The effect of water stress on photosynthetic carbon metabolism in four species grown under field conditions

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Abstract. The effect of gradually-developing water-stress has been studied in Lupinus albus L., Helianthus annuus L., Vitis vinifera cv. Rosaki and Eucalyptus globulus Labill. Water was withheld and diurnal rhythms were investigated 4–8d later, when
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  Plant Cell and Environment  (1992) 15, 25-35 The effect of water stress on photosynthetic carbonmetabolism in four species grown under field conditions W. p. QUICK,* M. M. CHAVES,t R. WENDLER,* M. DAVID,t M. L. RODRIGUES,t J. A.PASSAHARINHO,1 J. S. PEREIRA,t M.D. ADCOCK,1 R. C. LEEGOOD* M. STITT*  Lehrstutit fUr  Pftanzenphysiotogie,  Universitat  Bayreuth, D-8580 Bayreuth, Gcrtnany, tittstituto Superior deAgnonomia, Universidadc  Tccttica  dc Lisboa, P-1399 Lisboa,  Portugal and ^Robert Hitt  Institute,  epartment  of Animaland Platu Sciences,  Utuversity  of  Sheffield Sheffield  SJO  2TN, UK ABSTRACT The effect of gradually-developing water-stress hasbeen studied in  Lupinus albus  L. elianthus annuus  L., Vitis vinifera  cv. Rosaki  nd  Eucalyptus globulus  Labill.Water was withheld and diurnal rhythms were investi-gated 4-8 d later, when the predawn water deficit wasmore negative than in watered plants,  nd  the stomataclosed almost completely early during the photoper- iod.  The contribution of 'stomatal' and 'non-stomatal'components to the decrease of photosynthetic ratewas investigated by (1) comparing the changes of therate of photosynthesis in air with the changes ofstomatal conductance and (2) measuring photo-synthetic capacity in saturating irradiance and 15%CO2. Three species (lupin, eucalyptus and sunflower)showed larger changes of stomatal conductance thanphotosynthesis in air, and showed little or  no  decreaseof photosynthetic capacity in saturating CO2. Photo-synthesis in air also recovered fully overnight afterwatering the plants in the evening. In grapevines,stomatal conductance and photosynthesis in airchanged in parallel, there was a marked decrease ofphotosynthetic capacity, and photosynthesis andstomatal conductance did not recover overnight afterwatering water-stressed plants. Relative water con-tent remained above 90% in grapevine. We concludethat non-stomatai components do not play a signifi-cant role in lupins, sunflower or eucalyptus, but couldin grapevine. The effect of water-stress on partitioningof photosynthate was investigated by measuring theamounts of sucrose and starch in leaves during adiurnal rhythm, and by measuring the partitioning of^ C-carbon dioxide between sucrose and starch. In allfour species, starch was depleted in water-stressedleaves but sucrose was maintained at amounts similar to,  or higher  than,  those  in  watered plants. Partitioninginto sucrose was increased in lupins and eucalyptus,and remained unchanged in grapevine and sunflower.It is concluded that water-stressed leaves in all fourspecies maintain high levels of soluble sugars in their Correspotidetiee: R. C.  Leegood Robert Hitt histitute. DepartinentofAtiimal and Platit .Scietiees, University of  Sheffield Sheffield SW2TN, UK. leaves, despite having lower rates of field photosyn-thesis, decreased rates of export, and low amounts ofstarch in their leaves. Key-words:  pbotosynthesis; water stress;  Lupinus;  Heli- anthus; Vitis; Eucalyptus;  starch; sucrose; carbon par-titioning. Abbreviations: C CO2 concentration in the intracellularspaces in the  leaf;  D, water vapour pressure deficitbetween the air and the  leaf;  DW, dry weight; FW, freshweight; gs, stomatal conductance; RWC, relative  water content; Tr, transpiration rate;  ip water potential. INTRODUCTION The following experiments were carried out to charac-terize the effect of water stress on four different speciesgrowing in pots under field conditions. In particular, wehave asked (1) whether there is any evidence for a direct(non-stomatal) inhibition of CO2 fixation and (2)whether water stress has a selective effect on  th partitioning of photosynthate between starch and solu-ble sugars, and the atnounts of sucrose and starch in the le f The simplest explanation for the inhibition of photo-.synthesis during water stress would be that the stomataclose and the internal CO2 concentration (Q) decreases(Farquhar Sharkey 1982; Sehulze 1986). Thetefore, itwas rather surprising that photosynthesis oftendecreases in parallel with, or more than, stomatalconductance (g) (Wong, Cowan Farquhar 1985;Huber, Rogers Mowry 1984; Raschke Reeseman 1986;  Cornic  et al.  1989). These results were srcinallyinterpreted as evidence that Cj increases, implying thereis an additional direct or 'non-stomatal' effect of waterstress on photosynthesis. However, the calculation of Qassumes uniform stomatal behaviour (Laisk 1983) and itis now well established that stomata close non-uniformlyduring short-tenn water stress. This will lead to anoverestimate of Q during water-stress, especially inheterobaric leaves where lateral gas diffusion is moredifficult (Farquhar  ct al.  1987; Terashima  et al.  1988;Downton, Loveys Grant 1988). A similar explanation  5  26  W. P. Quick  et al.has been proposed for the apparent 'non-stomatal'effect of abscisic acid (Daley  et al.  1989). Therefore,stomatal closure is likely to play a major role in thedecrease of photosynthesis during water stress, buttechnical problems make it difficult to define its preciserole in many species.An alternative way to assess whether water stress hasa direct effect on biochemistry is to investigate the effectof water stress in experimental systems where stomatahave been removed; for example, in cell suspensions(Kaiser, Steppen & Urbach 1981; Sharkey & Badger1982) or leaf strips from which the epidermis has beenremoved (Dietz & Heber 1983; Kaiser 1982,1987). Suchexperiments indicate that the relative water content(R WC) must be decreased to 70-50%, corresponding toa very large water deficit, before photosynthesis isinhibited. Similar tesults have been obtained using veryhigh (5%) concentrations of external CO2 to allow rapiddiffusion into the spinach leaves, independent of thestomata (Kaiser 1987; Ouick  et al.  1988; Robinson,Grant & Loveys 1988).Ouite apart from direct effects on the  rale  of photo-synthesis, water stess could modify the immediate fate ofthe fixed carbon. In the following article, we restrictourselves to partitioning between starch and solublesugars. Many studies have reported that water stressleads to a general depletion of soluble sugars and starchin leaves (Wardlaw 1969; Hanson & Hitz 1982; Sung &Krieg 1979; Huber  et al.  1984) and have concluded thatwater stress has a larger effect on carbon assimilationthan on translocation and use. Nevertheless, somestudies have reported that soluble sugars accumulate inleaves during water stress (Turner, Begg   Tonnet 1978;Jones, Osmond & Turner 1980; Munns & Weir 1981),and propose that they might contribute to osmoregu-lation (Morgan 1984). Moreover, during short-termwater stress, starch synthesis is more strongly inhibitedthan sucrose synthesis in ambient CO2 (Ouick  et al. 1988;  Vassey & Sharkey 1989) and in saturating CO2(Ouick  et al.  1988). Short-term water stress also stimu-lates the conversion of starch to sucrose (Stewart 1971;Fox&Geiger 1985).In the following experiments, we have compared theeffect of water stress on the rate of photosynthesis andthe partitioning of photosynthate in four differentspecies including two annuals  {Lupinus albus  L. and Heliatuhus annuus  L.), and two woody perenials  {Vitisvinifera  cv. Rosaki and  Eucatyptusglobulus  Labill.). Wewill show that only one of these species shows asignificant non-stomatal inhibition of photosynthesis(grapevine). However, they all maintain or increasesoluble sugars in their leaves, even though field photo-synthesis is inhibited and starch is depleted. We con-clude that, when water stress develops under fieldconditions, there is an alteration in the balance betweensucrose synthesis and translocation which allows manyspecies to maintain or increase the pool of soluble sugarsin their leaves. M TERI LS ND METHODS The experiments described in this paper were carriedout during the months of May to July 1989 at theInstituto Superior de Agronotnia, Lisboa, Portugal.Four plant species were used during the study: (1) lupins {Lupinus albus  L.), which were sown on 3 March; (2)eucalyptus  {Eucalyptus globulus  Labill.), which wereone-year-old plants, with juvenile leaves, grown in 4dm ^pots; (3) sunflower  Helianthtis annuus  L.), which weresown on the 13 May 1989 in 10dm'' pots (four plants perpot) and used 4 weeks later; and (4).grapevine  {Vitisvinifera,  cv. Rosaki), which were one-year-old own-rooted plants grown in a 9dtn'' pots. All plants weregrown in a soil tnixture of peat and sand (1:1) and werewatered daily. Plants were also fed weekly with anutrient solution containing nitrogen, potassiutn, phos-phate and micronutrients, except for lupins where thenitrogen was omitted to allow symbiotic N-fixation.Other experiments were carried out with lupins andgrapevine during the same period of the years 1988 and1990.Water stress was induced by withholding water. Thedevelopment of water stress was monitored by continu-ous measurement of stotnatal conductance and leaf(predawn) water potential (i|)), experiments werestarted when the predawn  i  was lower than controls andstomata were closed early in the morning. Alltneasuretnents described were carried out on recentlyfully expanded leaves to reduce developtnental variationand at least three individual plants were used for eachdata point. For each data point, atnbient photosynthesiswas measured first and satnples of the same leaf werethen taken for further analysis (see below).Gas exchange tneasurements were obtained undernatural light conditions using a portable CO2/H2Oporometer (H. Walz, Mess und Regeltechnik, Effel-trich, Germany) described in detail by Lange &Tenhunen (1985; see Lange  et  al 1984). A portion of anattached leaf was inserted into a 70cm'' cuvette and  COT and H2O exchange and environmental conditions weredetermined as in Chaves  et  al (1987). Leaf conductanceto water vapour (gs) was calculated from transpirationrates (Tr) and the water vapour pressure differencebetween leaf and air (D), assuming saturation in theintercellular air spaces. The tneasuretnents were carriedout in a very well stirred cuvette to minimize boundaryresistance (ga), and ga is considered negligible. Leafinternal partial pressure for CO2 (Ci) was estimatedfrom gs and measured net photosynthesis as in Chaves  etal.  (1987). All values reported were obtained at irra-diances above saturation (photosynthetically activeradiation 1000 /j-mol m~- s~') and at leaf temperaturesranging from 28 to 35°C.Leaf water potential (i|j) was determined using apressure chamber (P.M.S. Instrument Co., Oregon,U.S.A.). Measurements were made on detached leavesadjacent to those used for gas exchange measurements.   ater stress and photosynthetic carbon metabolism  7 Figure 1 Dependence of the rate of CO^-dependent O2evolution on the CO2 concentration of the gas phase in a leafdisc Oi-eleetrode lor Ittpins for ttirgid (O) and water-stressed(•) leaf discs. Actinic illtnnination was saturing for photosynthesis 9WJ /xmol m ') and the chamber wasmaintained at 24°C. Each point is the average ol' threemeasurements; error bars indicate the standard error of themean. Water stressed material was obtained by allowing adetaehed leaf to wilt on the bench until its fresh weight haddecreased by 10-15%. Similar results were obtained usingnaturally stressed plant material. These leaves were then used for determination ofrelative water content (RWC). Ten leaf discs (0-7ctn-)were punched from each leaf and the fresh weightdetermined before (FW,) and alter (FW,) floating for  2 h on water to obtain fully turgid material. The dry weight(DW) was detertnined after drying the discs at 7()°C for24h (RWC % = 100 (FW,-DW)/(FW,-DW)).The CO2-saturated rate of photosynthesis wasdetermined in a leaf-disc oxygen electrode (HansatechLtd, Kings Lynn, U.K.); the chamber was used asdescribed by Bjorkman & Demmig (1987). Gas wasprovided by mixing air and CO2 from pressurizedcylinders and the CO2 concentration was varied usingflow controllers. Very high CO2 concentrations(10-20%) are needed to observe maximutn rates ofCO2-dependent O2 evolution in water stressed plants(Robinson  el al.  1988) but high CO2 concentrations arealso known to inhibit photosynthesis in non-stressedplants. The effect of CO2 concentration on the rate ofphotosynthesis was tested for all plants prior to experi-mentation. O2 evolution was measured for 20 min, andwas constant for the last 10-15 min. A typical CO2concentration dependence of photosynthesis forstressed and control plants is shown in Fig.   for lupins.The data show that 15-20% CO2 is required to saturatephotosynthesis for stressed plants (10-fold higher thanrequired for control plants) and that these concentra-tions cause only minimal inhibition in control plants.Similar data were obtained for other plants used in thisstudy (data not shown) and 15% CO2 was used in allfurther experiments.Chlorophyll  a  fluorescence was measured simultane-ously with oxygen evolution using a PAM fluorometer(H. Walz, Mess- und Regeltechnik, D-8521 Effeltrich,Germany) as in Quick & Stitt (1989). Photochemicaland non-photochemical quenching were determinedusing high intensity light pulses (Bradbuty   Baker1984; Quick & Stitt 1989; Schreiber, Schliwa & Bilger1986). The non-photochemical fluorescence quenchingwas further resolved by determining the fast cotnponent(qi.:) of the quenching relaxation kinetics upon removalof actinic illumination (Quick & Stitt 1989). This wasobtained by monitoring the F,, level in the presence offar red light (given by a Corning RG715 red-pass filter)after retnoving actinic illumination and applying a 1-slight pulse (4000 tnol m ^ s~') upon completion of thefast cotnponent of the rise observed in Fo, usually90-120s after removing actinic illumination.The recent partitioning of photosynthesis betweenstaich and sucrose was determined in a leaf-disc oxygenelectrode. Three leaf-disc electrodes were connected inparallel to a tnixing chatnber and a circulating air-pumpin series. The pump allowed the circulation of air in thetnixitig chatnber through the leaf chatnbers in a closedcircuit (systetn volutne approximately 2dm-') at a flow of0-5 dm^ min '. Valves included in the circuit allowed thewhole systetn to fie flushed with CQ2-enriched air (15%)once the leaf satnples were in place. During this period,actinic illumination was provided to allow steady-statephotosynthesis to develop. The system was then closedand the citculating pump turned on. A lOOmtn-' volutneof 2 ktnol m^' KHCO3 (pH 9-3, adjusted with K2CO,,)containing  4  x 10'Bq NaH''*CQ., was then injected witha hypodertnic syringe into an Eppendorf tube suspendedin the tnixing chamber containing 500mm'' saturatedcittic acid to release '•'CQ2. Circulation was continuedfor 5 tnin and then turned off, the gas pressure in theelectrodes was allowed to equilibrate and then the gasvalves to the leaf chatnbers were closed. PhotosyntheticQ2 evolution was monitored for  5  min and then the leafsatnples were frozen in liquid nittogen and stored at-80°C. Insoluble (starch) and neutral (sucrose) frac-tions were measuted for '''C incorporation as described in Quick et  at (1988).The current amounts of starch, sucrose, glucose andfructose in leaves in atnbient CQ2 were determined inparallel samples that were frozen in liquid nitrogen andstored at-80°C, as described in Stitt era/.  (1983,  1989). RESULTSExperimental design Plants were water-stressed hy withholding water. Sincethey were grown in telatively large pots, the water in thesoil was slowly depleted over several days and waterstress developed gradually. The experiments werecarried out with plants whose stomata closed early in the  28  W P Quick  et al. -02-  U l  -0.6--  -08- ^ -1.0- -1.2- 908070 X 9.00 13.0017.00 9.00 13.00 1700 900 13.00 17 00 4'"  Day6'" Day" Day Figure 2.  Daily time courses of (A) leaf water potential and (B)leaf relative water content for lupins for control (circles) andwater stressed (squares) plants. Stressed plants were not wateredbetween 12 May and the evening of 18 May, Leaf chlorophyllcontent was 559 and 6 8mg m  ^  for control and stress plants,respectively. The ambient light intensity varied between 1000and 200 /xmol m ' s ' which was saturating for photosynthesis.Leaf temperature increased from 20 to 35°C during the course ofa day. Each point is the average of three measurements; errorbars indicate the standard error of the mean. (corresponding to the first day on which a small depress-ion of the predawn water potential was found) and 6d,by which time the plants were visibly wilted and hadtheir stomata closed for most of the photoperiod. Theplants were rewatered on the evening of the sixth day,and measurements were carried on the seventh day tomonitor the recovery. Control plants were wateredthroughout the experiment. We report here experi-ments carried out in 1989, Similar results were obtainedin experiments in 1988, which are not shown.On the fourth day, t|/ (Fig, 2A) and RWC (Fig, 2B)were only marginally lower in non-watered plants thanin watered plants, except towards the end of the day.During the sixth day, i|j and RWC were much lower innon-watered plants (-t-2-35 MPa and 63%) than inwatered plants (-  I  -()() and 80%), The leaf temperaturewas lower on day 6 (35°C) than day 4 (38°C), accountingfor the smaller decline of  i| and RWC in the control thanon the fourth day. After rewatering on the evening ofday 6, (|/ and RWC recovered fully overnight,COi fixation by watered plants in ambient COTdecreased slightly during the morning hours (Fig, 3A),following the deerease of  i|;  and RWC, This deerease wasmarginally less on the sixth day, when the controls had asmaller water deficit. On both days, photosynthesis inmorning. The plants had not all reached the same waterdeficit, but in all cases, the water stress was severe. Theeffect on photosynthesis was investigated (1) bymeasuring CO2 fixation, stomatal conductance and Qunder natural conditions, and (2) by measuring photo-synthetic capacity in saturating light and 15% CO2 in aleaf-disc  O2  electrode. Comparison of these two approa-ches should allow us to assess whether field photosyn-thesis is being inhibited by stomatal or non-stomatalmechanisms. The effeet of water stress on carbohydratepartitioning was investigated both (3) by measuring thelevels of starch and soluble sugars in leaves of wholeplants growing under field conditions, and (4) bymeasuring the partitioning of '*C into starch and solublesugars in a leaf-disc electrode in saturating light and CO2,  Because photosynthesis and partitioning variesdiurnally and the severity of water stress also variesduring the day, these parameters were measuredthroughout an entire photoperiod. In some cases, plantswere also rewatered at the end of the day to investig-dtewhich parameters recovered rapidly overnight. Lupin The effeet of water-stress was investigated in lU-week-old lupins. Water was withheld from half of the plantsand the predawn water potential was monitored eaehday. Detailed measurements were made after 4d o fc  9 O   -s 30- 25- 20- 15- 10- 5- J 300- i  200- 100- 0 g  250- I 200- i 150- o 1 50- ConltolSlress ~i  ——  r 1 ——I r- A -1 1 r © 9 00 13.00 1700 9.00 13.00 17.00 9.00 13.00 17.00 4"'  Day 6'" Day 7'" Day Figure 3.  Daily time courses of (A) net photosynthesis, (B)stomatal condtictancc and (C) the calculated internal CO2concentration, lor lupins measured under ambient eonditions forcontrol (•) and water stressed (•) plants. See Fig, 2 and'Materials and methods' for further details. Each point is theaverage of three measurements; error bars indieate the standarderror of the mean.  Water stress and photosynthetic carbon metabolism  29 LUPINUS uo  r  - r 90013 001700  9 0013 001700  9,00  13 001700 4'^  Day S ^ Cay 7'^ Day Figure 4 Daily time eourses of (A) leaf soluble sugar (glueose  -H fruetose -I- suerose) eontent, and (B) stateh eontent, for lupinsfor eontrol (•) and water stressed (•) plants. See Fig, 2 forfurther details, Eaeh point is the avetage of three measurements;error bars indieate the standard error of the mean. the non-watered plants was only slightly lower than inwatered plants at the beginning of the day. It decreasedto low rates during the morning hours and remained lowfor the remainder of the day. The inhibition cotnpared tothe controls was already large on day 4, even though leafwater status was not tnarkedly different frotn thewatered plants.The decrease of the photosynthetic rate in air wasaccompanied by stomatal closure (Fig, 3B), Thedecrease of  g  was larger than the inhibition of photosyn-thesis (compare Fig, 3A & B) and the estimated internalCO2 concentration (Q) decreased (Fig, 3C), On day 4,photosynthesis did not recover at the end ofthe day (Fig,3A) even though the stomata reopened (Fig, 3B) and theapparent Cj increased (Fig, 3C),Watered plants contained considerable levels (20mmol  m ~,  equivalent in fresh weight to about 40 /xmolg-') of soluble sugars (Fig, 4A), These increased slightlyduring the day. Non-watered leaves maintained similarlevels of soluble sugars, even though their rate ofphotosynthesis was much lower (see above. Fig, 3A),Watered plants contained considerable amounts ofstarch (Fig, 4B), Staich increased during the photoper-iod, and was remobilized at night. In contrast, non-watered plants contained negligible amounts of starch.This  was  already apparent on the fourth day, and wasvery marked by the sixth day. The starch level did notrecover on the morning after rewatering.The rate of photosynthesis in saturating light and CO2was measured in a leaf-disc O2 electrode using a f5%CO2 atmosphere supplied from a gas mixing system.These measurements of 'photosynthetic capacity' willremove the effect of the sfomata, and reveal whetherthere is a direct effect of water stress on photosynthesis.We took care to supply enough CO2 to overcome thediffusional resistance in the  leaf which often increases inwater stressed material (see 'Methods and Fig, 1),Photosynthetic capacity was constant throughout theday, and was not decreased in non-watered plants (Fig, 5A),  This reinforces the evidence that the inhibition ofambient photosynthesis is primarily due to stomatalclosure (see above).Partitioning of ''*C into starch and soluble sugars wasinvestigated in leaf discs during 20 min in saturating lightand CO2 (Fig, 5B), In watered plants, an increasingportion ofthe photosynthate was partitioned into starchlater in the day, as is also seen in many other plants (Stitt et  al.  1987), The non-watered plants showed similar orslightly higher partitioning into sucrose than the wateredplants.Figure  6  shows the light response ofthe water-stressedleaves in tnore detail. Leaf discs were illuminated at high (1500),  medium (700) or low (200) photon flux density,O2 evolution (Fig, 6A), photochemical quenching (Fig,6B) and energy-dependent fluorescence quenching (Fig,6C) were measured. Identical results were obtained indiscs from watered and non-watered plants, emphasiz-ing how the water stress had not led to any significant UJ E E3,CO  5 o O cro I Ol Zi  In Sou 6050 LO 302010u-3- 2-   n- —1—•—•—/—1 r——•  Control  —•  Stress —1— —1 tf Y —r 1 —r I 1— i - —1 1 ' I (A). --  D- 900 13 00  1700  9 00  1300 1700 900 1300 1700 4' Day 6' Da)/ 7' Day Figure 5 Daily time eourses of (A) the rate of photosynthetieoxygen evolution, and (B) the ratio of radioactivity ineorporatedinto soluble and insoluble traetions, during a 20-min exposure to '• COT  in a leaf dise eleetrode in an atmosphere of L'i% CO;,Leaf samples were taken from the leaves used for measurementof ambient photosynthesis in Fig, 2, Light intensity was 950/xmol m - s ' and euvette temperature was 24°C, Analysis ofthe soluble fraction by ion exehange ehromatography showedthat 84, 8-7 and 7-3% of the radioactivity was incorporated intothe neuttal, anionie and cationie traetions respeetively (97%recovery of radioactivity) and that these proportions were notaffeeted by water stress, Eaeh point is the average of threemeasuretnents; error bars indieate the standard error of themean.
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