Fourth International Walnut Symposium, Bordeaux (France) 12-16 September 1999

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WATER RELATIONS IN WALNUT DURING WINTER T. Améglio, A. Lacointe, H. Cochard, G. Alves, C. Bodet, M. Vandame, V. Valentin, B. Saint-Joanis S. Ploquin, P. Cruiziat.

F. Ewers

U.A. PIAF INRA Site de Crouelle 234 av. du Brezet F-63039 ClermontFerrand Cedex 2 (France.)

Michigan State Univ. Dept. of. Botany and Plant Pathology. East Lansing, MI 48824 (USA)

J.L. Julien, A. Guilliot, G. Petel

U.A. PIAF Univ. Blaise Pascal 24 av. des Landais, F-63177 Aubière Cedex. (France)

Keywords: embolism, root pressure, stem pressure, vessel-associated cell, plasma membrane H+-ATPase, sugar fluxes. Abstract Studies dealing with the water status of the xylem sap in branches of trees during the winter period show that variation in degree of embolism can occur as a consequence of the frost-thaw alternation. At present only two known mechanisms are proposed to explain a resorption of embolism : sap pressurisation or cambial growth. Results show that walnut hydraulic conductivity varies considerably during the winter period and with the water status of xylem (tension/pressure). In winter, water status can be either under tension (negative water potential) or under pressure (positive, above atmospheric pressure), depending on the air temperature. Positive sap pressures are related to high sugar content (stem pressure) These studies show also clearly that important relations exist between xylem water status and dynamics of the carbon reserves (see also Valentin et al., this issue). Sugar content in the vascular sap is controlled by contact cells or vesselassociated cells (VACs: see also Alves et al. this issue a and b). Refilling of embolisms is most active in spring before budbreak (root pressure) and during this period, sugars in the vascular sap are retrieved for growth of buds and the vascular cambium.

1. Introduction Investigations in the water status of the xylem sap of trees during the winter period (when trees do not bear leaves), show that embolism (air blockage) can occur as a consequence of the frost-thaw alternation (Cochard and Tyree, 1990; Just and Sauter, 1991; Améglio et al., 1995; Pockman and Sperry, 1997). When the sap freezes, previously dissolved gases escape due to their very low solubility in ice (Sperry and Sullivan, 1992). The resulting micro-bubbles expand when temperatures increase and eventually initiate

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embolism. The resulting embolized conduit no longer contributes to water transport. There is growing evidence that freezing-induced embolism can limit the growth, survival, and geographic distribution of plant species (Tyree and Cochard, 1996). In this study, the authors, for some tree species (e.g. oak), embolism was total in spring and cambial growth started before budbreak in order to build new active vessels. For other tree species (e.g. grapevine, beech) embolism can be resorbed (Sperry et al., 1988a). The classical explanation (Pickard, 1989; Tyree and Yang, 1992; Yang and Tyree, 1992) involves an osmotic pressure difference between the xylem sap compartment and a neighbouring compartment (sap pressurisation: Améglio et al., 1995). As phloem is considered to be not functional in winter, it is suggested that xylem is involved as the long distance transport pathway available for water and nutrient at this time of the year (Lacointe et al., this issue). In this context, the present work was undertaken on walnut grown in orchard, to investigate the xylem water status during winter and the mechanisms involved in its changes, in order to determine whether water and nutrients are available for budbreak.

2. Materials and methods Measurements were made on excised one-year old twigs of Walnut (Juglans regia L. cv. Franquette scions on wild walnut rootstocks) from twelve-year-old trees in an orchard and three-year-old trees in containers, at the INRA PIAF station near Clermont-Ferrand, in south central France. Embolism rate, sugar fluxes and pH were measured in respectively 20, 30 and 12 cm long stem segments of the one-year-old twigs from the same tree. Loss of hydraulic conductivity (embolism) was measured by Sperry’s method (Sperry et al. 1988,b). Sap was extracted under moderate vacuum (Bollard, 1953) and its osmolarity was measured with a Roebling 13DR Automatic osmometer (Messtechnik, D-1000 Berlin, Germany). Using a spectrophotometer at 340 nm, sucrose, glucose and fructose contents were determinate after enzymatic assays (Boehringer, 1984). In order to investigate the effect of temperature on sugars efflux in xylem vessels, the segment were conditioned for 48h at either 1°C or 15°C (± 1°C) before measurements. Each segment was rinsed (perfused) with 3ml of a saline solution (Alves et al., this issue). The efflux rate was quantified as the enrichment of vessels in sugars, within one hour, in the saline solution, which was substituted for the original xylem sap (more details in Valentin et al., this issue). For influx rate measurements, stem segments were prepared as for efflux rate measurements. Each segment was rinsed (perfused) with 3ml of the previous saline solution. Then 0.5 ml of the previous solution with an admixture of 10 mM sucrose and 200 mM mannitol, containing 70 kBeq/ml 14C-sucrose and 400 kBeq/ml3H-mannitol was kept for half an hour in the stem segment before parenchyma 14C-sucrose and 3H-mannitol contents determination (more details in Valentin et al., this issue). Sugar influx is considered to occur via H+/sugars symports (Sauter, 1983 ; Fromard, 1990) depending on the pH gradient generated by a plasma membrane H+-ATPase. To test this hypothesis, we decided to measure pH gradient and ATPase activity. The effect of fusicoccin (FC) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), two effectors of the plasmalemma H+-ATPase, were investigated using the perfusion technique (with the same saline

Fourth International Walnut Symposium, Bordeaux (France) 12-16 September 1999

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solution). The perfused solution pH was measured continuously with a microelectrode (Mini Combo PH750, World Precision Instruments, USA) before and after the addition of 10 µM FC (ATPase activator). The same perfusion technique was used to test the effect of the uncoupler CCCP (10 µM) on the pH of the sap (more details in Alves et al., this issue, a).

% of loss of hydraulic conductivity

3. Results After the first freeze–thaw alternation, embolism increased rapidly (early December). During winter, embolism decreased and before budbreak hydraulic conductivity was totally re-established (fig. 1). 100 80 Fig. 1: Time course of percent hydraulic conductivity loss of a twig. The bars represent standard errors (n=6).

60 40 20 0

1/10 31/10 30/11 30/12 29/1 28/2 30/3 29/4

29/5 28/6 28/7

During winter, there were negative correlations between temperature and pressure, with pressures interpreted as stem pressure. In spring, the relationship was inverted, with higher pressures occurring at high temperatures (fig. 2). 30

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Pressure (kPa)

Fig. 2: Summary of the relationship between resting temperature (0 5 oC versus 17 oC) and xylem pressure. Means + SE are shown, n = 8 and 4 respectively for winter and spring.