Awang, Kamis

Zainudin, Siti Rubiah

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1997-2000, Section 2-Extended AbstractsStomatal Conductance in Relation to Xylem Sap Abscisic Acid Concentration in H. odorataand M. elengiSeedlings*Siti Rublah Zainudin and Kamis AwangFacultyof ForestryUniversitiPutraMalaysia43400 UPM,Serdang,SelangorMalaysiaE-mail ofCorrespondingAuthor:rubiah@forr.upm.edu.myKey words: H. odorata, M. e/engi,soil compaction, drought, xylem ABA.IntroductionStomata impose a critical control overwater loss and exchange of gases be-tween the atmosphere and leaf cells(Liang et al., 1996). Effective controlis important for plant growth and sur-vival especially when water supply islimited especially in the harsh urbanenvironment. Accumulated evidencehas shown that inhibition of leafgrowth and stomatal conductance areperhaps the first responses when rootsystems are exposed to stress condi-tions, such as drought, flooding andsoil compaction (Passioura et al., 1988;Davies et al. and Zhang et al. 1991;1992a; Tardieu et al., 1993; Hartung etal.,1994). Under these conditions,roots may respond by synthesising andexporting chemical signals through thetranspiration stream to shoots wherephysiological processes are regulated(Davies et al. and Zhang et al., 1991).Much evidence has indicated that root-derived abscisic acid (ABA) in thexylem is involved, and ABA is themost likely candidate as the chemicalsignal involved in communication be-tween root and shoot and in the regula-tion of leaf growth and stomatal con-ductance, especially under conditionsof soil drought and compaction (Zhanget al. and Davies et al. 1989; Gowing etal., 1993). Tardieu et al., (1992) foundthat the abscisic acid (ABA) content ofroot tips increased when maize (Zeamays L.) was grown in compacted soil,whilst the application of 1u'M ABA tounstressed seedlings promoted thegrowth of short thick roots similar tothose produced in response to me-chanical impedance (Hartung etal., 1994). Stypa et al. (1987) also ob-served increases in ABA content inboth the stunted roots and shoots ofmaize plants grown in compacted soilunder laboratory conditions. In con-trast, Lachno et al., (1982) and More et140al., (1988) failed to detect any increasein ABA concentration in the impededroots of maize. Although the effects ofsoil compaction have been extensivelystudied, few experiments have exam-ined the involvement of root-to-shootsignalling in mediating plant responses.Increased xylem sap ABA concentra-tions and associated reductions in sto-matal conductance have been recordedin the shoots of maize plants growingin compacted soil under field condi-tions (Tardieu et al., 1991). Hartung etal., (1994) also reported increases inxylem sap ABA concentrations inplants subjected to mechanical imped-ance. It is therefore possible that aroot-sourced signal such as ABA maybe involved in plant responses to soilcompaction.This experiment was designed to ex-amine the relations between stomatalconductance, xylem sap ABA and leafwater potential in two species grown incompacted and unwatered soil, and toinvestigate whether ABA plays a simi-lar role under these two differentstresses in its regulation of stomatalconductance of these two species.Materials and MethodsThe experiment was a 2x2x2 factorialdesign, i.e. two species, H. odorata andM. elengi, two soil compaction treat-ments (compacted and non-compacted), and two soil drying treat-ments (well-watered and at soil waterpotential 2! -1.5MPa). The soil usedbelongs to the Tropeptic haplorthoxand the seedlings were grown in a se-ries of soil columns consisting of 70-cm long sections of polyvinyl chloride(with internal diameter of 10 cm), witheach column containing two layers ofsoil. The top layer was packed to a drybulk density of 1.0 and 1.5 g/cm'' to adepth of 20 cm and only the first 20 cmof the soil in the column was com-pacted to simulate urban environment.In total, eight different treatments wereused with 24 plants each as replicates.Twelve days later, when the seedlingswere well-established (manifested bythe appearance of new leaves) droughttreatments was imposed by leaving halfof the plants unwatered, while the re-maining half were watered daily as thewell watered treatments.Soil sampling and determination of soilwater content: A 0 - 15 cm depth of thesoil was sampled with a punch (10 mmin diameter and 20 cm in length). Sixsamples were collected for each treat-ment. Fresh soil was weighed immedi-ately after sampling and then overdried at 105°C to a constant weight.The soil water content was calculatedbased on the following equation:Soil water content (%) = 100 x (freshweight - dry weight)! fresh weightMeasurements of stomatal conductanceand leaf water potential: For each plant,the physiological measurements weremade on fully expanded leaves with aLi Cor 6200 Photosynthesis System(Lincoln NE, USA). Twelve meas-urements were carried out, and stoma-tal conductance was expressed as apercentage of the control (well-wateredand non-compacted). Immediatelyafter measurements, the water potentialof the same leaf was measured. Leafdiscs, 6mm in diameter, were sampledand quickly sealed into thermocouplechambers, which were connected to aWescor HR-33T dew-point microvolt-meter. A standard 2-hour incubationtime at 25 QCwas adopted before waterpotential readings were taken.Collection of xylem sap: Xylem sapwas collected between 1400 and 1600hfrom detopped plants from the start ofthe experiment until the 12thday.Plants with intact root systems, takenout of PVC pipes using a plastic bag,were carefully put in a large pressurechamber and detopped about 20 cmAgricultural Sciencesfrom the base. The bark (with phloemtissues) 2cm below the cut surface wasremoved carefully with a razor bladeand the cut surface was then rinsedwith distilled water. Collection pres-sures varied from 0.5MPa (for well-watered plants) to l.5MPa (for unwa-tered plants). In order to avoid thecontamination of the broken cells at thecut surface with phloem sap, the first100 mnr' of sap was discarded and thesecond l00-200mm3 collected andstored in Epppendorf tubes at -70 QCpending ABA assay. After 12 days ofdrought, the collection of xylem sapwas not carried out because the pres-sure needed to obtain xylem sap fromunwatered plants were too high. ABAassay of leaf and xylem sap samples:Leaf samples for ABA analysis werealso collected after measurements ofstomatal conductance and water poten-tial everyday until the 21thday. Leaveswere harvested and plunged into liquidnitrogen. After being freeze dried,samples were stored in a dessicatoruntil assay. The concentration of ABAwas measured by a modification of thatof Loveys et al. (1984). The data weresubjected to three-way analysis of vari-ance (ANOV A) to determine the sig-nificance of the various factors andtheir interactions. The treatment meanvalues of each parameter were sub-jected to the Duncan's New MultipleRange Test (DMRT) to evaluate thedifferences among the treatments.Results and DiscussionEffects on Leaf Water Potential andStomatal Conductance: After beingtransplanted, tree seedlings did notgrow immediately owing to the distur-bance to their root system during trans-planting. They started to grow nor-mally after about 7-9 days of recoverywhen new roots and leaves appeared.These new roots could have been ex-posed to the compacted soil environ-ment (for soil compaction treatments)once they appeared. Soil dryingtreatment was applied 14 days aftertransplanting. After withholding wa-tering, soil water content decreasedrapidly to around 40 to 53% of initialsoil water content after 12 days and thisdecrease slowed down somewhat af-terwards. Compacted or non- com-pacted soil water content showed asimilar decline. Soil drying resulted inthe development of leaf water deficitsin all treatments but the response dif-fered between difference levels withthe decrease in leaf water potential ofM. elengi took place much earlier thanin H. odorata. Stomatal conductance ofwell-watered seedlings somewhatfluctuated throughout the experimentalperiod, presumably as the result ofsmall differences in ambient condi-tions. Three days after withholdingwater, there was a significant reduction(P<O.OOl) in stomatal conductancecompared to well-watered controls forboth species. With further decrease insoil water content, stomatal conduc-tance decreased progressively to aminimum value of about 20% of thecontrol for M. elengi and 40% of thecontrol for H. odorata seedlings re-spectively at the end of the experi-mental period.Effects on Leaf and Xylem Sap ABAConcentrations: For M. elengi, theincrease in ABA concentration oc-curred after 4 days of soil drying inmature leaves and withholding water-ing resulted in more leaf ABA beingproduced under soil compaction alone.A similar trend was observed in H.odorata, in which the ABA concentra-tion in the leaves increased with stresstreatments particularly by withholdingwatering. There was no significantchange in the leaf ABA concentrationof the control plants for both speciesthroughout the experimental period.The leaf ABA concentration of M.elengi seedlings fluctuated throughoutthe experimental period and for H.odorata seedling, a significant (P <0.05) increase of leaf ABA concentra-tion was established on day 5, and thiswas followed by further decline on day10. The ABA concentrations in thexylem sap of control plants fluctuatedthroughout the experimental periodthough not significantly. However, theABA concentration in the xylem sap ofthe stressed plants increased signifi-cantly (P<O.OOI) on day 4 after with-holding water relative to the control.The increase in ABA concentration inxylem sap of the two species was moresensitive to stress treatments than thatof leaf ABA. However after day 8, theABA concentration in the xylem sap ofM. elengi seedlings declined sharplywhile the xylem sap of H. odorata re-mained almost constant thereafter.Within 12 day of withholding watering,the concentration of ABA in xylem sapunder soil compaction and water stressincreased three - to seven fold, withvariation among species and treat-ments.Relationship between Stomatal Con-ductance, ABA in the Leaf and theXylem Sap: The increase in xylem sapABA concentration was associatedwith decline in stomatal conductance ofwater-stressed and compacted seed-lings. When stomatal conductance ofwater-stress plants and compactedplants was plotted against xylem sapABA and ABA, stomatal conductanceshowed a highly significant (r = 0.94for M. elengi and r ::0.98 for H. odo-rata, P<O.OOI) correlation with thexylem sap ABA, while it exhibitedonly a low relationship with the bulkleaf ABA (r =0.17 for M. elengi andr= 0.03 for H. odorata).ConclusionsWith soil drying, significant reductionof stomatal conductance occurredmuch earlier than the reduction of leafwater potential and furthermore, thesoil compaction treatment also reducedstomatal conductance. These resultssuggested that, at least at the initialstate of both treatments, stomatal con-ductance was controlled by something,other than leaf water potential. Furtheranalysis of xylem sap and bulk leafABA indicated that there existed a tightrelationship between stomatal conduc-tance and xylem ABA concentration inboth species but not with the leaf waterstatus or with the concentration ofABA concentration in the leaf. Whenplants grown in compacted but wellwatered soil, the leaf water potentialand leaf ABA concentration were al-most unchanged, but the ABA concen-tration in the xylem sap increasedabout three-to fivefold, and stomatalconductance was significantly inhib-ited. Therefore, as in the case of soildrying, xylem ABA could act as apositive stress signal to control theshoot physiology when plants aregrown in compacted soil and also con-trol the gas exchange of plants underfield conditions. A substantial increasein xylem ABA was observed at highbulk density and accompanied bysmaller, non-significant changes infoliar ABA. Mulholland et al., (1996b)reported a subcritical bulk density,which produced large differences inleaf expansion, rates and leaves areasbetween two barley genotypes, Steptoeand Az34. The inability of Az34 toproduce as much ABA compared toSteptoe in response to soil compactionmay have been crucial to its failure tomaintain near normal rates of leaf ex-141UPM Research Report 1997-2000, Section 2-Extended Abstractspansion at sub critical level of com-paction. A similar result was obtainedfrom this study whereby. xylem sapABA of M. elengi was comparativelylower than H. odorata seedlings. Andthis account for its failure to maintainnormal growth rate compared to H.odorata seedlings.To conclude. stomatal conductance isvery much controlled by ABA concen-tration in the xylem. These resultssuggested that ABA has a positive roleas a root to shoot signal in maintainingleaf expansion and aiding seedlingestablishment at this threshold level ofsoil compaction.Benefits from the studyUnderstanding the importance of ABAin the xylem sap in controlling stomatalconductance and as a growth hormoneand aiding seedling establishment atcertain level of soil compaction.Literature cited in the textDavies, W.J., and Zhang, J. 1991. Rootsignals and the development of plantsgrowing in drying soil. Ann. Rev. PlantPhysiol. Molec. BioI. 42: 55-76.Davies, W.J .• Tardieu, F.• and Trejo, C.L.1994. How do chemical signals work inplants that grow in drying soil? PlantPhysio!. 104: 309-314.Gowing, DJ.G., Jones, H.G., and Davies,WJ. 1993. Xylem-transported abscisicacid: the relative importance of its massand its concentration in the control ofstomatal aperature. Plant Cell Environ.16: 453-459.Hartung, W., Zhang J., and Davies. W.J.1994. Does abscisic acid of a stressphysiological role in maize plantsgrowing in heavily compacted soil? J.Expt. Bot. 45: 221-226.Lachno, D.R., Harrison-Murray, R.S., andAudus, LJ. 1982. The effects ofmechanical impedance to growth on thelevel of ABA and IAA in root tips of Zeamays L. J. Expt. Bot. 33: 943-951.Liang, J., Zhang, J., and Wong, M.H. 1996.Stomatal conductance in relation to xylemsap ABA concentrations in two tropicaltrees, Acacia confusa and Litseaglutinosa. Plant Cell Environ. 19: 93-lOO.Loveys, B.R. 1984a. Diurnal changes inwater relations and abscisic acid in field-grownMore, TA., Stevens, T., and Allen, P.G.1988. Valuation of urban parks.Landscape and Urban Plan IS: 139-152.Passioura, J.B. 1988. Root signals controlleaf expansion in wheat seedlingsgrowing in drying soil. Aust. J. PlantPhysiol. IS: 687-693.Stypa, M.L. 1987. Effects of soil bulkdensity on seedling physiology andgrowth of corn (Zea mays L.). Ph.D.Dissertation, University of Waterloo,Canada.Tardieu F., and Davies, WJ. 1992.Stomatal response to abscisic acid is afunction of current plant water status.Plant Physio!. 98: 540-549.Tardieu, F., and Davies, WJ. 1993.Integration of hydraulic and chemicalsignalling inthe control of stomatalconductance and water status ofdroughted plants. Plant, Cell Environ.16:341-349.142Tardieu, F., Katerji, N., Bethenod, 0.•Zhang, J., and Davies, WJ. 1991a. Maizestomatal conductance in the field: Itsrelationship with soil and plant waterpotentials, mechanical constraints andABA concentration in the xylem sap.Plant Cell Environ. 14: 308-314.Vitis vinifera cultivars. III. The influence ofxylem-derived acid on leaf gas exchange.New Phytol. 98: 563-573.Zhang, J., and Davies. W.J. 1989b.Sequential responses of whole plant waterrelations towards prolonged soil dryingand the mediation by xylem sap ABAconcentrations in the regulation ofstomatal.Project Publications inRefereed JournalsKnight, M.R., Smith S.M., and Trewavas,AJ. 1992. Wind-induced plant motionimmediately increases cytosolic calium.Proceeding of the National Academy ofSciences of the USA. 89: 4967-4971.Mullholland, BJ .•Taylor, LB .•Black, C.R.,and Roberts. J.A. 1996b. Effect of soilcompaction of barley (Hordeum vulgareL.) growth. II. Are increased xylem sapABA concentrations involved inmaintaining leaf expansion in compactedsoils? J. Expt. Bot. 47: 551-55.Project Publications inConference ProceedingNone.Graduate ResearchSiti Rubiah Zainudin. 2000. Urban Forestry[Ph. D.I Universiti Putra Malaysia.

Stomatal conductance in relation to xylem sap abscisic acid concentration in H. odorata and M. elengi seedlings

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Stomatal conductance in relation to xylem sap abscisic acid concentration in H. odorata and M. elengi seedlings

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