Materials and methods Directory UMM :Data Elmu:jurnal:E:Environmental and Experimental Botany:Vol44.Issue2.Oct2000:

physiological responses have received relatively little attention Mcdonald and Davies, 1996. In beans Shimshi, 1970, coffee Tesha and Kumar, 1978, and winter wheat Shangguan, 1997, the stomatal conductance g s increased with nitrogen nutrition under well-watered conditions and be- came more sensitive to leaf water potential. It decreased as soil water became less available. Other works on tea Nagarajah, 1981 and cotton Radin and Ackerson, 1989 have indicated an opposite response, i.e. the stomatal sensitivity to leaf water potential was decreased by high nitro- gen nutrition. A strong correlation was found between leaf conductance and leaf nitrogen con- tent; however, this relationship was weaker than that between stomatal conductance and water po- tential Radin and Parker, 1979; Bolton and Brown, 1980; Morgan, 1986; Shangguan and Chen, 1990; Ciompi et al., 1996. Sugiharto et al. 1990 found a significiant positive correlation between the photosynthetic capacity of leaves and their leaf nitrogen concentration suggesting that most of the nitrogen used for synthesis of compo- nents of the photosynthetic apparatus. In particu- lar, Rubisco, the leaf protein playing the major role in carbon assimilation, was strongly affected by nitrogen deficiency Seemann et al., 1987. Although CO 2 and NO 3 − assimilation are linked, it is not completely clear to what extent they are coupled Lawlor, 1995. Therefore, further eluci- dation of the relation of leaf nitrogen content to the gas exchange and water use is needed. Water use efficiency indicates the performance of a crop growing under any environmental con- straint. At the leaf level, intrinsic water use effi- ciency WUE i is defined as the ratio of photosynthetic rate P n to transpiration rate. To achieve the same P n at a lower g s , a higher Rubisco activity and capacity for electron trans- port is required and thus a higher concentration of nitrogen in the leaf does not necessarily mean a proportional increase in the rate of photosynthesis Shangguan et al., 1999. In crop plants with C 3 photosynthetic pathway, carbon isotope discrimi- nation D has been used to provide time-inte- grated estimates of plant intrinsic water use efficiency Farquhar and Richards, 1984; Far- quhar et al., 1989. Foliar D values of C 3 plants have also been used as an integrated measure of the response of photosynthetic gas exchange to environmental variables such as humidity Winter et al., 1982, salinity Guy et al., 1986, light intensity Zimmerman and Ehleringer, 1990, soil water availability Meinzer et al., 1992, and ele- vated CO 2 Picon et al., 1996. A high but nega- tive correlation was found between carbon isotope discrimination D and WUE t Ehdaie and Hall, 1991; Ismail and Hall, 1992 and D with WUE i Wright et al., 1988, 1994. Meinzer et al. 1990 reported simultaneous reductions in stomatal con- ductance and D with increased WUE for water stressed cowpea. Rao and Wright 1994 showed significant effects of location and water regime on D for cowpea. Ehleringer et al. 1991 reported a high correlation between D and C i C a . To our knowledge, there is little information on the ef- fects of the two major environmental constraints on photosynthetic gas exchange and D in winter wheat. Since different stress histories could signifi- cantly have effects on a number of physiological mechanisms in wheat Morgan, 1984, the present study was designed to eliminate the uncertain effect of nitrogen nutrition stress with growing plants in solution culture and imposing water stress with polyethylene glycol PEG 6000. In this study, it was hypothesized that: 1 there would be interaction between drought and nitrogen nutri- tion on photosynthetic gas exchange and water use efficiency, and the effects of nitrogen nutrition on plants depend on solution water status; 2 WUE t and WUE i would be increased by the nitrogen nutrition supply. For verifying the hy- potheses winter wheat was grown under different combinations of nitrogen nutrition and water sup- ply levels and the study was focused on the effects of nitrogen deficiency and water stress on leaf water status, photosynthesis, nitrogen content, and water use.

2. Materials and methods

2 . 1 . Plant material and growth conditions Seeds of winter wheat Triticum aesti6um L. cv Xiaoyuan 6 were initially germinated in dark- ness at a constant temperature of 25°C on moist filter paper in dishes for 5 days. Upon emer- gence, the uniform seedlings were transferred to plastic pots, 20 cm in height and 10 cm in di- ameter, containing a mixture of vermiculite and perlite 1:1, vv four seedlings per pot. All plants were irrigated weekly with a nutrient so- lution and placed in a growth chamber. The nu- trient solutions were modified half-strength Hoagland solutions containing either 15 mM NO 3 − high-N or 1.5 mM NO 3 − low-N. All other ions in the solutions were constant, except for SO 4 2 − and Cl − , which were used in equal portions to maintain charge balance. Between nutrient additions, deionized water was applied as needed. Pots were covered with plastic beads to reduce evaporation from the soil surface. The conditions in the growth chamber were: a photo- synthetic photon flux density of 700 mmol m − 2 s − 1 over plants; daynight temperature: 2518 9 2°C; midday relative humidity: 60 9 5; and a 12-h light period. The first measurement was performed 45 days after planting. In the following measurements, PEG 6000 was added to the solutions in both nitrogen treatments and the seedlings had been kept in the solutions for 9 days. Drought stress was achieved by adding different amount of PEG, the osmotic potential of the solutions were − 0.24 MPa well watered and − 1.25 MPa droughted, respectively. 2 . 2 . Leaf water potential and gas exchange mea- surements Leaf water potential 8 w was measured with a pressure chamber LI − COR 3005, Inc., Lincoln, NE on the first fully expanded leaves. A port- able infrared CO 2 analyzer LCA − 3, ADC, Hoddesdon, UK was used to measure P n , g s , E and intercellular CO 2 concentration C i during the 9 days. The leaf was then removed for carbon isotope analysis. The youngest fully expanded leaf of three plants of each treatment were enclosed into the gas exchange chamber between 10:00 and 11:30 h for gas exchange measurements. Before the gas exchange measurements, the print of the leaf was taken for leaf area measurement by an area meter. Plant WUE i was determined as the ratio of P n to E. 2 . 3 . Carbon isotope composition analysis In order to calculate WUE t , all leaves of the plants were used for d 13 C and nitrogen determi- nations within each water and nitrogen treatment. The leaves were oven-dried at 70°C for at least 48 h and then finely ground. Relative abundancies of 13 C and 12 C were analyzed by mass spectrometry Delta S, Finnigan Mat. Carbon isotope compo- sition d was expressed as the 13 C 12 C ratio rela- tive to that of the Pee Dee Belemnite standard PDB. The resulting d 13 C values were used to calculate D Farquhar and Richards, 1984: D ‰ = d a − d p 1000 + d p × 1000 1 where d a and d p refer to the isotopic compositions of air − 8.0‰ and the plant material, respectively. D is related to the time-integrated value of the ratio of the C i to ambient CO 2 concentration C a and thus to plant WUE i Farquhar et al., 1989: D = a + b − a C i C a = 1 − 1.6 × 10 − 3 6 C a P n E 2 where a and b are the discrimination coefficients against 13 CO 2 during diffusion into the leaf and carboxylation. The values of a and b are estimated to be 4.4 and 27.0‰, respectively Farquhar et al., 1989. 6 mmol mol − 1 is the mean value of leaf-to-air water vapor pressure difference during the growing period. Plant transpirational water use efficiency WUE t is related to D by Farquhar and Richards, 1984: WUE t = C a 1.6 × 10 − 3 6 b − D b − a 2 3k 1 − F 3 where C a mmol mol − 1 is the mean ambient CO 2 concentration, k is the ratio of plant carbon mass to plant mass, and F is the proportion of net daytime fixation of carbon lost by mitochondrial respiration, fine root mortality or exudates by the whole plant. 2 . 4 . Nitrogen measurements For determining leaf nitrogen concentration, 200 mg of powdered material was analyzed with a modified Kjeldahl analysis using concentrated sul- phuric and salicylic acid and Na 2 SO 4 , K 2 SO 4 and Se as a catalyst in a ratio of 62:1:1 ww. The N-concentration of the digests was determined on a continuous flow analyzer Skalar, Breda, Netherlands. 2 . 5 . Statistical analysis S.E., variance, regression and correlation coeffi- cients, and significant differences among regres- sion coefficients were calculated by standard methods with the DAPS statistical package Feng and Tang, 1997.

3. Results and discussion