P. Grieu, C. Robin and A. Guckert
Agronomie et Environnement INRA-ENSAIA BP 172 F-54505 Vandoeuvre les Nancy Cedex FRANCE.
Material and methods
Forage legumes represent a major component of permanent grasslands with economic importance. They may suffer from periods of drought and it is important to understand physiological mechanisms involved in their reaction to drought. Their growth potential can be expressed in late spring and summer if rains or soil water supply are not restricted. Studies reported the effect of drought on white clover (Engin and Sprent, 1973; Shamsun-Noor et al., 1989) but little is known about the mechanisms responsible for the drought resistance of this species. The knowledge of these mechanisms would allow a better understanding of the origin of drought resistance in white clover and then, to establish the bases of choosing varieties. Decreasing soil water content is thought to be responsible for drought-induced stomatal closure and also for limitations of mesophyll photosynthesis (Grieu et al., 1988). However, the underlying mechanisms of these effects have not yet been clearly established. To evaluate the nature of disorders induced by drought on photosynthetic processes in white clover cv. Crau, we analysed gas exchange rates under ambient conditions, oxygen evolution rates and chlorophyll a fluorescence measurements under saturating light and CO2 conditions.
Plant material and experimental conditions. Plants of white clover cv Crau were grown in a naturally illuminated greenhouse, in 3 litre pots filled with peat-sand-vermiculite potting mix. Plants were irrigated daily with tap water and fertilized once a week with a nitrogen free nutrient solution (Robin et al, 1987). Two weeks before the onset of the experiments, 40 seedlings were transferred to a climate chamber with following day/night conditions: 14/10 h; relative humidity, 60/95 %; air temperature, 21/15°C; photon flux density at the top of the plants, 500 m mol.m-2. s-1, CO2 molar fraction in air, 400 m mol. mol-1. Thereafter, drought was imposed by withholding water for up to 15 days.
Measurements. Pre-dawn leaf water potential (PB) was monitored with a pressure chamber. Net CO2 assimilation rate (A) and stomatal conductance for water vapour (Gw) were measured with a portable gas exchange system (LI 6200; Li-cor USA) inside another climate chamber with a photon flux density of 1000 m mol. m-2. s-1 after one hour of acclimation of the plants. Fluorescence measurements were carried out with a modulated fluorometer (PAM 101, Walz Germany) on leaf disks (10 cm2) enclosed in an oxygen electrode chamber (LD2/2, Hansatech UK) flushed with humidified air containing 5% CO2. Further details are given in Figure 1 and in Epron and Dreyer (1993).
Calculation of Amax and calibration were determined according to Delieu and Walker (1981).
The response of leaf gas exchange of Trifolium repens cv Crau to increasing drought is shown in Figure 2. Both net CO2 assimilation rate (Fig. 2a) and stomatal conductance to water vapour (Fig. 2b) decreased in response to the drought-induced decline in leaf predawn water potential: until -1 MPa, the soil drought is relatively moderate, beyond this value, the water deficit becomes more severe. Rates of O2 evolution under saturating conditions of CO2 and light (Amax) were maintained at much higher rates than A (Fig. 3). Only a very limited decline in Amax could be observed. Neither the photochemical efficiency of PSII in the dark (Fv/Fm) nor the photochemical efficiency of open PSII reaction centres (Fv'/Fm') were significantly reduced in response to decreasing PB (Fig. 4).
Stomata closed dramatically in white clover in response to water supply being withheld (Fig. 2) in ambient CO2 and limiting irradiance. The decline in A was more gradual, and significant rate of A was recorded at PB near -2, 5 MPa, indicating that Trifolium repens cv. Crau is rather resistant to drought, as had already been described by Guckert et al. (1993) and Shamsun Noor et al. (1989) on the same species.
On the other hand, measurements of O2 evolution (Amax, Fig. 4) and of chlorophyll a fluorescence (Fig. 3) at 5% CO2 and saturating irradiance showed the absence of any effect of drought on the capacity of photosynthetic apparatus, which is rather resistant to leaf water deficits imposed by soil water depletion, and is able to perform at high rates provided there is a sufficient supply of CO2 to the chloroplasts. In fact, the limited decrease in Amax observed in water stressed leaves was not correlated to the intensity of drought. This had already be shown by Epron and Dreyer (1993) on Quercus petraea. Maintenance of high and constant values of Fv/Fm and Fv'/Fm' during all the drought cycle demonstrated that photochemistry of PSII, light-driven electron transport and enzymatic reactions requiring ATP and NADPH from chloroplasts were not significantly affected by the leaf water deficits induced by soil water depletion.
We may therefore conclude that water deficits imposed during a few days limit photosynthesis in Trifolium repens cv. Crau by reducing the supply of CO2 to chloroplasts when stomatal conductance decreases in response to soil drought. The photosynthetic apparatus was not affected by soil drought. The drought resistance of cv Crau was due to the stomatal closure which also involved a decrease of biomass production. These methodological tools could allow for the breeding of new cultivars more resistant to water deficits.
DELIEU T., WALKER DA., (1981): Polarographic measurement of photosynthetic oxygen evolution by leaf disks. New Phytologist. 89: 165-178.
ENGIN M., SPRENT JI., (1973): Effects of water stress on growth and nitrogen fixing activity of Trifolium repens. New Phytologist. 72, 117-126.
EPRON D., DREYER E., (1993): Photosynthesis of oak leaves under water stress: maintenance of high photochemical efficiency of PSII and occurence of non-uniform CO2 assimilation. Tree Physiology, (in press).
GENTY B., BRIANTAIS JM., BAKER NR., (1989): The relationship between the quantum yield of photosynthesic electron transport and quenching of chlorophyll fluorescence. Biochem Biophys Acta 990: 87-92.
GRIEU P., GUEHL JM., AUSSENAC G., (1988). The effect of soil and atmospheric drought on photosynthesis and stomatal control of gas exchange in three coniferous species. Physiologia Plantarum 73: 97-104.
GUCKERT A., ROBIN C., GRIEU P., GRAS F., (1993): Influence du deficit hydrique sur le trèfle blanc. Fourrages. (in press).
ROBIN C., CHONE T., GUCKERT A., (1987): Rôle de la position de la feuille dans l'assimilation et le transport du carbone chez le trèfle blanc (Trifololium repens L.). Agronomie. 7 (8), 599-605.
SHAMSUN NOOR L, ROBIN C, SCHONTZ D, HEULIN T, GUCKERT A, (1989): Effet d'un deficit hydrique sur le trèfle blanc (Trifolium repens). I. Importance du cultivar. Agronomie. 9, 251-257.
Figure 1: Fluorescence signals and measured parameters. After dark adaptated leaf disks were first illuminated with a modulated light (Lm) and initial fluorescence (Fo) was recorded. A pulse (Ls) was used to determine maximal fluorescence (Fm). Then, the leaf disks were continuously illuminated with an actinic white light (La) for 20 min. F' was recorded and a second pulse was imposed to determine maximal fluorescence (Fm'). The actinic light was removed and Fo' recorded.
Figure 2: Relationships between leaf pre-dawn water potential and (a) net CO2 assimilation rate and (b) leaf conductance to water vapour.
Figure 3: Relationships between leaf pre-dawn water potential and photochemical efficiency of PSII in the dark (Fv/Fm) and in the light (Fv'/Fm').
Figure 4: Relationships between leaf pre-dawn water potential and oxygen evolution rate (Amax).