaptedto their target growing regions. However, plant hy-draulic properties form a complex interactive networkso that decoupling of a single trait is unlikely to makea dramatic impact on crop productivity or droughtresistance (Sinclair 2012).

The hydration status of a plant incorporates all of itshydraulic traits. The hydration status here is defined ashow well the plant maintains the water balance of itsleaves, whether it is through stomatal regulation, wateruptake and transport capacity of roots, desiccationresistance, volumetric elasticity (water holding capac-ity), osmotic adjustment or any combination of theseand other hydraulic traits. Therefore, being able tomonitor the hydration status of a plant would be par-ticularly useful to breeding programs and also provideinsight into adaptation to particular environments orresponses to abiotic stress.

Many methods have been used previously to mea-sure plant water use or water balance. One of the moststandard techniques is the determination of leaf waterpotential using a pressure chamber (Scholander et al.1965). However, this method is destructive, usingdetached leaves, and temporal and spatial variationslimit sampling (O’Toole et al. 1984). Stomatal con-ductance and transpiration are commonly measuredusing porometry and gas exchange equipment andalthough these measurements can be carried out onintact leaves, they are disruptive and suffer from thesame temporal and spatial resolution problems as leafwater potential measurements (O’Toole et al. 1984).Thermal imaging using infra-red technology to mea-sure leaf and canopy temperatures, as a surrogate forstomatal conductance, has been used for several dec-ades (Ehrler et al. 1978), but recent advances in remote

sensing and methods to normalise spatial and temporalvariation has led to rapid adoption of thermal imagingin modern phenotyping programs (Jones et al. 2009;Munns et al. 2010; Reynolds 2002). While thermalimaging has obvious advantages in scaling fromleaves to whole fields, proponents of the technologyhave suggested that accompanying measurements ofleaf water status, especially turgor, would provide theextra information needed to understand the effect ofstomatal behaviour on plant adaptation and growthrate (Munns et al. 2010).

New magnetic patch-clamp pressure probes (ZIM-probes) potentially provide an alternative or comple-mentary method of directly monitoring leaf hydration.ZIM-probes are non-invasive and continuouslymonitor the hydration status of leaves in real-time(Zimmermann et al. 2008, 2010). Because the devicescommunicate wirelessly to a remote controllerconnected by GSM to an Internet server, the data canbe viewed online over the Internet at any time. TheZIM-probe technology uses miniature pressure sen-sors that are clamped to leaves via magnets, improvingon the spring-loaded connection mechanism in theoriginal prototype (Zimmermann et al. 2008). Themagnets apply a constant clamp pressure to the leaf,so that the pressure sensors are able to detect relativechanges in leaf turgidity. Turgor is related to the hy-dration status as cell and bulk leaf turgor pressuredecline when leaves dehydrate during transpirationand in response to drought (Kramer and Boyer1995). The ZIM-probe technology has been used formonitoring water status of trees and irrigation sched-uling of horticultural crops (Ehrenberger et al. 2012;Rüger et al. 2010; Zimmermann et al. 2010), but canalso potentially be adapted to monitoring the water useof agricultural crops. In this paper we evaluate ZIM-probes for monitoring the hydration status of wheat.Adaptation of the technology for crops such as wheatposes a greater challenge than that of trees becausewheat leaves are more delicate and less rigid than treeleaves. Wheat leaves are also narrow; typically 5–20 mm depending on age and genotype, and haveprominent mid-ribs that limit the region where the10 mm diameter ZIM-probes can be clamped. Weclamped the probes to mature leaves of a popularAustralian wheat (Triticum aestivum L.) cultivar whenthe plants were at the stem elongation stage (Z30 inZadok’s growth stage for cereals) (Zadoks et al. 1974)and compared plant water use/balance measured with

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traditional methods against the output from ZIM-probes under controlled environmental conditions.

Materials and methods

Plant material

Seeds of the bread wheat (Triticum aestivum L.) culti-var Wyalkatchem were kindly provided by Dr. BenBiddulph (Department of Agriculture and Food, West-ern Australia). Seeds were surface sterilised with com-mercial bleach (active constituent 2.1 % sodiumhypochlorite) and then pre-germinated on moist filterpaper in Petri-dishes. Seedlings were transferred topots (75 mm diameter, 350 mm depth) filled withcommercial potting mix (Doublegee cocopeat mixwith macrominerals and trace elements, Richgro Gar-den Products, Canningvale, Western Australia) whenthe roots were 2–3 cm long. Plants were watered everythird day with water and once every 2 weeks withwater soluble fertiliser (Thrive Soluble All PurposePlant Food, N:P:K 27:5.5:9). Plants were grown in agrowth cabinet with 12 h light/dark and 60–75 %relative humidity. The temperature and light intensityin the cabinet were programmed to gradually increaseand decrease during the day to mimic diurnal changes(Supplementary Fig. 1).

Experimental protocol

ZIM-probes (ZIM Plant Technology GmbH, Hennigs-dorf, Germany) were clamped to the penultimate leafof the main stem of six individual plants when theplants were at the stem elongation stage of develop-ment. All probes were located 6–7 cm from the leaftip, where the leaf blade was smooth and flat. ZIM-probes were clamped with the sensor facing the abax-ial leaf surface. Although the probes only weigh 5.5 g,wheat leaves could not support a probe by themselvesunlike previous studies on trees (Ehrenberger et al.2012; Rüger et al. 2010) or vines (Westhoff et al.2009; Zimmermann et al. 2008). Therefore, eachprobe was supported by an iron rod impaled in thesoil, which allowed the clamped leaf to maintain itsoriginal orientation (Fig. 1a).

The output from the probes was monitored for2 days to ensure that there was no damage to theleaves during clamping. All plants were then watered

thoroughly and the traditional plant water relationsmeasurements conducted over a diurnal cycle the fol-lowing day (day 5).

After the diurnal cycle measurements, the ZIM-probes continued monitoring for a further 9 days.Three plants were watered after 3 days (day 8), where-as water was withheld from the other three plants until6 days (day 11), when all plants were rewatered. Theprobes were removed from the plants after 14 daysmonitoring and the leaves inspected for damage.

Plant water relations

Because of the destructive nature of some of the plantwater relations measurements, these measurementswere conducted on plants grown and treated identical-ly as those clamped with the ZIM-probes.

Whole plant transpiration (Eplant) was measuredgravimetrically. The evening before measurementsand after all plants had been thoroughly watered andallowed to drain, the plant pots were covered withplastic bags and sealed to the base of the plant. Fourindividual plants were weighed at each time point andthen again 1 h later. Eplant was standardised by totalleaf area, which was measured with a LI-COR LI-3000A Portable Leaf Area Meter (LI-COR Environ-mental, Nebraska, USA). Eplant was also measured onplants clamped with ZIM-probes, to ensure that therewas no difference between clamped and unclampedplants.

After measuring Eplant, gas exchange (Eleaf, CO2

assimilation and stomatal conductance) was measuredon the same plant with a portable differential gasanalyser (CIRAS-2 Portable Photosynthesis System,PP Systems, Amesbury, US) on the penultimate leavesof the main stem. Measurements were only conductedwhen the lights were on in the growth cabinet.

The water potential (Ψleaf) of the same leaf used forgas exchange was measured using a Scholander-typepressure chamber fitted with a specimen holder forgrass blades (Soil Moisture Equipment Corp., SantaBarbara, USA). The leaf was enclosed in a plastic bagbefore excision from the plant and during themeasurement.

Leaf patch clamp pressure probes (ZIM-probes)

The measuring principle of the magnetic leaf patchclamp pressure probe (commercial name: ZIM-probe)

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is described in detail elsewhere (Zimmermann et al.2008, 2010). Briefly, an intact leaf is positioned be-tween the two pads of the probe (diameter 10 mm),each of which is connected with magnets. The probemeasures the pressure transfer exerted by the twomagnets through the leaf patch. The leaf patch isassumed to be in hydraulic contact with the surround-ing unclamped leaf tissue. The output pressure signal(i.e. the so-called patch pressure Pp) is sensed by apressure sensor that is integrated in one of the pads.The clamp pressure (Pclamp) that is exerted by the twomagnets onto the leaf patch can be adjusted to therigidity of the leaf by varying the distance betweenthe two magnets and is constant during the measure-ments. Essentially, leaf turgor opposes the clamp pres-sure and the pressure sensor detects changes in turgorby monitoring the change in pressure opposing themagnetic force (i.e. turgor). Therefore, Pp is inverselycorrelated with leaf turgor pressure (Zimmermann etal. 2008, 2010), such that when the leaf dehydratesduring stomatal opening and in response to waterdeficit Pp increases and conversely, decreases againwhen the leaf rehydrates.

The Pp signals were transmitted via wireless trans-mitters to a controller (ZIM-transmitter and ZIMradio-controller, ZIM Plant Technology GmbH, Hen-nigsdorf, Germany), which sent the data by a GPRSmodem (fitted with a commercial prepaid SIM card) toan Internet server, from which the data were down-loaded. Ambient temperature and relative humidity of

the growth cabinet were simultaneously recorded bymicroclimate probes (ZIM Plant Technology GmbH,Hennigsdorf, Germany) that were sent to the controllervia the same wireless transmitters as the Pp signals.

Tests under the same conditions as the experiments,but without leaves connected between the magnetsshowed that ZIM-probes were relatively independentof temperature (Supplementary Fig. 2).

Statistical analyses

Statistical analyses were performed using GraphPadPrism version 5.02. Diurnal values of Ψleaf, Eplant andgas exchange were compared with One-Way ANOVAand Tukey multiple comparison post-test. The rela-tionship between Pp and traditional plant water rela-tions measurements were compared with correlationanalyses.

Results

ZIM-probes were clamped to wheat leaves where theblade was smooth and flat, 6–7 cm from the tip(Fig. 1a). The prominent mid-rib prevented clampingfurther from the tip.

Typical diurnal Pp recordings on well-wateredwheat plants are shown in Fig. 2, together with thecorresponding changes in ambient temperature (T) andrelative humidity (RH) of the growth cabinet. Pp

a bFig. 1 A ZIM-probeclamped to a wheat leaf (a)and examples of leaf patchesbeneath the probes at theend of the experiment(2 weeks) (b)

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increased when the plants were illuminated during theday and decreased again during the afternoon. Apartfrom variation in amplitude, the kinetics of the Pp

curves from different plants were the same; the riseand fall in Pp occurred at the same time, and reachedthe plateau at the same time (Fig. 2). Fluctuations in Pp

followed similar patterns to changes in T, but did notcoincide exactly. For example, the midday plateau inPp was reached 10 min earlier than the correspondingT plateau value. In addition, small fluctuations in Pp

occurred during the midday plateau, when T was con-stant (24 °C) and during the afternoon hours, thecontinuous drop of Pp was interrupted twice by a smallincrease (at 17:00 and at 18:30) whereas the drop of Twas only interrupted once for 55 min at 17:00 when Twas constant at 15 °C. Sudden decrease in Pp whenplants were watered was not seen in T recordings andnoisy fluctuations in T recorded during the night werealso not reflected by the Pp recordings.

Leaf water potential (Ψleaf) decreased during theday, with the minimum Ψleaf values occurring overthe same time period as maximum Pp (Fig. 3a). Cor-relation between Ψleaf and Pp was highly significant

(P<0.0001, r=−0.973) (Fig. 3b), although slopes andintercepts for individual leaves varied (P<0.0001).Ψleaf was also highly correlated with ambient temper-ature (P<0.0001, r=−0.87) (Fig. 4). Eplant and Eleaf

(data not shown) and Pp also correlated well(P=0.0002, r=0.9591) (Fig. 3c and d), whereas Gs

and Pp were less related (P=0.0115, r=0.9113)(Fig. 3f). Although Gs appeared to show a diurnaltrend, values during illumination hours were not sig-nificantly different (P=0.1596) (Fig. 3e). Gs in thedark was too low for the CIRAS machine to detect.

Half of the plants connected with ZIM-probes werewatered on days 4, 8 and 11, whereas the other halfwere only watered on days 4 and 11. Typical Pp

recordings of these drying and re-watering cycles areshown in Fig. 5a and b, respectively. Pp peak values atnoon and, to a lesser extent, minimum Pp values atnight continuously increased when plants were notwatered (Fig. 5a). Re-watering caused an instanta-neous fluctuation in Pp (Fig. 5a inset) and lower Pp

peak values on subsequent days. Pp peak and nightvalues were constant with only 2 days between water-ing events (Fig. 5a). Longer periods without water(7 days) caused continuous increase of Pp peak andnight values until the sixth day (Fig. 5b). The drop inPp during the afternoon also continuously occurredlater until on the sixth day without water, half-inversed curves of Pp occurred during the end of thelight where Pp increased again instead of declining tothe night value (Fig. 5b, day 10). Pp dropped againduring the night but to higher values than on precedingdays. Pp curves returned to their original shape withwatering the next morning (day 11), i.e. peaking atnoon and continuous increase in peak Pp on the fol-lowing days when the plants were not watered(Fig. 5b).

The rate of leaf rehydration may take longer when thesoil is dry. To analyse whether this could be detected bythe probes, single exponential decay curves were fit toeach afternoon’s data between peak Pp and minimum Pp

night values. Best fits for comparison of the curvesparameters incorporated 67 % of the data points (be-tween 16:00 and 02:00 h). The rate constant (K) of Pp

relaxations during afternoon hours decreased with on-going water deficit between watering events, while thehalf-time (T1/2) of Pp relaxations increased (Fig. 6).Watering three plants on day 11 reduced T1/2 from 188to 90 min and increased K from 4 to 8 min−1. T1/2 of theremaining unwatered plants could not be determined

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Fig. 2 Typical patch pressure (Pp) recordings from penultimateleaves of the main stems of well-watered wheat plants (a) andthe corresponding ambient temperature (T; solid line) and rela-tive humidity (RH; dashed line) of the growth cabinet (b). Eachline in (a) represents the recording from a different plant/probe.The arrow indicates when the plants were watered. Pp wasnormalised to the value at 07:00 h on day 4 to give ΔPp

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due to the development of the half-inversed curvesduring the end of the light period (Fig. 5).

Similar to the afternoon recovery phase analysis,comparison of plots of the Pp values against T for

morning and afternoon hours also relates to the waterstatus of the wheat plants (Fig. 7). When plants werewell-watered, Pp and T exhibited an inverse hysteresis(Fig. 7b). Pp and Twere linearly related in the morningbut the afternoon data was curvilinear as the decline inPp occurred much faster than the decline in T. Con-versely, when water uptake was limited, Pp and Twerelinearly related and exhibited no hysteresis betweenmorning and afternoon (Fig. 7a). The intensity of theinverse hysteresis loops was related to the plant watersupply, decreasing as the area between the morningand afternoon Pp = f (T) curves decreased. In order toquantify the level of hysteresis, the area betweenthe two Pp = f (T) curves was calculated andnormalised to the rectangular area given by theco-ordinates of the lowest and highest Pp valuesthat encompass the hysteresis loop (inset ofFig. 7b). In between watering events the area ofthe inverse hysteresis loop decreased and increasedagain upon watering (Table 1).

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Fig. 3 Concomitant diurnal changes in patch pressure (Pp; solidline) (a, c and e) and correlation with traditional leaf waterrelations measurements (b, d and f; filled circles, dashed line).ΔPp shown is for a single ZIM-probe normalised to the value at07:00 h. Leaf water potential (Ψleaf) was measured with a

pressure chamber (a and b), transpiration was measured gravi-metrically and normalised by leaf area (Eplant) (c and d) andstomatal conductance (Gs) was measured with a portable differ-ential gas analyser (e and f). Values for Ψleaf, Eplant and Gs aremeans ± SEM, n=4

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At the end of the experiment (2 weeks), the probeswere removed from the leaves. The leaf patches be-neath the probes were yellow and only slight

impressions from the probe pads could be seen onthe leaf surface with closer inspection under the mi-croscope (Fig. 1b).

To examine the variability in Pp between individualprobes and the dependence of temperature in moredetail a separate experiment was conducted by clamp-ing probes to three individual plants within the same7.5 L pots. Each pot represented a different clampingpressure by accurately adjusting the distance betweenthe magnets (Fig. 8). Diurnal fluctuations in Pp weremuch larger when the magnets were wound fully tothe base of the probe pad (i.e. distance between mag-nets=0 mm) than when the magnets were wound outfrom the probe pad 1.6 or 2.6 mm (Fig. 8a–c). How-ever, the variability between the individual probes wasalso largest when Pclamp was maximum (mean Pp

SEM in 8a 5.02, 1.41 in 8b and 0.88 in 8c).Figures 8 and 9 also show the response of Pp

when plants were placed in the dark (covered with

Fig. 5 Diurnal changes inpatch pressure (Pp) ofplants subjected to differentwatering regimes (a and b)and concomitant changes inambient temperature (T;solid line) and relative hu-midity (RH; dotted line) (c).Plants were watered two (b)or three (a) times (indicatedby arrows). Noon and nightPp values gradually in-creased between wateringevents. Inset in (a) is an en-largement of the immediatefluctuation in Pp due towatering on day 4

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Fig. 6 Half-times (T1/2; open circles, dashed line) and rateconstants (K; filled circles, solid line) of the patch clamp recov-ery phases during the afternoons after watering on day 4. T1/2and K were determined by fitting exponential decay curves tothe afternoon/night data between 16:00 and 02:00. Values aremeans ± SEM, n=6

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aluminium foil) and subsequently re-illuminatedwhile ambient temperature and relative humidityvaried diurnally. Pp rapidly decreased when theplants were placed in the dark while temperaturewas constant and conversely, rapidly increasedwhen the plants were reilluminated (Fig. 9). Themaximum diurnal amplitude of ΔPp was smallerin the dark than when the plants were illuminated(Fig. 9). In addition, Pp increased more rapidlythan ambient temperature at the start of the daywhen the first lights came on in the growth cabi-net (PAR 155 μmols−1m−2), but when the plantswere covered the rate of change in Pp at the startof the day was slower than ambient temperature(Fig. 9).

Discussion

This is the first report of the application of the leaf patch-clamp pressure probes (ZIM-probes) to agricultural cropplants. We chose to evaluate the application on wheatplants because wheat is the most important agriculturalcrop in Australia (Australian Bureau of Statistics 2011).Cereal crops also dominate the world’s agricultural pro-duction (FAOSTAT, http://faostat.fao.org/site/339/default.aspx, accessed 13/6/12). Water stress is oftenthe most limiting factor for cereal yield production,particularly in southern Australia (Loss and Siddique1994). On-line monitoring of plant water status willnot only aid farmers of horticultural crops in determin-ing when to irrigate, but could also potentially

Fig. 7 Typical examples of the dependency of the leaf patchpressure (Pp) values against T measured for the morning (filledsquares) and afternoon hours (open circles). a Pp = f (T) curvetogether with the diurnal changes of Pp and T measured on awater-limited wheat plant (no inverse hysteresis). b Pp = f (T)curve together with the diurnal changes of Pp and T measured

on a well-watered wheat (occurrence of inverse hysteresis). Thearea of the inverse hysteresis was normalised by referring to theco-ordinates of the lowest and highest Pp readings of the probesand determining the rectangular area which encompassed theelliptical inverse hysteresis (see inset in b)

Table 1 Area ratios of the hysteresis between patch pressure (Pp) vs. ambient temperature (T), corresponding to the recordings shownin Fig. 5. The plant in Fig. 5a was watered on days 4, 8 and 11, whereas the plant in Fig. 5b was only watered on days 4 and 11

Day 2 3 4 5 6 7 8 9 10 11 12 13

Fig. 5a: area ratio (%) 24 15 34 16 17 8 13 23 20 21 16 18

Fig. 5b: area ratio (%) 20 14 24 11 14 9 1 1 12 46 10 5

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complement existing technologies in cereal phenotyp-ing programs for drought resistance. We tested applica-tion of the technology on wheat to determine first,whether it is possible to clamp the devices on the smallleaves of wheat and second, how well the output

correlates with plant water relations using traditionalmethods. Our study demonstrates that ZIM-probes canbe adapted to wheat and that the probes monitor leafhydration status non-destructively.

ZIM-probes were relatively easy to clamp to wheatleaves despite their narrow shape and more delicatestructure compared with tree leaves, as long as theprobes were supported. The loss of chlorophyll beneaththe ZIM-probes patches after 2 weeks of clamping is tobe expected, but the hydraulic connection with the restof the leaf was maintained. Longer-term monitoring ispotentially possible, but reclamping may be neededunder severe stress and as leaves age and senesce.

The output from the probes correlated well withtraditional measurements of leaf water relations on in-dividual plants, particularly leaf water potential andtranspiration. Pp did not correlate so well with stomatalconductance, but stomatal conductance did not varymuch during daylight hours. The leaf water potentialthreshold for stomatal closure in wheat is low and evenunder water deficit wheat plants tend to keep theirstomata open (Henson et al. 1989). Consequently, in-creasing transpiration with increasing ambient tempera-ture led to a decrease in leaf water potential andconcomitant increase in Pp. ZIM-probes also detecteddifferences between watered and unwatered plants andPp profiles during drying and rewatering cycles demon-strated dehydration and rehydration kinetics, respective-ly. These combined elements indicate that the probesmonitor the relative hydration status of wheat leaves.

Variation in the diurnal amplitude of Pp on differentplants was due to differences in initial clamp pressureand variation in the compressibility and thickness ofindividual leaves, although all clamped leaves were ofthe same age and the probes were clamped a similardistance from each leaf tip. Variation in the diurnal Pp

amplitude consequently led to variation from leaf to leafin the slopes and intercepts of the Pp and Ψleaf correla-tions. Pp can obviously be normalised so that all valuesfall within the same max-min range. Initial clamp pres-sure can also be coarsely adjusted by increasing thedistance between the magnets. A screw thread has beenincorporated onto the counter-pad, so that the magnetcan be wound closer to or further away from the sensormagnet. Variability in Pp amplitudes between individualprobes on wheat leaves was reduced when the magnetswere 1.6 to 2.6 mm apart. This optimum distance islikely to vary between leaf types. Nevertheless, clamppressure will not be identical without prior knowledge

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Fig. 8 Relative change in leaf patch pressure (ΔPp) for differ-ent clamp pressures. Different clamp pressures were applied bywinding the opposing magnets fully to the stamp base (a), orwinding out the magnets 1.6 mm (b) and 2.6 mm from the stampbase (c). Each figure shows the mean ± SEM for three plants.Note the different ΔPp-axes. ΔPp was calculated by normal-ising to the first value at time=1.00. The shaded blocks indicatewhen the plants were in the dark (covered with aluminium foil).Corresponding ambient temperature (T) and relative humidity(RH) are shown in D

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of the leaf properties and a measurement of the forceapplied by the magnets. The probes therefore, show therelative changes in leaf hydration. If more detailed in-formation is needed, such as predicting absolute turgorpressure values, calibration would be needed. Severalreports on tree species showed a correlation between Pcmeasured with a cell pressure probe and Pp that could bepredicted by the original theoretical basis (Ehrenbergeret al. 2012; Rüger et al. 2010). The usefulness of abso-lute values will depend on the application. For genotypiccomparisons, relative changes in leaf hydration willmostly be sufficient.

Despite variation in Pp minimum and maximumamplitudes, the Pp profiles reflected changes in theenvironment. All leaves responded at the same timeto changes in ambient temperature and light intensity.Pp of all plants started to increase at the same timeeach morning when the lights came on and tempera-ture began to increase, with illumination causing morerapid changes. All Pp profiles reached the peak at thesame time. Any temporal differences that were ob-served in Pp were due to the level of the water stress.The Pp profiles were similar to the changes in ambienttemperature, particularly under well-watered condi-tions, but there were disparities between Pp and Tthroughout the experiment. Moreover, illuminationcaused dramatic changes in the Pp profiles that is tobe expected with irradiance-induced changes in sto-matal opening and hence, leaf hydration. The under-lying diurnal fluctuations of Pp are not an artefact ofthe probe as demonstrated when there are no leavesclamped between the magnets, but are due to changesin leaf properties in response to ambient conditions.The volumetric elasticity of leaves is temperature de-pendent (Woodward and Friend 1988), but it is alsodependent on the hydration of cell walls and cellturgor pressure (Murphy and Ortega 1996; Rascio etal. 1988; Steudle and Zimmermann 1977). It is impos-sible to separate these interacting components, but

knowledge of the absolute contribution of each com-ponent is not needed for comparing the overall relativehydration status of different plants and temporalresponses to environmental conditions.

The drying and rewatering cycles identified similarPp responses in wheat that has been seen for other plantspecies (Ehrenberger et al. 2012; Zimmermann et al.2008). Peak and minimum Pp gradually increased be-tween watering events as it is more difficult for the plantto fully rehydrate with increasing soil water deficit.Osmotic adjustment in the leaves of some wheat geno-types can occur in response to water deficit (Johnson etal. 1984), which will aid in maintaining turgor andhence, leaf hydration. However, with increasing timebetween watering events rehydration was slower asindicated by the slower recovery rates in the afternoon(increasing half-times). With-holding water for 6 daysinhibited the afternoon recovery in Pp, but generated aninverse curve during part of the night. Inverse curvesduring the night have been attributed to complete turgorloss (Ehrenberger et al. 2012). Assuming that this wasthe case for wheat, the process was reversible uponrewatering. In addition, the afternoon recoveryhalf-times for Pp also decreased again upon rewa-tering. Another method of analysing the Pp pro-files that demonstrated dehydration/rehydrationkinetics was the relationship between Pp and Tduring the drying and rewatering cycles. Whenplants were well hydrated, Pp = f(T) exhibitedhysteresis as Pp recovered quicker than T duringthe afternoon. The hysteresis declined betweenwatering events and increased again upon water-ing, i.e. the area between the morning and after-noon plots decreased and then increased again.

In conclusion, the ZIM-probe technology is a po-tentially useful tool for assessing relative temporalvariations in hydration status of wheat. The methodsof analysing the Pp profiles in this study will be usefulfor genotypic comparisons of hydration status under

2.2 2.4 2.6 2.8 3.0

0

5

10

15

20

Time (d) P

p (k

Pa)

4.2 4.4 4.6 4.8 5.010

15

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25

Time (d)

T (C

)Fig. 9 Typical example ofthe effects of illuminationand ambient temperature (T;grey line) on mean leaf patchpressure (ΔPp; black line).Arrows indicate when theplants were covered with al-uminium foil and then un-covered. The example shownis a zoomed in view of Fig. 8c

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water deficit. Afternoon recovery times may differbetween genotypes, as well as the hysteresis relation-ship between Pp and T. The timing of the onset ofinverse curves due to turgor loss may differ withdrought resistance and speed of the decline in soilwater content.

Acknowledgments Funding for HB and research costs wereprovided by The UWA Institute of Agriculture and researchfacilities by CSIRO Plant Industry as part of the UWA/CSIROPI partnership. This project is supported by the Group of EightAustralia - DAAD German Research Cooperation Scheme toKS and ZIM Plant Technology (Project-ID: 54387088) and agrant of the Europäischer Fonds für regionale Entwicklung(EFRE 80145650; State Brandenburg) to ZIM Plant TechnologyGmbH. Sam Henty provided technical assistance. We are grate-ful to Professor Steve Tyerman for comments on the manuscript.

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