Modulation of leaf conductance by root to shoot signaling under water stress in Arabidopsis

For. Stud. China, 2006, 8(4): 1-5DOI 10. 1007/s1 1632-006-0027-2Modulation of leaf conductance by root to shoot signaling under waterstress in ArabidopsisFan Yi-juanLiu QingWei Kai-faLi Bing- bingRen Hui boGao Zhi-huiJia Wen-suoCollege of Agonomy and Biotechnology, State Key Laboratory of Plant Phyiology and Bichemist, China Agicutural Universit,Beijing 00094, P. R. ChinaAbstract Signal communication betwen root and shoot plays a crucial role in plant rsistance to water stess. While many studies onroot to shoot signals have been carried out in many plant species, n) information is available for the model plant, Arabidopsis, whoseadoption has great sigificance for further probing the molecular aspects of long distance stress signals. Here, we introduced the es-tabishment of technigues for ivestiationis of root to shoot signals in Arabidopsis. Stomatal movecments in rlation to root signalswere probed by using these techniques. The results show that Arabidopsis is a sutable plant species for parial roots drying (RD)experiments. n the PRD system, while no significart diferences were found in leaf water potential between well-watered andstressed plants, water sress led to a decrease in leaf conductance, which suggests a rgulation of stomatal movements by root toshoot sigals. While water stess caused a sgnifcant increse in the concntration of sap abcisic acid (ABA) of xylem, no increasein xylem sap pH was observed. Moreover, the increase in the ABA content of xylem coineided with the derase in leaf conductance,which sggests a pssble role of ABA in the regulation of stomatal movements. Ifared temperature images showed that leaf tem-peraturs of PRD plant were higher compared with those of well watered plants, which further indicates that stomatal movementscan be modulated by root signals. The confirmation of rot to shoo signaling in Arabidopsis has etalihled a basis for further inves-tigation into the molecular mechanisms of the root to shoot signaling under water stess.Key words Arabidopsis, water stess, ABA, leaf conductance1 Introductionhand many signals may be involved in the process andon the other hand, for different plant species the naturePlants have evolved different kinds of mechanisms forof the signals may be different, e.g, while ABA hasplant resistance to water stress. No matter what kindbeen proven to be a key signal in the root to shootof mechanism, a prerequisite for stress-tolerance is asignaling in maize, it is not able to act as a root torapid sense and response to the initial water stressshoot signal to mediate the regulation of stomatalsignal. Stomatal closure is well known to be a keymovement in wheat (Muns and King, 1988; Muns,response to water stress. For many_ years it has been1992; Muns and Kramer, 1996). Besides ABA, moregenerally thought that stomatal closure is directlyattention has been paid to some other signals in root tocaused by a decline in leaf water status in response toshoot signals (Wilkinson and Davies, 1997; Bacon etsoil drying (Slayter and Taylor, 1960; Kramer, 1969;al, 1998; Wilkinson et al, 1998; Bahrun et al, 2002;Davies and Zhang, 1991). At present it has been ac-Gilliham and Tester, 2005). Although investigations incepted that, besides hydraulic signals, chemical sig-root to shoot signals have been carried out in differentnals originating from roots may play a crucial role inkinds of crop species, no information is available inthe regulation of stomatal movements under waterthe model plant Arabidopsis. Adoption of Arabidopsisstress (Henson et al., 1989; Zhang and Davies,1989;for studies of root to shoot signals has particular sig-Gowing et al, 1990; Davies and Gowing, 1999; An-nificance for a probe into the root to shoot signals ingela et al, 2001). Evidence suggests that root signalsrela中国煤化工)f plant resistance tonormally come from partial root drying (PRD) ex-watriggered in roots? Howperiments, in which PRD is able to induce a decline indo rYHCNMHGn root to shoot signalsleaf conductance while no detectable change occurs inbmanipulated,thereby manipulating plantleaf water status (Davies and Zhang, 1991).stress-tolerance and so on? Adoption of ArabidopsisRoot to shoot signaling is complicated. On the onewould greatly contribute to thorough investigations ofAutho力户数据dence. E-mail: jiaws@cau edu.cn2Forestry Studies in China, Vol.8, No.4, 2006these questions because Arabidopsis is the best plant2.3 Xylem sap collectionspecies for molecular rescarch.PRD is a commonly adopted system for studies ofXylem sap was collected using a pressure chamber.root to shoot signaling. It is dificult to perform suchWhen plant stems were about 10 cm, all leaves werestudies using Arabidopsis. One reason is the smallerremoved from the petiole bases and the stem base wassize of Arabidopsis, which makes it difficult to dotightly wrapped with parafilm. The whole plant wasPRD experiments; another reason is that Arabidopsissealed in a pressure chamber with the stem extrudedis a rosette plant, which makes it difficult to obtainfrom the sealing pad. A suitable pressure was appliedxylem sap. In our present study we have first estab-and xylem sap was collected at a rate basically thelished a method for PRD experiment to obtain sapsame as the transpiration rate, which was assessed byafter which investigations on the root to shoot signal-weighing water loss over a specific time period.ing were carried out in Arabidopsis.2.4 pH measurement2 Materials and methodsTwo μL xylem sap was mixed with 2 μL DM-NERF2.1 Plant materials(500 μgmL~' in distilled water) and then placed on aslide. Samples were assayed using a confocal micro-Arabidopsis seeds were sown in pots (15 cmx 10 cm)scope system (Leica SP, Leica). The excitation wascontaining nutrition soil mixed with sand (3:1); fol-respectively set to 514 and 488 nm at power intensitieslowing that, seeds were kept at a temperatures of 4°Cof 5% and fluorescence emission windows were set tofor two days. Plants grew under the following condi-525 to 545 nm.tions: light intensity 300 μmolm s，day/night tem-Fluorescent intensity was measured at excitations ofperature 23°C/20°C, relative humidity 80%, light/dark514 and 488 nm and the ratio of 514/488 nm was cal-period 12 hours/12 hours. After germination, plantsculated. Calibration was performed with MES buffersfurther grew for four to seven weeks until being used.(pH 4.2, 4.4, 4.6, 4.8 5.0, 5.2, 5.4) and the sample pHwas calculated according to a calibration curve.2.2 PRD experiment and measurement ofphysiological parameters2.5 ABA analysisArabidopsis seeds were sown and grown as describedABA analyses were carried out using the radioimmu-above. About 10 to 15 days after germination, plantsmoassay (RIA) method as described by Quarrie et alwere subjected to PRD experiments. Seedling trays(1988). The monoclonal antibody (Mac 252) was pro-with cells of 5 cmx5 cmx8 cm were used and for eachvided by Dr. S. A. Quarrie and 50 μL of it was mixedindividual plant two adjacent cells were selected forwith a 200 μL phosphat-buffered saline (pH 6.0), 100the PRD system. Briefly, in the middle site of the wallμL diluted antibody solution and a 100 μL H-ABAedge of two adjacent cells a breach with 2 mm in(about 20,000 dpm, approximately 8,000 cpm) solu-width and 5 mm in depth was cut and the edges of thetion. The reaction mixture was incubated at 4°C for 45breach were sealed with glue bands. Plants were care-min and the bound radioactivity was measured in 50%fully transplanted right in the breach with its root sys-saturated (NH4)2SO4-precipitated pellets with a liquidtem split into two parts and each part was placed inscintillation counter.each individual cell. After the plants were grown foranother three weeks, water stress treatments wereperformed by withholding watering in one cell while3 Resultsanother cell remained watered. Leaf conductance wasmeasured using a prometer (AP4, Delta-T Devices,Figure 1 shows the development of root systems in theBurwell, UK) at various times during the process ofPRD experiment. It can be seen that both the dried andPRD. Leaf water potential was measured with a C-52wet parts of roots developed very well, although itthermocouple psychrometer chamber and a HR-33Tseems that the dried part grew better than the wet partdew point microvoltmeter (Wescor Inc., Logan, UT,and also grew better than the plants well-watered inUSA). Briefly, leaf discs were cut and placed insideboth parts. When water was withheld from partialthe psychrometer chamber. After sample temperatureroots中国煤化Tan to decline on theand water vapour reached equilibrium in about 30 min,third:antime, no detectablemeasurements were taken by the dew point method.chanYHC N M H Gere observed betweenThree weeks after PRD, lcaf temperature images werePRD plants and the control plants (Fig. 2B). The re-taken with a Thermacam PM250 infrared camera (In-sults suggests that partial root watering is able to re-frametrics, North Billerica, MA, USA).tain the leaf water status which did not decline whileFan Yi-juan et al: Modulation of leaf conductance by root to shoot signaling under water stress in Arabidopsisconcentration of xylem and this increase in ABA con-Acentration coincided with the decline in leaf conduc-tance, which showed that ABA might be able to act asa root signal to regulate stomatal movements (Fig. 4A).In recent years it has been increasingly suggested thatan increase in pH of xylem may be able to act as aroot signal to regulate stomatal movements and leafgrowth; therefore, we analyzed the effect of waterstress on xylem pH. In well- watered plants, the pH ofxylem is normally around 5.0 Fig. 4B); serious waterBstresses led to a minor decrease in the pH of xylem. Itseems that pH signals are not involved in root to shootsignals.Infra-red imaging is a recently developed technique,which can be used to monitor sensitive changes inplant temperature. Compared with well-watered plants,the leaf temperature in PRD plants is higher (Fig.5),which indicates that the transpiration rate in PRDplants is lower than that of well-watered plants. ThisFig. 1 Root development in root split system. A: PRD, the leftresult provided alternative evidence that stomatalpart is water-stressed, the right part is well watered; B: con-moment can be regulated by root signals, such as soiltrol, both parts are well watered.drying.other part of roots dried, indicating that the PRD sys-tem was suitable for studies of stomatal movements in4 Discussionrelation to root-shoot signaling.Interestingly, a significant decline in leaf conduc-It is well known that soil drying inevitably leads to atance was observed on the fourth day of withholdingdecline in leaf water status; hence, for many years itwater (Fig.3), while no decline in leaf water potentialhas been generally thought that the changes in leafoccurred (Fig. 2B), which strongly suggests that leafwater status play a central regulatory role in plant re-conductance might be regulated by chemical signalssponses to soil drying (Slayter and Taylor, 1960;coming from roots in drying soils.Kramer, 1969). Until recently it has been increasinglyTo identify further the nature of the signals, we de-indicated that shoot responses to soil drying can occur.termined changes in ABA concentrations of xylem.in the absence of any detectable changes in leaf waterWater stress led to a considerable increase in the ABAstatus (Davies and Zhang, 1991). An example of thisis the response of stomata or growth to early soil dry-ing. In an investigation of the leaf growth of apple600500-鲤稣8δ至05互trees in response to soil drying, root systems of indi-vidual plants were split between containers. With-400holding water from one container significantly re-300duced leaf growth (Gowing et al, 1990). In another200experiment, the roots of sunflower plants were sealedin a pressure chamber and a pressure was applied,100- Awhich could retain the leaf water status as the soil46810 12dried (Saab and Sharp, 1989). Leaf conductance of the-0.3-0.4S50 1-0.5500-0.6t08@8鲤鲤，， 450-0.7#和互-0.8350-0.9- B中国煤化工-1.024681012MHCN MHG810 12Days after watering withheldDays after watering witheldFig. 2 Effects of water stress on soil water content (A) and leafFig. 3 Effect of water stress on leaf conductance.●: waterconductance (B).●: water stressed; o: well watered.stressed; o: well watered.4Forestry Studies in China, Vol.8, No.4, 2006ments. Actually, the root to shoot signaling is not so0Asimple. In many cases, the stomatal responses to soil75 tdrying cannot be explained by ABA signals. In wheat,50 tfor example, xylem sap ABA did not cause any in-萬昌45-hibitory activity to stomatal movements even though文30 tsoil drying led to an increase in the ABA concentra-tion of xylem of about 50 times (Muns and King,1988). Many other studies also showed that stomatal01214movement was not related to xylem ABA althoughsoil drying normally induced a considerable increasein the ABA concentration of xylem. The root to shoot.4上signaling is complicated. It is now clear that for dif-5.2 tferent plant species and at different cases the nature of●the root signals may vary. Therefore, it is important to香5.0probe widely the root to shoot signaling in different4.8plant species and Arabidopsis, as a kind of special[Bmodel plant, has particular significance in further re-vealing the mechanisms for root to shoot signaling.468101214As mentioned above, a key point to demonstrate theshoot responses to root signals is to assure a constantFig. 4 Effect of water stress on ABA content of xylem (A) andleaf water status as the soil is drying and the PRDpH (B).●: water stressed; o: well watered.system is a normally adopted approach to fit such anaim. Arabidopsis is a small rosette plant, which makespressurized plants declined as the soil dried, evenit very difficult to do PRD experiments and obtainthough the leaf water potential did not decline. Thesexylem sap. On the basis of a successfully establishedearly investigations provided direct evidence that ei-PRD system and a novel way of colleting sap, thether the responses of leaf growth or conductance topresent investigation has provided evidence thatsoil drying is not related to the changes in leaf waterstomatal movements can be regulated by root signals.status.A sole explanation for this is that shoot re-Many molecular databases are now available insponses can be mediated by some signals coming fromArabidopsis, which makes it possible to probe furtherthe dried root system.into molecular aspects, such as the molecular mecha-It has been widely accepted that ABA can act as anisms for drought-triggered signal production in rootsstress signal. The reason for ABA to act as the stressand the manipulation of root signals to asses the ef-signal is that water stress can induce a considerablefects of root signals on shoot response or stress toler-accumulation of ABA. Extensive studies indicated thatance. Further investigations of the molecular aspectswater stress was able to induce ABA accumulation inin relation to root to shoot signaling are clearly veryxylem sap. Because ABA is a well-known regulator ofimportant for a thorough understanding of the pro-stomatal movement, hence, ABA is generally thoughtfound mystery for the mechanisms of plantas the root to shoot signal, mediating stomatal move-stress-resistance.ControlPRC21C中国煤化工HCNMHGABFig. 5 Effects of water stress on leaf surface temperatures. A: temperature images; B: normal pictures.Fan Yi-juan et al: Modulation of leaf conductance by root to shoot signaling under water stress in Arabidopsis5Acknow ledgementsthe maize root xylem. Plant Physiol, 137:819 -828Munns R. 1992. A leaf elongation assay detects an unknownThis work was supported by the State Basic Researchgrowth inhibitor in xylem sap from wheat and barley. Aust. J.and Development Plan of China (2003CB114300) andPlant Physiol, 19: 127-135he National Natural Science Foundation of ChinaMunns R, Cramer G R.1996. Is coordination of leaf and root(Grant No. 30470160).growth mediated by abscisic acid? Opin. Plant Soil, 185:33- 49Munns R, King R W. 1988. Abscisic acid is not the onlyReferencesstomatal inhibitor in the transpiration stream of wheat plants.Plant Physiol, 88: 703-708Angela S, Davies W J, Hartung W. 2001. The long-distanceQuarrie s A, Whitford P N, Appleford N E J, Wang T L, Cookabscisic acid signal in the droughted plant: the fate of theS K, HensonI E, Loveys B R.1988. A monoclonal antibodyhormone on its way from root to shoot. J. Exp. Bot, 52:to (s)-abscisic acid: its characterization and use in a radio-1,991-1,997immunoassay for measuring abscisic acid in crude extracts ofBacon M A Wilkinson s, Davies W J. 1998. pH-regulated leafcereal and lupin leaves. Planta, 173: 330-339cell expansion in droughted plants is abscisic acid dependent.Saab I N, Sharp R E. 1989. Non-hydraulic signals from maizePlant Physiol, 118: 1,507-1,515roots in drying soil: inhibition of leaf elongation but notBahrunJensen C R, Asch F, Mogensen V O. 2002.stomatal conductance. Planta, 179: 466 474Drought- induced changes in xylem pH, ionic composition,Slayter R O, Taylor S A. 1960. Terminology in plant andand ABA concentration act as early signals in field-grownsoil-water relations. Nature, 187: 992maize (Zea mays L.).J Exp. Bot, 53: 251-263Wilkinson s, Corlet J E, Oger L, Davies W J.1998. Effects ofDavies W J, Zhang J.1991. Root signals and the regulation ofxylem pH on transpiration from wild-type and flacca tomatogrowth and development of plants in drying soil. Ann. Rev.leaves: a vital role for abscisic acid in preventing excessivePlant Physiol. Plant Mol. Biol, 42: 55-76water loss even from well- watered plants. Plant Physiol, 117:，Davies W J, Gowing D J G. 1999. Plant responses to small703- -709perturbations in soil water status. In: Press M C, ScholesJD,Wilkinson S, Davies W J. 1997. Xylem sap pH increase: aBarker M G (eds.), Physiological Plant Ecology. Oxford:drought signal received at the apoplastic face of the guardBlackwells, 67- -89cell that involves the suppression of saturable abscisic acidGowing D J G, Davies W J, Jones H G. 1990. A positiveuptake by the epidermal symplast. Plant Physiol, 113:root-sourced signal as an indicator of soil drying in apple559- -573Malus x domestica Borkh. J. Exp. Bot, 41: 1,535 -1,540Zhang J, Davies W J. 1989. Sequential responses of wholeHenson I E, Jenson C R, Turmer N C.1989. Leaf gas exchangeplant water relations towards prolonged soil drying and theand water relations of lupins and wheat. I Shoot responses tomediation by xylem sap ABA concentration in the regulationsoil water deficits. Aust. J. Plant Physiol, 16 401-413of stomatal behaviour of sunflower plants. New Phytol, 113:Kramer P J. 1969. Plant and Soil Water Relationships: A Mod-167-174ern Synthesis. New York: Mc-Graw-Hil, 482Gilliham M, Tester M.2005. The regulation of anion loading to(Received July 12, 2006 Accepted September 10, 2006)中国煤化工MYHCNMHG