Changes in the physiological response between leaves and fruits during a moderate water stress in table olive trees

1 CHANGES IN THE PHYSIOLOGICAL RESPONSE BETWEEN LEAVES AND 1 FRUITS DURING A MODERATE WATER STRESS IN TABLE OLIVE TREES 2 3 I.F. Girón a,b M. Corell c, b , A. Galindo d , E. Torrecillas e , D. Morales f , J. Dell’Amico f , A. 4 Torrecillas d , F. Moreno a,b , A. Moriana c,b* 5 6 a Instituto de Recursos Naturales y Agrobiología (CSIC), P.O. Box 1052, E-41080 Sevilla, 7 Spain. 8 b Unidad Asociada al CSIC de Uso sostenible del suelo y el agua en la agricultura (US- 9 IRNAS). Crta de Utrera Km 1, 41013, Sevilla, Spain. 10 c Departamento de Ciencias Agroforestales, ETSIA, Universidad de Sevilla, Crta de Utrera 11 Km 1. 41013 Sevilla, Spain. 12 d Dpto. Riego. Centro de Edafología y Biología Aplicada del Segura (CSIC). P.O. Box 164. 13 E-301000 Espinardo, Murcia, Spain 14 e Dpto. Conservación de suelos y Agua. Centro de Edafología y Biología Aplicada del 15 Segura (CSIC). P.O. Box 164. E-301000 Espinardo, Murcia, Spain 16 f Instituto Nacional de Ciencias Agrícolas, Cuba 17 18 19 *Corresponding author: [email protected] Phone: (+34)954486456; Fax: (+34)954486436 20 21 22 2 Abstract 23 Pit hardening period is the phenological stage when water stress is recommended in 24 regulated deficit irrigation (RDI) in olive trees. In table olive trees, fruit growth is a very 25 important process which could affect the final profit of the yield. RDI scheduling based on 26 water status measurements could improve water management, but accurate threshold values 27 are needed. Previous works in low fruit load conditions suggested -1.8 MPa of midday stem 28 water potential as “first step” of water stress level where no variations of fruit growth have 29 been detected. The aim of this work is to describe the physiological response of table olive 30 trees with a significant yield in a moderate water stress conditions during pit hardening 31 period. Water relations of Control (no water stress) trees and Stressed trees were studied in 32 a mature table olive orchard in Seville (Spain). Control trees were irrigated with 100% of 33 ETc and values around field capacity were measured. Irrigation in Stressed trees was 34 withdrawn during pit hardening period, and they were irrigated as Control in the rest of the 35 experiment. Fruit growth was not affected until the last days of the deficit period, though 36 midday stem water potential and maximum leaf conductance measurements reached 37 minimum values a few days after the beginning of the water stress period. Such responses 38 suggest two phases in the water stress period. At the beginning of the experiment, the 39 physiological response of the trees (osmotic adjustment and trunk dehydration in the 40 present work) compensated the decrease in water potential. In this phase, leaves and fruits 41 are similar water sink in the shoots. During the last days of the drought period, the 42 reduction of the osmotic adjustment and the greater decrease of fruit water potential 43 transform fruits in more strength water sink than leaves. These changes produced a 44 decrease in the fruit growth. The recovery, though it was not complete, increase fruit size as 45 the same level than Control.. 46 3 Keywords: Regulated deficit irrigation, recovery, stress integral, water potential, water 47 relations. 48 1. Introduction 49 Regulated deficit irrigation (RDI) in olive trees is scheduling with a water deficit 50 period during pit hardening (Goldhamer, 1999). This phenological stage is a dynamic 51 period which can change in length with the water status of the tree (Hammami et al., 2013). 52 In addition, in conditions of significant fruit load in the tree, vegetative growth is stopped 53 (Rallo and Suárez, 1989) and water relations are clearly changed in comparison with low 54 fruit load conditions (Martin-Vertedor et al., 2011). Therefore, pit hardening is a complex 55 phenological stage from the point of view of water relations. 56 Irrigation scheduling in deficit conditions has been changed in recent decades and 57 there are several works that suggest water status measurements as a more efficient tool than 58 the traditional water balance (i.e. Goldhamer and Fereres, 2001). But all the parameters 59 related directly to the plant physiology could be altered by the drought adaptation process. 60 In olive trees, osmotic adjustment has been suggested as one of the first responses of trees 61 to drought conditions (Dichio et al., 2006). Great dehydration capacity has also been 62 reported as a physiological respond to water stress (Fereres, 1984). Midday stem water 63 potential has been considered the best indicator even in low water stress level (Moriana et 64 al., 2010). 65 Moriana et al (2012) suggested -1.4 MPa as an adequate threshold value midday 66 stem water potential during the pit hardening period in no water stress conditions. 67 Dell’Amico et al. (2012) in table olive trees reported no decrease in fruit volume in low 68 water stress conditions with minimum values around -1.8 MPa. The low fruit load in this 69 4 latter experiment is likely to have limited the level of water stress. According to the 70 literatures, such values of water potential are too high for deficit irrigation in olive trees. No 71 clear reduction of fruit yield has been reported with values around -3.5 MPa during pit 72 hardening (Moriana et al., 2003; Iniesta et al., 2009), though fruit growth has been reduced 73 with values higher than -3.0 MPa (Moriana et al., 2013). 74 The aim of this work is to describe the physiological response of table olive trees in 75 moderate water stress conditions during the pit hardening period. This is the first step to 76 establish a more accurate threshold values or indicators of water potential for irrigation 77 scheduling. We hypothesize that a significant fruit load on the tree will control the process 78 and that shoots water relations would tend to allow fruit growth during the water deficit 79 period. 80 81 2. Materials and Methods 82 2.1. Description of the experiment 83 Experiments were conducted at La Hampa, the experimental farm of the Instituto de 84 Recursos Naturales y Agrobiología (CSIC). This orchard is located at Coria del Río near 85 Seville (Spain) (37º17’N, 6º3’W, 30 m altitude). The sandy loam soil (about 2 m deep) of 86 the experimental site was characterized by a volumetric water content of 0.33 m 3 m -3 at 87 saturation, 0.21 m 3 m -3 at field capacity and 0.1 m 3 m -3 at permanent wilting point, and 1.30 88 (0-10cm) and 1.50 (10-120 cm) g cm -3 bulk density. The experiment was performed on 44- 89 year-old table olive trees (Olea europaea L cv Manzanillo) during 2012. Tree spacing 90 followed a 7 m x 5 m square pattern. Pest control and fertilization practices were those 91 commonly used by growers and no weeds were allowed to develop within the orchard. 92 Irrigation was carried out during the night by drip using one lateral pipe per tree row and 93 5 five emitters per plant, delivering 8 L h -1 each and spacing 1 m. Irrigation requirements 94 were determined according to daily reference evapotranspiration (ET o ) and a crop factor 95 based on the time of year and the percentage of ground area shaded (40%) by the tree 96 canopy (Kr=0.8). The crop coefficient values (Kc) considered were 0.76 in May, 0.70 in 97 June, 0.63 in July and August, 0.72 in September and 0.77 in October (Fernández et al. 98 2006). 99 Trees were irrigated with 100% of crop evapotranspiration (ET c ) in order to obtain 100 non-limiting soil water conditions until the beginning of pit hardening (Phase I). The 101 beginning of the pit hardening was estimated according to Rapoport et al. (2013) around 102 day of the year (DOY) 173. From this date until DOY 233 irrigation was withdrawn in a 103 Stressed treatment (Phase II). All measurements were made in 6 olives irrigated at 100% 104 ET c throughout the experiment (Control trees) and 6 olives where irrigation was withdrawn 105 (Stressed trees). After DOY 233 trees were irrigated with the same amount of water as 106 Control trees (Recovery). The experiment was stopped at DOY 256 because the harvest had 107 taken place. 108 2.2 Measurements 109 Micrometeorological 30 min data, namely air temperature, solar radiation, relative 110 humidity of air and wind speed at 2 m above the soil surface were collected by an 111 automatic weather station located some 40 m from the experimental site. Daily reference 112 evapotranspiration (ET o ) was calculated using the Penman-Monteith equation (Allen et al., 113 1998). 114 Soil moisture was measured with a portable FDR sensor (HH2, Delta-T, U.K.) with 115 a calibration obtained in previous works (Fernández and Díaz, unpublished data). This 116 calibration was performed according to the instructions of the sensor and compared the soil 117 6 moisture measured gravimetrically and the output voltage of the sensor (Equation 1). 118 Equation 1 permits the estimation of the dielectric constant. 119 θ=0.4437∗Volt−0.1697 (1); (r 2 =0.76***;n=59;RMSE=0.017) 120 Where: 121 θ: soil moisture measured gravimetrically 122 Volt: output voltage of the sensor 123 The measurements were made in four plots per treatment (one access tube per plot). 124 The access tubes for the FDR sensor were placed in the irrigation line around 30 cm from 125 an emitter (Fernández et al., 1991). The data were obtained at 1 m depth with a 10 cm 126 interval. 127 The drought cycle was characterized by weekly measurements of maximum leaf 128 conductance (g) and midday stem water potential (ψ stem ). Abaxial leaf conductance was 129 measured in two full expanded and well illuminated leaves per tree in each treatment with a 130 steady state porometer (LICOR-1600, LICOR, UK) around 10:00 GMT, when maximum 131 values are expected (Xiloyannis et al., 1988). Midday stem water potential in one leaf per 132 tree was measured with a pressure chamber (Model 1000, PMS, USA) around 13:00 GMT. 133 Leaves near the main trunk for ψ stem measurements were covered with aluminium foil two 134 hours before measuring. After the leaf excision, two small cuts parallel to the main nerve 135 were done. These cuts permitted more length in the leaf base to insert in the pressure 136 chamber and increase the grip with the rubber. 137 In order to describe the cumulative effect of the water deficit, the water stress 138 integral was calculated from the Ψ stem data (Myers, 1988) during the period of water stress 139 (equation 2). In this publication, the integral is estimated with the sum of the surfaces 140 7 calculated as the average Ψ of two consecutive dates multiplied by the number of days 141 between this dates (Equation 2). Myers (1988) referred the surface to the maximum Ψ 142 measured during the experiment (Equation 2, “c”). Moriana et al (2012) suggested -1.4 143 MPa of midday stem water potential as reference value in olive trees during pit hardening 144 period. Stress integral was calculated with both reference values in order to compare the 145 usefulness of the Moriana et al (2012) reference. All the values higher than the reference 146 were considered as equal to this. . The expression used was: 147 148   |∑Ψ ∗ | (2) 149 where:  is the stress integral 150 Ψ is the average midday stem water potential for any interval 151 c is the maximum value of midday stem water potential in the experiment 152 (traditional use) or the value -1.4 MPa. 153 n is the number of the days in the interval 154 155 The water relations of the leaves and fruits were measured around the time of 156 maximum leaf conductance. Two fully expanded and shaded leaves per tree were randomly 157 cut. Leaf water potential ( ψ leaf ) was measured with the pressure chamber (Model 1000, 158 PMS, USA) in one of them. This leaf was then covered with aluminium foil and 159 immediately frozen in liquid nitrogen and stored at -80 o C. This sample was used to measure 160 actual osmotic potential ( ψ π ) . The second leaf was put in a test tube with distilled water, in 161 which only the petiole was in contact with the water. The test tube was covered with 162 aluminium foil and put into a portable freezer until arrival at the laboratory. Then the test 163 8 tubes were kept in the dark for 24 hours at 6-8 o C and then frozen in liquid nitrogen and 164 stored at -80ºC. This sample was used to measure leaf saturated osmotic potential. Fruit 165 water potential was measured with the pressure chamber (Model 1000, PMS, USA) in one 166 fruit per tree in the same shoot where leaf water potential was measured. The fruit was then 167 covered with aluminum foil and immediately frozen in liquid nitrogen and stored at -80 o C. 168 This sample was used to measure actual fruit osmotic potential. All frozen tissues (leaf and 169 fruit) were equilibrated at 20ºC for 15 min before determination of osmotic potentials. In 170 the leaf samples, the central nerve was separated from the rest of tissue. Then the tissue was 171 used for the determination of osmotic potential. The osmotic potential was measured after 172 thawing the samples and expressing the sap, using a vapour pressure osmometer (Wescor 173 5600, Logan, USA). Apoplastic dilution was not considered in any of the osmotic potential 174 measurements. According to Dichio et al (2003), the amount of apoplastic water is very low 175 (lower than 15%) and similar until water stress values of -3.3 MPa of predawn leaf water 176 potential. 177 Values of turgor pressure (Ψ p ) were calculated as: 178 Ψ p =Ψ-Ψ π (3) 179 Where: 180 Ψ p is the turgor pressure 181 Ψ is the water potential 182 Ψ π is the osmotic potential 183 Trunk diameter fluctuations were measured throughout the experimental periods, 184 using a set of linear variable displacement transducers (LVDT) (model DF±2.5 mm, 185 accuracy ±10 µm, Solartron Metrology, Bognor Regis, UK) attached to the main trunk, 186 9 with a special bracket made of Invar, an alloy of Ni and Fe with a thermal expansion 187 coefficient close to zero (Katerji et al., 1994). Measurements were taken every 10 s and the 188 datalogger (model CR10X with AM 416 multiplexer, Campbell Sci. Ltd., Logan, USA) 189 was programmed to report 15 min means. Maximum diameter was measured at the 190 beginning of the day. Trunk growth rate (TGR) in day “n” was calculated as the difference 191 between the maximum daily diameter of day “n+1” minus those of day “n” (Cuevas et al., 192 2010). TGR is the slope of the figure of maximum diameter. Both parameters, Maximum 193 diameter and TGR, was used as descriptors of the water relations. 194 The fruit volume was estimated periodically throughout the experiment from a 195 survey of ten fruits per tree (60 fruits per treatment). Two measurements were made for 196 each fruit: the longitudinal dimension and the transversal (at the equatorial point) 197 dimension. At the beginning of the pit hardening period six shoots with fruits per tree were 198 selected randomly. For each shoot the number of fruits were measured at the beginning, the 199 end and in the middle of the period of water stress. 200 The orchard was divided in two blocks following the slope, then trees inside each 201 block was at the same height in the orchard. Six trees per block (three Control and three 202 Stressed were measured). This design was imposed for the small experimental surface 203 available. Two blocks and two treatments provide no enough repetition to make an 204 ANOVA. Therefore, data of the present work are presented only with the average and 205 standard error because it is no possible to do any significant test. The present work only 206 describe the physiological processes which occur during the water stress but cannot 207 conclude definitively the level of the selected indicators. 208 209 3. Results 210 16 However, cumulative effect of water stress is usually not considered in irrigation 351 scheduling likely because there is not a reference to compare. The suggested change in the 352 present work that used the same midday stem water potential as a reference in the Myers’s 353 equation supposes a clearer differentiations between Control and Stressed trees than using 354 the traditional parameters (Fig. 9). In addition, the S Ψ values obtained using the value -1.4 355 MPa could be more comparable between different works. 356 357 5. Conclusions 358 Moderate water stress conditions produced a progressive physiological response in olive 359 trees that divided the water stress period in two phases. At the beginning (from DOY 190 to 360 214), a group of physiological processes (fruit and leaf osmotic adjustment, fruit drop and 361 trunk shrinkage) delayed the effect of drought in the turgor pressure and fruit growth. At 362 this level of water stress (in some days around -2.5 MPa) fruits and leaves had the same 363 strength as water sink. In the second phase of the period of water stress (from DOY 214 to 364 232), the reduction of leaf osmotic adjustment likely increased of Ψ leaf- Ψ fruit and fruits 365 received the water mainly via xylem. Such changes produced a reduction in the fruit and 366 leaves turgor pressure and a decrease in the fruit growth. The recovery, although it was not 367 completed, permitted an increase in the fruit size until values similar to Control. 368 369 Acknowledge 370 This research was supported by the Spanish Ministerio de Economía y Competitividad 371 (MINECO), (AGL2010-19201-CO4-03 y AGL2013-45922-C2-1-R). The stay of D. 372 Morales and J. Dell’Amico was supported by the AECID project A1/035430/11. Thanks 373 17 are due to Professor R Fernández-Escobar, M Cera and D Pérez-López for help in the 374 statistical analysis. Thanks are due to J. Rodriguez and A. Montero for help with field 375 measurements. 376 377 378 379 380 381 382 383 384 References 385 Allen, R.G., Pereira, L.S., Raes, D., Smith. M., 1998. Crop evaportranspiration. Guideline 386 for computing crop water requirements. FAO irrigation and drainage paper nº 56. 387 Roma. FAO. 388 Bradford, K.J. and Hsiao, T.C. 1982. Physiological responses to moderate water stress. In: 389 Physiological Plant Ecology II. Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, 390 H.(Eds.). Springer-Verlag, New York, USA, pp 264-324. 391 18 Caruso, G., Raoport, H.F., Gucci, R. 2013. Long-term evaluation of yield components of 392 young olive trees during onset of fruit production under deficit irrigation regimes. 393 Irrig. Sci. 31:37-47. 394 Cermák, J., Kucera, J., Bauerle, W.L., Phillips, N., Hinckley, T.M. 2007. Tree water 395 storage and its diurnal dynamics related to sap flow and changes in stem volume in 396 old-growth Douglas-fir trees. Tree Physiol. 27, 181-198 397 Cuevas, M.V., Torres-Ruiz, J,M,, Álvarez, R,, Jiménez, M.D., Cuerva, J., Fernández, J.E. 398 2010. Usefulness of trunk diameter variations for irrigation scheduling in a mature 399 olive tree orchard. Agric. Water Manage. 97, 1293–1302. 400 Dell’Amico, J., Moriana, A., Corell, M., Girón, I.F., Morales, D., Torrecillas, A., Moreno, 401 F. 2012. Low water stress conditions in table olive trees (Olea europaea L) during 402 pit hardening produced a different response of fruit and leaf water relations. Agric. 403 Water Manage. 114,11-17. 404 Dichio, B., Xiloyannis, C., Angelopoulos, K., Nuzzo, V., Bufo, S.A., Celano, G. 2003. 405 Drought-induced variation of water relations parameter in Olea europea.Plant Soil 406 257:381-389 407 Dichio, B., Xiloyannis, C., Sofo, A., Montarano, G., 2006. Osmotic regulation in leaves and 408 roots of olive trees during water deficit and rewatering. Tree Physiol. 26,179-185. 409 Fereres, E. 1984. Variability in Adaptive Mechanisms to Water Deficits in Annual and 410 Perennial Crop Plants. Bull. Soc. Bot. Fr., Actualit‚s Botaniques 131, 17-32 411 Fernández, J. E., Díaz-Espejo, A., Infante, J. M., Duran, P., Palomo, M. J., Chamorro, V., 412 Giron, I. F., Villagarcía, L., 2006. Water relations and gas exchange in olive trees 413 under regulated deficit irrigation and partial root zone drying. Plant Soil 284, 273- 414 291 415 19 Goldhamer, D.A., 1999. Regulated deficit irrigation for California canning olives. Acta 416 Hortic. 474, 369-372. 417 Goldhamer, D.A., Fereres, E., 2001. Irrigation scheduling protocols using continuously 418 recorded trunk diameter measurements. Irrig. Sci. 20,155-125. 419 Greenspan, M.D., Schultz,.H.R., Matthews, M.A. 1996. Field evaluation of water transport 420 in grape during water deficits. Physiol. Plant. 97, 55-62. 421 Hammani, S.B.M., Costagli, G., Rapoport, H.F. 2013. Cell and tissue δψnamics of olive 422 endocarp sclerification vary according to water availability. Physiol. Plant. 149, 423 571-582. 424 Iniesta, F., Testi, L., Orgaz, F., Villalobos, F.J. 2009. The effects of regulated and 425 continuous deficit irrigation on thw¡e water use, oil and yield of olive trees. Eur. J. 426 Agr. 30, 258-265. 427 Katerji, N., Tardieu, F., Bethenod, O., Quetin, P., 1994. Behavior of Maize stem diameter 428 during drying cycles: comparison of two methods for detecting water stress. Crop 429 Sci. 34, 165-169. 430 Martin-Vertedor, A.I., Perez-Rodriguez, J.M., Prieto-Losada, H., Fereres-Castiel, E. 2011. 431 Interactive responses to water deficits and crop load in olive (Olea europaea L., cv. 432 Morisca) I. - Growth and water relations. Agric. Water Manage. 98, 941-949. 433 Moriana, A. and Fereres, E. 2002. Plant indicators for scheduling irrigation for young olive 434 trees. Irrig. Sci. 21, 83-90 435 Moriana, A., Orgaz, F., Fereres, E., Pastor, M. 2003. Yield responses of a mature olive 436 orchard to water deficits. J. Amer. Soc. Hort. Sci.. 128:425-431 437 20 Moriana, A., Girón, I., Martín-Palomo, M.J., Conejero, W., Ortuño; M.F., Torrecillas, A., 438 Moreno, F. 2010. New approach for olive trees irrigation schedulings using trunk 439 diameter sensors. Agric. Water Manage 97, 1822-1828. 440 Moriana, A., Pérez-López, D., Prieto, M.H., Ramírez-Santa-Pau, M., Pérez-Rodriguez, J.M. 441 2012. Midday stem water potential as a useful tool for estimating irrigation 442 requirements in olive trees. Agric. Water Manage. 112:43-54. 443 Moriana, A., Corell, M., Girón, I.F., Conejero, W., Morales, D., Torrecillas, A., Moreno, F. 444 2013. Regulated deficit irrigation based on threshold values of trunk diameter 445 fluctuation indicators in table olive trees. Sci Hort 164, 102-111. 446 Myers, B.J. 1988. Water stress integral a link between short term stress and long term 447 growth. Tree Physiol. 4, 315-323. 448 Nobel, P.S. and De la Barrera, E., 2000. Carbon and water balances for young fruits of 449 platyopuntias. Physiol. Plant. 109, 160-166. 450 Pomper, K. W. and Breen, P. J. 1997. Expansion an osmotic adjustment of Strawberry fruit 451 during water stress. J.Am.Soc.Hort.Sci. 122, 183-189 452 Rallo, L. and Suarez,M.P. 1989.Seasonal distribution of dry matter within the olive fruit- 453 bearing limb. Adv. Hortic. Sci. 3, 55-59. 454 Rapoport, H.F., Pérez-López, D., Hammami, S.B.M., Aguera, J., Moriana, A. 2013. Fruit 455 pit hardening: physical measurements during olive growth. Ann. Appl. Biol. 163, 456 200-208. 457 21 De Swaef, T., Steppe, K., Lemeur, R. 2009. Determining reference values for stem water 458 potential and maximum daily trunk shrinkage in young apple based on plant 459 responses to water deficit. Agric. Water Manage. 96, 541-550. 460 Xiloyannis, C., Pezzarossa, B., Jorba, J., Angelini, P. 1988. Effects of soil water content on 461 gas exchange in olive trees. Adv. Hort. Sci. 2, 58-63. 462 463 464 22 Table 1. Average of trunk growth rate (TGR) during the three period of the experiment in 465 Control and Stressed trees. The experiment was divided in three phases, which are equal to 466 the ones presented in the rest of figures. Phase I: from the beginning of the experiment until 467 pit hardening (DOY 173). Phase II: from the beginning of pit hardening until recovery 468 (DOY 232). Recovery: which finished at harvest. 469 470 471 472 473 474 475 Control ( µ m día - 1 ) Stressed ( µ m día - 1 ) Phase I -0.9±3.2 -5.4±3.3 Phase II 3.2±2.6 -20.8±3.1 173-192 -1.0±3.3 -16.2±3.3 193-201 -11.0±5.6 -41.9±6.1 202-209 23.9±6.1 6.8±5.1 210-224 -10.2±6.3 -35.0±6.7 225-232 25.7±7.9 -9.8±16.0 Recovery 4.5±3.8 22.2±7.4 23 Figure Captions 476 Air Temperature (ºC) 10 15 20 25 30 35 40 45 50 DOY 140 160 180 200 220 240 260 ETo (mm) 0 2 4 6 8 10 Rainfall (mm) 0 2 4 6 8 10 477 478 Fig. 1. Climatological data during the experiment. (a) Maximum (dot line) and medium 479 (solid line) temperature. (b) Reference evapotranspiration (ETo, solid line) and rainfall 480 (bar). There was only a one rainfall event at date 233. Vertical lines indicate the period of 481 water deficit. 482 483 484 24 Midday Stem Water Potential (MPa) -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 DOY 160 180 200 220 240 260 g max (mmol m-2 s-1) 0 200 400 600 800 a b Soil Moisture (m3 m-3) 0.05 0.10 0.15 0.20 0.25 0.30 a WP FC c 485 Fig. 2. Pattern of the soil moisture (a), midday stem water potential (b) and maximum leaf 486 conductance (gmax), c) in Control (solid symbols) and Stressed trees (empty symbols). 487 Each symbol is the average of 3 (a), 6 (b) and 12 (c) data respectively. Vertical bars 488 represent the standard error. Vertical lines indicate the period of water deficit. The 489 horizontal line in midday stem water potential graph shows the level used as a reference in 490 stress integral (-1.4 MPa). 491 492 493 25 Leaf Water Potential (MPa) -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 Fruit Water Potential (MPa) -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 DOY 160 180 200 220 240 260 Ψleaf-Ψfruit (MPa) -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 c b a 494 Fig.3. Pattern of leaf water potential (a), fruit water potential (b) and difference between 495 leaf and fruit water potential (∆Ψ, leaf-fruit water potential), c) in Control (solid symbols) 496 and Stressed trees (empty symbols). Each symbol is the average of 6 data. Vertical bars 497 represent the standard error. Vertical lines indicate the period of water deficit. 498 499 500 501