Environmental and Experimental Botany 48 (2002) 169–175

Effect of mechanically-simulated hail on photosynthesis,dark respiration and transpiration of apple leaves

Iryna Tartachnyk, Michael M. Blanke *Obst�ersuchsanlage Klein-Altendorf der Uni�ersitat Bonn, Meckenheimer Str. 42, D-53359 Rheinbach, Germany

Received 13 June 2001; received in revised form 20 March 2002; accepted 21 March 2002

Abstract

Mechanical hail injury, as a source of abiotic stress, was examined using non-destructive in-situ gas exchangemeasurements and stomatal imprints of affected apple leaves. Apple leaves closed their stomata, as shown by stomatalimprints, within 3 min after induced damage. Water vapour efflux (evaporation) increased in the light, in spite ofstomatal closure, by 16% from 3.9 to 4.5 mmol H2O m−2 s−1 for approximately 10 min after injury due toevaporation from ruptured tissue, with a subsequent decrease to 2.3 mmol H2O m−2 s−1 after 2.5 h. After 3 h,hail-damaged leaves re-opened their stomata to a certain extent and recovered in terms of transpiration. Photosynthe-sis decreased from 13.5 �mol CO2 m−2 s−1 to 9.1 �mol CO2 m−2 s−1 15 min after hail damage with a concomitantincrease in intercellular CO2 from approximately 250 to 340 ppm CO2 and recovered to 73% of its control within 5h. No difference in dark respiration was observed between control and hail-damaged leaves. Water loss, measured inthe dark to separate stomatal transpiration from hail-induced (non-stomatal) evaporation, of apple leaves alsoincreased in the dark after simulated hail damage within 10 min from 1 to 1.3 mmol H2O m−2 s−1 and then declinedslowly from 1.3 to 0.9 mmol H2O m−2 s−1 within 1 h. It is concluded that approximately 0.27 mmol H2O m−2 s−1

or 20–27% of the recorded water loss after the hail damage was evaporation from ruptured tissue. © 2002 ElsevierScience B.V. All rights reserved.

Keywords: Stomata; Mechanical stress; Wounding

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1. Introduction

Hail is one of the abiotic sources of plant stresswhich is mostly unpredictable in both timing andmagnitude and can injure plants at any time.

Hailstones of approximately 3–6 mm diameterwere found in the local hail storms of 3 July 2000and 13 June 2001 and damaged plants by ruptur-ing their leaves resulting in damage to the leaves(Blanke, 2000). Reports in the literature of theeffects of hail on plant physiology are scarce(Weber, 1955; Popovski, 1984; Henson and He-ichel, 1986; Obaga, 1984; Dwyer et al., 1994;MingRong, 1998). This may be, because hail dam-age in most cases does not puncture, but rupture

* Corresponding author. Tel.: +49-228-735-142; fax: +49-228-735-764.

E-mail address: mmblanke@uni-bonn.de (M.M. Blanke).

S0098-8472/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S0098 -8472 (02 )00022 -9

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leaf tissue, an injury which is difficult to induceand to standardise. For estimates of damages andfinancial claims, the largest German hail in-surance recommends experiments with a stapleremover which is unique in applying the rupturingin an identical way as hailstones (VereinigteHagelversicherung, pers. commun., 2000).

The effects of mechanical stress on photosyn-thesis remain unclear (Biddington, 1984). To ourknowledge, no studies examined the effect of hailon evapotranspiration, and only a few hail reportsexist on photosynthesis (Obaga, 1984; Popovski,1984), including 14CO2 assimilation experiments(MingRong, 1998).

This study tested our hypothesis that hail, likeother mechanical injuries, such as bird or insectdamage, (a) reduces photosynthetic activity, (b)increases dark respiration, (c) increases waterefflux from injured leaves despite induced tempo-rary stomatal closure.

Hence, the present work assessed the effects ofhail damage on plant physiology at the leaf levelby silicone imprints of stomatal responses and bymeasuring photosynthesis, dark respiration andtranspiration in-situ using apple as a modelspecies.

2. Materials and methods

2.1. Apple trees

Five 4-year-old apple trees (Malus domesticaBorkh. cv. ‘Golden Delicious’) were chosen forthe present experiment in the field and anotherfive in 25 l pots at Klein–Altendorf ExperimentalStation near Bonn, Germany.

2.2. Hail damage simulation

Hail damage was mechanically simulated, fol-lowing the recommendations of the hail in-surance, by rupturing apple leaves on one side ofthe midrib to standardise the procedure. Each leafwas ruptured by two 6–7 mm long slots in thecentre between the midrib and leaf margin using astandard stationery staple remover. The hail in-jury was simulated at approximately 11:00 h andmeasurements conducted for 5 h on 4 sunny daysin July 2000 in Klein–Altendorf near Bonn, Ger-many. Subsequent measurements were taken atthe same position on the leaves, with the injury inthe centre of the 2.5 cm2 PLC leaf chamber. Threetypes of control were used for the physiologicalmeasurements (Table 1). The first type of controlemployed measurements on the leaves immedi-ately before mechanical damage (control 1 forlight, control 3 for dark CO2 measurements).Leaves without induced hail damage were alsomeasured simultaneously with damaged leavesand served as control 2.

2.3. Porometry and stomatal imprints

Photosynthesis and transpiration were mea-sured with a portable porometer type CIRAS-1with automated gas mixing and a Parkinson leafchamber type PLC-B from PPSystems, Hitchin,UK. Flow rates into and out of the PLC werecontrolled by two mass flowmeters of the CIRAS-1 and kept at 220 ml min−1 and the boundarylayer resistance of the chamber reduced to lessthan Rb=0.34 m2 s mol−1 by vigorous stirring.The CO2 concentration in the PLC-B was set to360 ppm using CO2 soda cartridges and soda lime

Table 1Experimental design

Type of experiment 11:05–16:00 h09:00–10:55 h 11:00 h

Hail induction no Start of hail effectPhotosynthesis measurements forLight photosynthesis,leaf selection=control 1 measurementsstomatal transpiration and hail-control 2

stomatal imprints every 10 min onDark respiration and the injured spotsDark respiration=control 3 Hail induction

of the leavescuticular transpiration

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Fig. 1. Imprints of abaxial apple leaf surfaces in the light showing primarily open stomata before simulated hail damage (top), closedstomata directly after injury (middle line) and partially re-opened stomata after 5 h (bottom). Space bars denote 25 �m.

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absorber columns and the humidity kept at theambient concentration (Hamacher et al., 1994).Three fully expanded leaves were selected fromthe middle of long shoots of each of ten treeson the basis of identical orientation on the treeand similar photosynthetic rate, measured in themorning before hail damage was imposed at ap-proximately 11:00 h. Photosynthesis and transpi-ration were measured for 5 h during the dayafter hail damage with measurements alternatingbetween hail damaged and unaffected leaves toovercome variations in light or temperaturethroughout the 5 h period. Conditions for thelight measurements were a saturating PAR anda temperature range from 26 °C at 11:00 h to35 °C at 16:00 h resulting in VPDs of 1.8–2.6kPa. Respiration and transpiration in the darkwere measured on leaves of five potted trees in adark room at a VPD of 1 kPa and 21 °C for 5h following the simulated hail damage at 11:00h. and setting the porometer to record automati-cally at 10 min intervals.

Five potted apple trees were exposed to anirradiance of 200 �mol PAR m−2 s−1 duringthe day in a phytotron for 2 weeks (Table 1)before hail damage was induced. Silicone im-prints of abaxial stomata were obtained from150 apple leaves employing the method ofCapellades et al. (1990). Before and every 3 minafter the hail injury for the first hour and every10 min for the next 3 h, affected leaf areas ofthree ruptured and three control leaves werecoated with transparent nail polish. The resul-tant coating, when dry, was peeled off withhighly transparent scotch tape type ‘Tesa klar’.The strips were placed with the imprint on theupper side on microscope slides and pho-tographed in a light microscope type Zeiss Axio-scope 50 using a colour slide film (Agfa RSX II,100 ASA) at magnifications of 100× (leftcolumn in Fig. 1) or 50× (right column in Fig.1).

2.4. Experimental procedure

Five field-grown apple trees were used for thelight measurements and five potted apple trees

Fig. 2. Photosynthesis and dark respiration (a, top diagram)and transpiration (b, bottom diagram) of apple leaves asaffected by hail on 27 July 2000. Measurements in the light areopen symbols, those in the dark closed symbols. Arrowsdenote time of hail induction and numbers the photosyntheti-cally active radiation in �mol PFD m−2 s−1.

for the dark measurements (Table 1). A range ofleaves with the same orientation on the tree wasmonitored for commensurate photosynthesis andtranspiration in the morning. One of these leavesper tree was used to measure photosynthesis andtranspiration before hail damage was simulatedat 11:00 h and another of these leaves used asconcomitant control 2. Dark respiration andevapotranspiration were measured before haildamage was induced as control 3. The curvegiven in Fig. 2 for 1 day represents the typicalresponse on the 4 days measurements. Valuesare averages (�S.D). of five leaves from the fivetrees in the experiment.

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3. Results

3.1. Effects of simulated hail damage on stomatalclosure and apple leaf photosynthesis

Each of the two ruptures from simulated haildamage was 0.5–1×6–7 mm2, resulting in a netoverall loss of 10 mm2 leaf area. When the hail-in-jured leaf section was enclosed in the cuvettecovering an area of 2.5 cm2 for the photosynthesisand transpiration measurements, the proportionof the ruptured area was approximately 4% of thecuvette area.

Stomatal imprints of control leaves in the lightshowed stomata fully open before the hail damage(Fig. 1top). However, plants closed their stomatawithin 3 min after hail damage (Fig. 1middle) andremained mostly closed for up to 3 h in the light,but with a subsequent partial re-opening after 3 h(Fig. 1bottom).

The stomatal closure following hail injury waslocalised in concentric rings around the injury. Aninner ring of 3 mm was characterised by almostcomplete closure and an outer ring by partialstomatal closure, with the magnitude of closuredecreasing with distance from the injury (resultsnot shown).

Photosynthesis of apple leaves declined by 33%from approximately 13.5 �mol CO2 m−2 s−1

before hail damage to 9.1 �mol CO2 m−2 s−1

within 15 min of applying the hail damage (Fig.2a). Similarly, the intercellular CO2 concentrationincreased from approximately 250–340 �l l−1 (re-sults not shown). This increase was due to stom-atal closure after hail damage as indicated bystomatal imprints (Fig. 1, middle) induced by theapplied stress in contrast to fully open stomatabefore hail injury (Fig. 1top). After 10–15 min,photosynthesis remained constant for the rest ofthe day at approximately 30% below that of con-trol leaves (Fig. 2a) due to stomatal limitation(Fig. 2b, Fig. 1bottom).

3.2. Transpiration in the light

Transpiration in the light from the rupturedapple leaves was increased rapidly but transientlyby the injury. The increase from 3.9 to 4.5 mmol

H2O m−2 s−1 was approximately 16% and oc-curred 10 min after the induced hail damage witha subsequent decrease to a trough of 2.7 mmolH2O m−2 s−1 at 2.5 h (Fig. 2b). After 2.5 h,transpiration from hail-injured leaves recovered tovalues of approximately 3.4 mmol H2O m−2 s−1

(Fig. 2b), a reaction similar to that found withphotosynthesis and stomatal reopening (Fig. 2a,Fig. 1 bottom). Overall, the transpiration rate wassignificantly reduced compared with the controlsthroughout 4–5 h of the experiment (Fig. 2b).

3.3. Dark respiration and transpiration in thedark

There was no marked difference in dark respi-ration, measured at a constant temperature of21 °C, between hail damaged and control leaves.

Water vapour loss from apple leaves with simu-lated hail damage initially increased by 20–27%within 1 h in the darkness from approximately 1to 1.27 mmol H2O m−2 s−1 and then declinedslowly to 0.93 mmol H2O m−2 s−1 (Fig. 2),measured at a constant temperature of 21 °C.

4. Discussion

Photosynthesis of apple leaves declined by 33%within 15 min of applying the mechanical haildamage which confirms part (a) of the hypothesis.However, there was no marked difference in darkrespiration between hail damaged and controlleaves in contrary to Popovski (1984) and part (b)of the hypothesis, but agrees with results on me-chanically-stressed (dwarfed) tomato (Mitchell etal., 1977). The initial increase in transpirationafter the hail simulation was as expected andhence confirms part (c) of the hypothesis, but was,to our knowledge, previously not documented inthe literature. While we expected an overall in-crease in water vapour efflux from injured leavesof 2–3-fold due to ruptured tissue, the observedresults were much less dramatic in magnitude(16% in the light, 20–27% in the dark) and onlylasted 10 min on a typical summer day (Fig. 2).

The recorded water loss from the leaf in thelight was initially, for 10 min, enhanced with a

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subsequent decrease. The initial increase in waterefflux is probably due to a combination of anover-proportional increase in evaporation fromruptured tissue associated with malfunction ofwater conduction within the leaf and inactivationof water channel proteins (Blanke, 1998). Thesubsequent decrease in transpiration may be dueto changes in water pressure (Malone et al., 1994)causing stomatal closure as a result of loss ofturgor of guard cells due to water loss from thetissue surrounding the wounds. To help distin-guish between these opposing effects (stomata vs.wounding), gas exchange of apple leaves was mea-sured in the dark when stomata close (Blanke etal., 1994; Blanke and Leyhe, 1988).

The relative initial increase of 27% in water lossin the dark (Fig. 2b) was larger than the 10% inthe light (Fig. 2a). This initial increase in darktranspiration (Fig. 2b) is attributed to evapora-tion only from ruptured tissue, while stomata areclosed in the dark. The subsequent drop in darktranspiration to the initial value after 1 h (Fig. 2b)indicated the cessation of water flux from rup-tured tissue. In the light, the difference in transpi-ration between affected and unaffected leavesincreased within 1 h, alike dark transpiration,from inducing the injury (Fig. 2b), indicating thesame response time of 1 h for the cessation ofwater efflux from ruptured tissue. Light transpira-tion continued to decrease (Fig. 2b) due to stom-ata closure (Fig. 1b). The subsequent slowincrease in light transpiration (Fig. 2b) may be aresult of healing of the injury and partial stomatareopening.

Plant responses to mechanical stress such ashail injury include the induction of expression ofcalmodulin (Fig. 3) related genes. Injured tissuehas an immediate but temporary, 10–30 min (Fig.3), requirement for Ca-binding proteins whichcomplex with free cytoplasmic Ca2+ as secondarymessenger (Braam and Davis, 1990).

5. Conclusion

Hence, we conclude that1. leaf tissue surrounding the wound first after

simulated hail damage increase its water va-

Fig. 3. Schematic of the time course of events followingmechanical stress or hail injury indicating the underlying regu-latory mechanisms.

pour loss for approximately 15 (light) to 60(dark) min by approximately 0.27 mmol H2Om−2 s−1 or approximately 27% of therecorded water vapour efflux after the haildamage from approximately 10 mm2 is evapo-ration from ruptured tissue;

2. leaf tissue surrounding the wound after simu-lated hail damage partially but not fully closetheir stomata for at least 3 h which reducesboth its water vapour loss and photosynthesis;

3. both photosynthesis and transpiration wererecovered by, respectively, 73 and 84% of theircontrols within 5 h.

Acknowledgements

We thank Tudor Thomas for revising the En-glish, Michael B. Jackson, Long Ashton, UK forstimulating discussion of mechanical stress andDeutscher Akademischer Austauschdienst(DAAD), Bonn for a 1 year scholarship to thefirst author (I. Tartachnyk).

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