Vorgabe neu Journal of Applied Botany and Food Quality 80, 88 - 92 (2006) Laboratory of Plant Ecology, Ghent University, Ghent, Belgium Effects of severe water stress on partitioning of 14C-assimilates in tomato plants H. Zgallaï, K. Steppe, R. Lemeur (Received March 27, 2006) Summary Tomato plants (Lycopersicon esculentum Mill. cv Nikita) were grown hydroponically and subjected to severe water stress induced by addition of PEG-6000 to the nutrient solution. The PEG-treatment clearly impaired growth. Leaf photosynthesis decreased during the experiment. Moreover, the decrease in photosynthesis was associated with a decrease in dry weight of the shoot compared to the root. Also leaf area expansion, stomatal conductance and transpiration decreased. Water stress enhanced the transport of 14C-assimilates from the source leaf to the lower parts of the plant where the assimilates were incorporated in the lower stem, the leaves below the source leaf and the roots. It was observed that 14C was much more concentrated in the roots compared to the other plant parts. Introduction Water stress is one of the most important environmental constraints that limit plant productivity (BRADFORD and HSIAO, 1982). Plant productivity is determined by a complex series of events leading from CO 2 fixation in the chloroplasts, formation of phloem-mobile and storage metabolites, and delivery of these metabolites to sink tissue (MADORES and LUCAS, 1995). This partioning of carbon determines plant health and vigor during times of stress and sub- sequently leads to good or bad harvests (HOUPIS et al., 1999). The relationship between carbon translocation and water stress is interesting to study as water is a major constituent of tissue, a reagent in chemical reactions, and a solvent for and mode of translocation for metabolites and minerals. With the occurrence of water stress many of the physiological processes associated with growth are affected. Photosynthetic rate reductions due to increased water stress have been observed in a wide variety of plant species. Stomatal effects are usually considered to be the first and the major limitation to CO 2 fixation (CORNIC and MASSACCI, 1996; ZGALLAÏ et al., 2005). However, leaf photosynthesis may also be affected by accumulation of photosynthates due to changes in translocation or utilization of the assimilates (SHINOHARA et al., 1995). Inhibition of assimilate translocation by water stress has been observed in a number of plant species (GEIGER and FONDY, 1991). WARDLAW (1990) has indicated that the major effect of water stress on translocation is the delay and the reduction of transfer rate of assimilates from the assimilating tissues to the sink tissues. MUNNS (1988) reported that the reductions in translocation observed under water stress are probably the result from the reduction in growth and the slowing down in carbohydrate utilization prior to a reduction in photosynthesis. Disagreement exists as to the relative sensitivity of photosynthesis and translocation to water stress (SUNG and KRIEG, 1979). Determination of translocation or the carbon balance of the whole plant requires that the carbon balance of the source leaf is determined by the 14CO 2 steady-state feeding method (SHISHIDO et al., 1999). This method allows calculation of the proportion of total carbon accumulated in the sink organ that was supplied by the source leaf, as well as the growth contribution of the source leaf to the main plant sinks. These data are vital to determine the productivity of the source leaf under severe water stress. The modified sink-source relationship caused by water stress has been largely neglected in the past, although the assessment is of high importance to gain fundamental insights into the carbon metabolism during stress conditions (SHISHIDO et al., 1999). In this paper, we examined the effects of severe water stress induced by polyethylene glycol-6000 (PEG-6000) on the carbon metabolism in tomato plants (Lycopersicon esculentum Mill. cv. Nikita). Both photosynthesis and carbon translocation were measured in this study to elucidate the effects of severe water stress on the carbon metabolism. To investigate the relationship between translocation and water stress, the 14C incorporation of a leaf exposed to 14CO 2 , and the subsequent dis- tribution of the C-assimilates in different parts of the plants were analysed. Materials and methods Plant material and treatment Tomato (Lycopersicon esculentum Mill. cv. Nikita) seeds were germinated in pots containing sand and peat. Seedlings were grown in a greenhouse at 20 / 32 °C, a relative humidity of 60 % / 70 %, and under normal day-light conditions. After 14 d, plants were trans- planted to vermiculite and irrigated with a nutrient solution. The nutrient solution was aerated using an air compressor. The pH of the nutrient solution was monitored daily and kept at 6.0 with addition of HCl (0.1 mol/l) or NaOH (0.1 mol/l). At this time, the plant material was subdivided into two sets: one control and one treatment group. Each group consisted of 16 plants, with the plants being randomly arranged over two nutrient film drains. The osmotic potential of the nutrient solution (Ψ s ) of the treatment group was changed gradually at a rate of -0.04 MPa/d by addition of polyethylene glycol-6000 (PEG-6000). The addition of PEG was suspended when Ψ s reached -1.22 MPa (after 32 d; progressive severe stress) and a series of comparative investigations on treated and non-treated plants were performed. The concentration of PEG-6000 (in g/g water) needed for this level of water stress was determined using the equation given in MICHEL and KAUFMANN (1973). The water potential of the nutrient solution (Ψ s ) was measured using filter paper discs (0.25 cm2). Measurements were made with a thermocouple psychrometer (sample chambers type C52; Wescor Inc, Logan, Utah, USA) following the method described by SPANNER (1951). Measurement of photosynthesis, transpiration and stomatal conductance Photosynthesis (P n ), transpiration (E) and stomatal conductance (g s ) of four fully expanded mature leaves of different control (Ψ s = -0.16 MPa) and treated plants (Ψ s = -1.22 MPa) were determined using an infrared gas analysis system for CO 2 and H 2 O respectively (Walz compact CO 2 /H 2 O porometer; Heinz Walz, Efeltrich, Ger- many). The analyzer was connected to a leaf chamber in which the leaf received a photosynthetic photon flux density of 900 µmol m-2 s-1. The flow of the air through the chamber was 600 ml min-1. After the measurement the leaf areas were measured using an automatic leaf area meter (Li-Cor, Walz, Germany) calibrated to ± 0.1 cm2. Spatial distribution of photosynthetic assimilates Translocation was measured using the 14CO 2 steady-state feeding method (SHISHIDO et al., 1999). The radioactive pulse was given to the fourth leaf counted from the base of the shoot of tomato plants (Fig. 1). The treated leaf consisted of only the three terminal leaflets (the other leaflets were removed just before treatment). The remain- ing leaflets were confined hermetically in a transparent cylindrical cuvette (CRS087, PP systems, UK). 15 µl Na 2 14CO 3 (specific activity 53 mCi mmol-1) (Amersham, UK) was put in a reservoir inside the cuvette and 100 µl HCl (10-5 M) was added to the carbonate to form 14CO 2 . The 14CO 2 pulse was given during 15 min under natural light conditions. At the end of the translocation period (23 h), the plants were quickly divided into different parts (Fig.1). For quantitative analysis of the amount of radioactivity in the different plant parts the sample was placed in a scintillation counter. The sample was combusted in oxygen (dry combustion) by a sample oxidizer (Packard Instrument Co. Model 306 Tri-carb Sample Oxidizer) by which the carbon is evolved as 14CO 2 . Extracts were counted in a liquid scintillation counter (Nuclear Chicago Isocap. 300) using a toluene-base scintillation liquid containing 0.015 % POPOP (W/V), 0.6 PPO (W/V) and Triton X (2 parts of toluene / 1 part Triton X). The radioactivity in the various parts of the plants was expressed in two ways as described by MOR and HALEVY (1979): (a) as the percentage of radioactivity recovered in the plant, excluding the source leaf, and (b) as a relative specific activity (RSA), which is the ratio of the specific activity in a given part (soluble radioactivity/g dry weight) to specific activity of the whole plant. Fresh and dry weight of different parts of the plant were determined with a digital balance (Sartorious B310S, Göttingen, Germany), calibrated to ± 0.001 g. Results Effect of severe water stress on photosynthesis, transpiration and stomatal conductance The effect of severe water stress on the assimilation rate (P n ), transpiration rate (E) and stomatal conductance (g s ) are shown in Tab. 1. After the severe water stress, the assimilation rate and the stomatal conductance decreased to about 71 % of the control, but the intercellular partial pressure of CO 2 was identical (data not shown). The results indicate that the stomata of the leaves of the PEG-treated plants were nearly completely closed, causing stomatal limitation of photosynthesis. The decrease in photosynthesis and stomatal conductance was associated with a reduction in transpiration rate (Tab. 1). Tab. 1: Effects of severe water stress on the net photosynthesis rate (P n ), the stomatal conductance (g s ), the transpiration rate (E) and the shoot to root ratio of tomato plant grown in a nutrient solution. Values indicate the mean (n = 4). Different letters within the column show sig- nificantly differences using Duncan’s test. Treatment P n g s E Shoot/Root (µmol m-2 s-1) (mmol m-2 s-1) (mmol m-2 s-1) Control 13.02±0.47a 131.72±12.65a 30.00±1.45b 1.70±0.01a Stressed 3.87±0.02b 38.20±3.01b 17.19±4.14a 1.38±0.01b Fig. 1: Diagram showing the site of application of the solution containing the radiolabelled compound and the cut plant parts of tomato plant before counting. Symbols used: R = roots, Sb = stem below source leaf, Lb = leaves below source leaf, Sa = stem above source leaf, La = leaves above source leaf. Cuvette Site of application Reservoir R La Sa La Sb Lb Lb Lb The dry weight of the shoot, the root, the ratio shoot/root and the leaf area were significantly (P< 0.05) reduced by the imposed water stress (Fig. 2, Tab. 1). Effects of water stress on translocation 89 Effect of severe water stress on the distribution of photosynthetic assimilates The radioactive pulse was given to the fourth leaf counted from the base of the shoot (Fig. 1). The relative distribution of total 14C radioactivity 23 h after 14CO 2 exposure recovered in each plant part is shown in Fig. 3. The pattern of 14C distribution exhibited some marked differences between control and PEG-treated plants. For control plants, 14C was found mainly in the upper and lower stem and in the roots. The leaves contained less than 4 % of the recovered radioactivity (Fig. 3) which was equally distributed between the leaves below and above the source leaf. The main difference in the radioactivity pattern found after application of severe water stress concerned the distribution of the 14C in the lower parts of the plants. The largest increase was observed in the roots (an increase of 10.2 % compared to control plant), but also in the stem and the leaves below the source leaf an increase was detected (6.2 and 2.7 % re- spectively). Fig. 4 also shows the distribution of the radioactivity expressed in terms of relative specific activity (RSA) in the different parts of the tomato plants 23 h after application of the pulse to the source leaf. The RSA indicates that the stem and the roots are labelled most (the source leaf excluded). Compared to the control plant, the distribution of radioactivity in the whole plant was significantly different for tomato plants exposed to severe water stress. Except for the stem part above the source leaf, the RSA was higher in all distinguished parts of the PEG-treated plants compared to the control plants. Discussion In this study we investigated the response of hydroponically grown tomato plants to long-term severe water stress. Addition of PEG- 6000 to the nutrient solution was used to impose gradual water stress on the plants by exposing their root system to the solution. No toxic effects of PEG were observed and, hence, this osmoticum was considered as suitable to induce water stress (KOCHEVA et al., 2005; ZGALLAÏ et al., 2005). The water-stressed plants were clearly re- tarded in growth. Both photosynthesis and transpiration decreased significantly (Tab. 1) due to the rapid response of stomata to de- creasing water availability (BUCKLEY, 2005). The closure of the stomata was observed by the sharp decrease in g s during severe water stress. This was also demonstrated by BÜSSIS et al. (1998) and ZGALLAÏ et al. (2005). Hence, photosynthesis and transpiration are very sensitive to severe water stress. Another well-observed adaptation response in relation with water stress is a reduction in shoot growth relative to root growth (TURNER and BEGG, 1981; BOTA et al., 2004). Also our study indicates a de- crease in shoot / root ratio for the water-stressed tomato plants (Tab. 1). MEYER and BOYER (1981) and SHINOHARA et al. (1995), Fig. 2: Changes in shoot and roots dry weight and leaf area expressed as a percentage of the same parameters in the control plants. 0 20 40 60 80 100 120 Control Stressed Tr e atm e nt s P e rc e n ta g e to t h e c o n tr o l Shoot root Leaf area P er ce n ta ge o f co n tr ol ( % ) Control Stressed Control plant Stressed plant Fig. 3: Effect of severe water stress on the distribution pattern of (14C) carbon accumulated in each sink from a common source leaf. Distribution values of the sinks are expressed as percentages of the total 14C accumulated in the source leaf. Same symbols are used as in Fig. 1. Control plant Stressed plant R = 11.4 % Lb = 1.8 % Sa = 48.8 % La = 1.3 % Sb = 36.7 % Lb = 4.5 % Sa = 25 % La = 6 % R = 21.6 % Sb = 42.9 % 90 H. Zgallaï, K. Steppe, R. Lemeur reported that the decrease in shoot / root ratio mostly resulted from relative reductions in leaf growth. This significant shift in the shoot/ root ratio during water stress supports the idea of changes in biomass allocation (SHARP and DAVIES, 1985; SHINOHARA et al., 1995; BOTA et al., 2004). Changes in translocation were studied using the 14CO 2 steady-state feeding method (SHISHIDO et al., 1999). The total 14CO 2 fixation in control and treated source leaves was set equal to 100%. The pro- portion of carbon supplied by the source leaf to sink organs could be analysed. It was observed that water stress clearly affected the export of photosynthetic assimilates from the source leaf in the plant. After 23 h of translocation, the portion of 14C moved out of the source leaf in the control plant was equally distributed between the upper (50.1 %) and lower parts (49.9 %) (Fig. 3). In contrast, in severe stressed plants, the percentages of radioactive distribution were 31 % and 69 % for the upper and lower plant parts, respectively. Hence, the largest portion of 14C fixed by the source leaf was trans- ferred to the lower parts of the plant (Fig. 3) and there was much less movement of 14C towards the upper parts. This was confirmed by the relative specific activity measurements (Fig. 4). Hence, severe water stress had a significant effect on the movement of C-assimilates, whereby the roots received more photosynthetic assimilates compared to the control plants. These results suggest that tomato plants under severe water stress translocate more C-assimilates towards the roots in order to maintain root growth, which means survival (or uptake of water), even during conditions of severe water stress. These findings were supported by SHINOHARA et al. (1995) and OHASHI et al. (2000) who also observed changes in the distribution of assimilates, together with a decrease in shoot/root ratio and an increase of assimilates in the roots during water stress. In this study we used 14C to estimate the relative distribution of radiocarbon in tomato plants under severe water stress. The para- meters we used were the percentage of radioactivity recovered in the plant and the relative specific activity (RSA), which emphasized the ability of various organs to draw metabolites (MOR et al., 1981). This method enabled us to analyse the carbon balance of each individual sink, thereby obtaining information necessary to evaluate the productivity of plants under water stress condition. The higher translocation of photosynthetic assimilates towards the roots indicated that water-stressed tomato plants tried to maintain root growth and, hence, tried to survive even during the unfavourable growing con- ditions imposed. Acknowledgements The authors wish to thank Helga Pien and Philip Deman for their technical assistance, their enthusiastic support and highly valuable contribution to the experimental set-up. References BOTA, J., STASYK, O., FLEXAS, J., MEDRANO, H., 2004: Effect of water stress on partioning of 14C-labelled photosynthates in Vitis vinifera. Funct. Plant Biol. 31, 697-708. BRADFORD, K.J., HSIAO, T.C., 1982: Physiological responses to moderate water stress. In: Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H. (eds.), Encyclopedia of plant physiology, new series, 12B, 264-324. Springer, Berlin. BUCKLEY, T.N., 2005: The control of stomata by water balance. New Phytol. 168, 275-292. BÜSSIS, D., KAUDER, F., HEINEKE, D., 1998: Acclimation of potato plants to polyethylene glycol-induced water deficit. I: Photosynthesis and metabolism. J. Exp. Bot. 49, 1349-1360. CORNIC, G., MASSACCI, A., 1996: Leaf photosynthesis under drought stress. In: Baker, N.R. (eds.), Photosynthesis and the environment, 347-366. Kluwer Academic Publishers, The Netherlands. GEIGER, D.R., FONDY, D.B.R., 1991: Regulation of carbon allocation and partitioning: status and research agenda. In: Bonnemain, J.L., Delrot, S., Lucas, W.J., Dainty, J. (eds.), Recent advances in phloem transport and assimilate compartimentation, 1-9. Ouest Editions, Nantes. HOUPIS, J.L.J., ROGERS, H.H., HELMS, J.A., COSTELLA, M.P., 1999: Trans- location of carbon-14 in Pinus ponderosa seedlings subjected to long- term sulphur dioxide and water stress. Trans Illinois State Academy of Science 92, 191-202. KOCHEV, K.V., BUSHEVA, M.C., GEORGREV, G.I., LAMBREV, P.H., GOLTSEV, V.N., 2005: Influence of short-term osmotic stress on the photosynthetic activity of barley seedlings. Biol. Plant 49, 145-148. MADORES, M.A., LUCAS, W.J., 1995: Carbon partitioning and source-sink interactions in plants. Current Topics in Plant Physiology, Vol. 13. ASPP, Rockville, MD. MEYER, R.F., BOYER, J.S., 1981: Osmoregulation, solute distribution, and growth in soybean and seedlings having low water potentials. Planta 151, 482-489. MICHEL, B.E., KAUFMANN, M.R., 1973: The osmotic potential of polyethylene glycol 6000. Plant Physiol. 51, 914-916. MOR, Y., HALEVY, A.H., 1979: Translocation of 14C-assimilate in roses. I: The effect of the age of the shoot and location of the source leaf. Physiol. Plant 45, 117-182. MOR, Y., SPIEGELSTEIN, H., HALEVY, A.H., 1981: Translocation of 14C- assimilate in roses. II: The effect of shoot darkening and cytokinin application. Physiol. Plant 52, 197-200. MUNNS, R., 1988: Why measure osmotic adjustment? J. Plant Biol. 15, 717- 726. OHASHI, Y., SANEOKA, H., FUJITA, K., 2000: The effect of water stress on growth, photosynthesis, and photoassimilate translocation in soybean and tropical pasture legume siratro. Soil Sci. Plant Nutr. 46, 417-425. SHARP, R.E., DAVIES, W.J., 1985: Root growth and water uptake by maize plants in drying soil. J. Exp. Bot. 36, 1441-1456. SHINOHARA, Y., AKIDA, K., MARUO, T., ITO, T., 1995: Effect of water stress on Fig. 4: Effect of severe water stress on the distribution of radioactivity (expressed in terms of relative specific activity) in tomato plants 23 h after exposure to 14CO 2 . The radioactive pulse was given to the fourth leaf counted from the base of the plant. Averages of three replications are presented. The different letters within the histogram indicate significantly differences at a 5 % level. Same symbols are used as in Fig. 1. 0 2 4 6 8 La Lb Sa Sb R Plants parts Control Stressed d C d B a b c C A R el at iv e sp ec if ic a ct iv it y (R S A ) Plants parts B Plant parts Effects of water stress on translocation 91 the fruit yield, quality, and physiological condition of tomato plants using the gravel culture. Acta Hort. 396, 211-218. SHISHIDO, Y., KUMAKURA, H., NISHIZAWA, T., 1999: Carbon balance of a whole plant and the contribution of source leaves to sink growth using the 14CO 2 steady-state feeding method. Physiol. Plant 106, 402-408. SPANNER, D.C., 1951: The peltier effect and its use in the measurement of suction pressure. J. Exp. Bot. 2, 145-168. SUNG, F.J.M., KRIEG, D., 1979: Relative sensitivity of photosynthetic assi- milation and translocation of 14carbon to water stress. Plant Physiol. 64, 852-856. TURNER, N.C., BEGG, J.E., 1981: Plant-water relations and adaptation to stress. Plant and Soil 58, 97-131. WARDLAW, I.F., 1990: The control of carbon partitioning in plants. New Phytol. 116, 341-381. ZGALLAÏ, H., STEPPE, K., LEMEUR, R., 2005: Photosynthetic, physiological and biochemical responses of tomato plants to polyethylene glycol- induced water deficit. J. Integrat. Plant Biol. 47, 1470-1478. Address of the authors: Laboratory of Plant Ecology, Ghent University, Coupure links 653, B-9000 Ghent, Belgium E-mail: kathy.steppe@UGent.be 92 H. Zgallaï, K. Steppe, R. 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