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Environmental and Experimental Botany 59 (2007) 188–192

Transpiration response of Arabidopsis, maize, and soybeanto drying of artificial and mineral soil

Ammar Wahbi a, Thomas R. Sinclair b,∗a Soil Science Department, Faculty of Agriculture, University of Aleppo, P.O. Box 8047, Aleppo, Syria

b Agronomy Physiology Laboratory, P.O. Box 110965, University of Florida, Gainesville, FL 32611-0965, USA

Received 24 May 2005; received in revised form 15 September 2005; accepted 20 December 2005

bstract

Water-deficit stress is a major constraint on plant productivity and consequently, is a major focus of much research. Stress is often imposed onlants in these experiments by withholding water from the artificial potting media on which the plants are grown. No attention has been given,owever, to the possibility of differences in the dynamics of stress imposition between that resulting from dehydration of the artificial rootingedia and that of drying of mineral soil. The objective of this research was to compare transpiration rates during drying of a mineral soil and

f an artificial potting mixture for three test species: Arabidopsis thaliana, maize (Zea mays), and soybean (Glycine max). These results showedajor differences in transpiration response between the two soil media. Drying of mineral soil confirmed previous observations that no decrease

n transpiration rates occurred until 0.27–0.34 of the extractable water remained in the soil. Thereafter, there was essentially a linear decrease inranspiration with further soil drying. In contrast, transpiration rates of plants grown on the potting mixture began to decrease when about 0.6–0.7

f the extractable water still remained in the soil. Consequently, plants grown on the potting mixture as compared to the mineral soil were exposedo stress very early in the drying cycle and the stress was much more prolonged over a wide range of soil moistures. Caution is warranted inxtrapolating to natural, mineral soils the results obtained from plants subjected to water-deficits using artificial potting mixtures.

2006 Elsevier B.V. All rights reserved.

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eywords: Water-deficit stress; Transpiration; Maize; Soybean; Arabidopsis

. Introduction

Soil water-deficit is frequently the environmental factor inany natural environments that imposes the greatest constraint

n plant growth. Not surprisingly, a great deal of research haseen targeted to identifying and understanding plant responseso the imposition of water-deficit. Experimental approachesubjecting plants to water-deficit stress have, however, variedidely. In extreme cases, analysis of gene expression in response

o stress has been done with plants that have been physicallyemoved from their source of water and allowed to desiccateor a few hours before collecting tissue samples (e.g. Seki etl., 2002; Llorente et al., 2002). A more common approach is

o impose a ‘natural’ drought by withholding water from the

edium in which the plants are being grown (e.g. Merlot et al.,002; Taji et al., 2002).

∗ Corresponding author. Tel.: +1 352 392 6180; fax: +1 352 392 6139.E-mail address: trsincl@ifas.ufl.edu (T.R. Sinclair).

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098-8472/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2005.12.004

A potential difficulty often overlooked in the ‘natural dry-ng’ experiments, however, is that the nature of the water-deficitmposed on the plants is dependent on the properties of the root-ng medium. Experiments with transformed plants, for example,re almost universally done by using artificial rooting mediumecause it is readily available, appears to be of consistent quality,nd offers a ‘clean’ rooting medium. The artificial potting mediare commonly a mixture of organic matter, such as peat, and ver-iculite or perlite. Water release characteristics and hydraulic

onductivity changes during drying of artificial media (Da Silvat al., 1993; Heiskanen, 1995) can, however, be substantiallyifferent from the physical characteristics of mineral soils (e.g.lapp and Hornberger, 1978). As an example, the water releasend hydraulic conductivity of a peat medium and a sandy loamoil are compared in Fig. 1. While the water release curves areot greatly different, during much of soil drying the hydraulic

onductivity of peat is much lower than that of the sandy loamoil. The comparatively low hydraulic conductivity of the peatay be inhibitory to water transport to the plant even in the early

tages of soil drying.

A. Wahbi, T.R. Sinclair / Environmental and E

Fig. 1. Water release curves and hydraulic conductivities as a function of volu-mm

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planting in Experiments 1 and 2, respectively. The drained upper

etric water content for sandy loam soil (Clapp and Hornberger, 1978) and peatoss mix (Da Silva et al., 1993).

Plant gas exchange response to decreases in the volumetricater content of mineral soils has been well documented. Whileariability in the threshold volumetric water content for decreas-ng transpiration rate has been reported (Sadras and Milroy,996), in many studies the threshold has been reported to ben the range of 0.25–0.40 fraction extractable soil water across

number of plant species, various environmental conditions,nd a range of soil textures. Once the threshold fraction ofxtractable water has been reached, gas exchange declined in anpproximately linear manner with further soil drying, approach-ng a minimal level when the extractable soil water is exhausted.

hile plants growing on coarse soils also show the two-phaseinear response to soil drying, the decline in gas exchange iseported to occur at somewhat greater relative water contentshan the usual 0.35 value (Sadras and Milroy, 1996). Sinclairt al. (1998) confirmed the two-phase linear response for sev-ral sandy soils but the determination of the specific thresholdor the decline in transpiration was confounded for these soilsecause of the difficulty in defining the endpoints of extractableoil water.

Direct measures of the impact of the unusual hydraulic char-cteristics of the artificial potting mixture (Fig. 1) on plantranspiration as the medium dries have, however, not been docu-

ented. If the water-deficit imposed on plants in the experimentssed to induce gene expression, for example, is unlike the stressxperienced by plants under natural conditions, there is a ques-

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xperimental Botany 59 (2007) 188–192 189

ion about the relevance of results from experiments based onrying of artificial rooting medium.

The objective of this study was to compare directly the rel-tive transpiration rates of plants grown on mineral soil andrtificial medium during a drying cycle. The key question ishether plant gas exchange response to decreases in volumet-

ic water content of the artificial medium matches the commonesponse observed with mineral soils. Transpiration responsesere studied for three species: Arabidopsis thaliana, which is

he species often used in many transgenic studies, maize (Zeaays), and soybean (Glycine max).

. Materials and methods

.1. Rooting media

A commercial, sandy-clay top soil (0.74 sandy, 0.06 silt, 0.20lay), which is widely available in the U.S. (Top Soil, Wal-art, Inc.), was selected as the mineral soil in these experiments.he artificial potting mixture was Terra-Lite Agricultural Mix

#92873, Scotts Company, Marysville, OH), which has good aer-tion and drainage, and is essentially sterile. The potting mixtureas 0.35–0.45 g g−1 medium grade horticultural vermiculite,.55–0.65 g g−1 choice grade Canadian sphagnum peat moss,nd a nutrient starter.

.2. Arabidopsis dry-down experiments

Arabidopsis plants were grown in small pots that were con-tructed from end caps of 2-in. polyvinylchloride pipes. Theseots had an internal diameter of 48 mm and a depth of 35 mmiving a volume of about 60 cm3. Holes were drilled into the bot-om of the caps to allow free water drainage. The pots were filledith rooting medium, which required 48 g of dry mineral soil

nd 14 g of dry potting mixture. Consequently, the bulk densityf the two rooting media was quite different but, as indicated inhe volumetric extraction curve in Fig. 1, it was expected that thewo media had similar amounts of extractable water. Two Ara-idopsis experiments were performed with 11 pots in the firstxperiment and 12 pots in the second for each rooting media.our pots were maintained as the well-watered treatment in eachxperiment.

The genotype Landsberg erecta was used in these experi-ents. Seeds were surface sterilized by placing them in 50%

leach for 5 min and then rinsing in sterile water. Seeds werencubated for 2–4 d at 4 ◦C to break dormancy. Then, the seedsere placed on a gel medium (minerals, MES, sucrose and Phy-

agel at pH of 5.7–5.8) and kept under continuous light at 22 ◦Cor 14 d. At the 3- to 4-leaf stage, one or two plants were trans-lanted to each pot. The plants were grown in the laboratorynder an artificial light of 250–350 �mol m−2 s−1, a photope-iod of 12 h, and the temperature was approximately 22 ◦C.

Dry-down experiments were begun 10 and 7 d after trans-

eight of each pot was measured at the beginning of the exper-ments by over watering the pots in the late afternoon andllowing them to drain over night. The following morning the

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op and the bottom of the pots were closed with parafilm so thathe water loss was only a result of plant transpiration. The potsere weighed to the nearest 0.01 g to obtain the drained upper

imit. Subsequently, pots were weighed each afternoon over theollowing 3–4 wks. Four pots were kept well-watered by addingater daily to return the fraction of extractable water to approx-

mately 0.75 once the pots had initially dried below this level.o water was added to the drying pots.The daily relative transpiration rate (RT) was calculated for

ach plant subjected to water-deficit by dividing their transpi-ation rate determined from the differences in daily pot weight,y the mean transpiration rate of the four well-watered plants.he relative rates for each plant on drying rooting media were

urther normalized to a value of 1.0 for the first few days ofhe experiment when the medium for each plant was still wetnd there was no evidence of decreased transpiration rate. Thisecond normalization facilitated comparisons among plants ofiffering size.

Transpirable soil water for each pot was calculated by sub-racting the weight when normalized RT was first less than 0.1rom the drained upper limit weight. No attempt was made toorrect for the small increase in plant mass during the experi-ent, which would have resulted in a small error causing a small

nderestimate of the lower end point. This small error wouldave been the same for the two soil media since the growingonditions and duration of the experiments were equal. Fractionf transpirable soil water (FTSW) was calculated for each potn each day. This was done by subtracting for each pot the lowerimit weight from the weight measured each day, and dividing byhe total transpirable soil water for the pot (Sinclair and Ludlow,986). Data collection for each plant was stopped when the RTalue decreased to less than 0.1.

.3. Soybean and maize dry-down experiments

Soybean (cv. Biloxi) and maize (cv. Reids yellow dent) wererown in 3.2-l pots with holes in the bottom to allow free waterrainage. The pots were packed uniformly with either 2850 g ofhe dry top soil or 975 g of the dry potting mixture. Again, theeight difference was a result from differences in bulk density.leven pots were prepared for each species and each rootingedia, and placed in a greenhouse with day/night temperatures

f approximately 28/20 ◦C. The rooting media were initiallyver-watered, and after drainage, five seeds were sown in eachot on 26 September 2002 for maize and 1 October 2002 foroybean. Following germination, the plants were maintained inwell-watered condition. Two weeks after sowing the pots were

hinned to a single plant per pot.Dry-down experiments were initiated about 19 d after sowing.

ate on the afternoon of 13 October for maize and 20 October foroybean all pots were over watered. The following morning eachot was enclosed in a 16-l white plastic bag, which was sealedround the base of the stem of the plant with a tie. The pots were

eighed to the nearest g to obtain the weight at the drained upper

imit for each pot. Each afternoon the pots were reweighed. Fourots of each species and each rooting media were maintained inwell-watered condition by watering the medium once it had

ivpi

xperimental Botany 59 (2007) 188–192

ried to less than 0.75 of extractable soil water. Water was addedaily to the well-watered pots to return the rooting media to about.75 of extractable soil water. Seven pots of each rooting mediaere allowed to dry over approximately a 3-wk-period. To avoid

apid rooting media dehydration, water was added to the dryingots if needed so that there was only a maximum of 70 g netoss of water each day. Calculations of RT, normalized RT, andTSW were the same as described previously. The experimentas terminated for each plant subjected to water-deficit whenT was less than 0.1.

.4. Analysis of dry-down data

Values of normalized RT and FTSW obtained during the dry-own experiments for each plant on each day were all combinedo obtain the RT response curve for each soil medium in eachxperiment as a function of FTSW. The response data were ana-yzed by two procedures. First, a linear-plateau analysis wasttempted. A plateau region, where normalized RT was approx-mately 1.0, was defined for the region where the soil mediumas wet. The region of the linear decline in normalized RT as the

oil medium dried further was fitted by linear regression. Datahere RT was less than 0.8 were included in the linear regres-

ion for the stressed phase. The intersection of the linear regionith normalized RT = 1.0 was calculated as the plateau intercepthere drying induced a response in plant gas exchange. Thisodel has been one that has been shown to fit well the response

f plants grown on mineral soil, and the FTSW at which RTeparted from the plateau has been found to be approximately.35 (e.g. Ray et al., 2002).

In addition to the linear-plateau model, a simple exponentialodel was fitted to the data:

T = 1 − exp(−k × FTSW) (1)

n this exponential model, the value of k reflected the rate ofecrease in RT as FTSW decreased.

All regressions were done using GraphPad Prism Ver. 3.0GraphPad Software, Inc., San Diego, CA 92121, USA).

. Results

The mean amount of water available to the plants was, asxpected, roughly similar between the mineral soil and the arti-cial potting mixture in all experiments (Table 1). Therefore, theormalization of the extractable soil water to calculate FTSWid not disguise any major differences in the total amount ofater to which the plants had access in each experiment. TheTSW values represent roughly equivalent amounts of wateremoval from the two rooting media.

The response of Arabidopsis to drying of the mineral top soilas consistent with that reported for a range of crop species

Sadras and Milroy, 1996). That is, there was little or no change

n normalized RT in Experiment 1 until FTSW decreased to aalue less than 0.35 (Fig. 2a). In this experiment, the linear-lateau model represented these data very well with an r2 = 0.91n the linear decrease portion of the response. FTSW was equal

A. Wahbi, T.R. Sinclair / Environmental and Experimental Botany 59 (2007) 188–192 191

Table 1Results of regression analysis using a linear-plateau model for the top soil and an exponential decay model for the potting mixture

Variable Maize Soybean Arabidopsis I Arabidopsis II

Top soilTotal transpirable H2O (g) 1304 1204 36 34Plateau intercept (FTSW) 0.344 0.310 0.282 0.27595% confid. int. 0.32–0.37 0.28–0.35 0.26–0.30 0.26–0.29r2 for linear portion 0.95 0.92 0.91 0.97n 90 35 73 62No. plants 7 7 7 8

Potting mixtureTotal transpirable H2O (g) 1364 1507 47 35k in Eq. (1) 2.56 3.40 2.30 5.8595% confid. int. 2.10–3.01 2.96–3.84 1.87–2.74 5.31–6.40r2 0.95 0.95 0.95 0.98

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o 0.28 at the intercept of the linear portion with the plateau.

he artificial potting mixture, on the other hand, resulted in an

nitial decrease in normalized RT before FTSW had declined to.60 (Fig. 2b). There was a gradual decline in normalized RT asTSW decreased.

ig. 2. Arabidopsis I normalized relative transpiration rate (RT) plotted againstraction of transpirable soil water (FTSW) for: (a) top soil and (b) potting mix-ure. The data were obtained from seven plants for each rooting media and theegression lines were based on 73 data for the top soil and 125 data for theooting media.

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The results with the larger maize and soybean plants wereirtually the same as those for Arabidopsis. When grown on top

oil there was no change in RT until the soil had dried to a FTSWf less than 0.4 as illustrated in Fig. 3a with the data from maize.gain, the linear plateau model described these data well. The

egression for the linear-increase segment in Fig. 2a had an r2 of

ig. 3. Maize normalized relative transpiration rate (RT) plotted against fractionf transpirable soil water (FTSW) for: (a) top soil and (b) potting mixture. Theata were obtained from seven plants for each rooting media and the regressionines were based on 90 data for the top soil and 123 data for the rooting media.

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.95, and the FTSW for the intercept with the plateau was 0.34.he responses to water-deficit for both maize and soybean grownn top soil were poorly modeled by the exponential equationEq. (1)) because the exponential equation could not representell the sharp transition from the plateau to the linear decrease

Sinclair and Ludlow, 1986).Results from the dry-down experiment when maize and soy-

ean were grown on the artificial potting mixture were substan-ially different from the results commonly reported on mineraloil (Sadras and Milroy, 1996). There was a gradual decline inormalized RT over a wide range of FTSW beginning at FTSWalues greater than 0.60 in the case of maize illustrated in Fig. 3b.he exponential model described well the decay in RT observedith the artificial potting mixture with r2 values of 0.95 for both

pecies.A summary of regression results from all experiments is pre-

ented in Table 1. Drying of the top soil gave much the sameesults across species. The linear-plateau model described theesponse well and the plateau intercept for all species rangedrom 0.28 to 0.34. This is within the usual range for many of theesults reported in the literature (Sadras and Milroy, 1996). Inontrast, the response from drying of the artificial potting mix-ure was not represented by the linear-plateau model and insteadn exponential decay model was required.

. Discussion

The results of these dry-down experiments with mineral soilhowed transpiration responses to soil drying by Arabidop-is, maize, and soybean plants that were fully consistent withany previous reports for plants grown on mineral soil (Sadras

nd Milroy, 1996). There was no influence of top soil dryingn transpiration rate until FTSW reached 0.28–0.34 (Table 1).he decrease in normalized RT was highly linear once FTSWecreased to values less than a threshold value.

On the other hand, the artificial potting mixture resulted inlant responses to drying that were unlike any results reportedor mineral soils in that transpiration rates began to decline veryarly in the dehydration of the potting mixture. For example,ormalized RT values for maize on the drying potting mixturead decreased to 0.80 when FTSW was still 0.60, and to 0.65hen FTSW equaled 0.40. Normalized RT for plants grown on

he top soil were unaffected by decreases in FTSW to 0.60 and.40. At a lower FTSW, the normalized RT of plants on theotting mixture continued to be less than that on the mineraloil.

Once transpiration rates began to decline, there were differentatterns between the two soil media. In the potting mixture theecline was described by an exponential model (Eq. (1)) over aide range of FTSW. The decline in transpiration rate with top

oil, on the other hand, could be represented by a linear modelnce the threshold had been reached. Therefore, the duration andate of imposition of water-deficit stress imposed on the plants

as substantially different between rooting media.There was no substantial difference among the three species

n the transpiration response within either soil medium. That is,ery small Arabidopsis plants responded to drying of the rooting

T

xperimental Botany 59 (2007) 188–192

edium in the same manner as did maize and soybean plants.lant size or species had little impact on the overall pattern.

These results indicate that considerable caution is requiredn extrapolating results characterizing transpiration response toeveloping water-deficit stress with plants grown on an artificialotting mixture. The pattern of stress induction and subsequentntensity are clearly unlike that when plants are grown on min-ral soils. Instead of a long period of no change in transpira-ion as a mineral soil dries, plants grown on the artificial soilere subjected early in the drying cycle to a stress that influ-

nced plant gas exchange. The caution in using artificial pottingedium likely extends to gene expression studies that rely on the

mposition of stress by drying of the artificial medium. Futurenvestigations of water-deficit stress might be better served byrowing plants on mineral soils, or at least include confirmationsf plant and gene behavior identified using artificial soil mediay including tests using a mineral soil.

cknowledgments

The senior author is grateful to the Council for Internationalxchange of Scholars, visiting Fulbright Scholar Program, USA,

or providing part of the support funds for a sabbatical leave tondertake this research.

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