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Effects of temporary drought on nitrate-fed and nitrogen-fixing alfalfa plants
AN p
s
CiENCE
Plant Science 107 (1995) 159-165
F:LSEVIER
Effects of temporary drought on nitrate-fed and nitrogen-fixing alfalfa plants M.C. Antolin*, J. Yoller, M. Sinchez-Diaz Departamento de Fisiologia Vegetal, Universidnd de Navarra. Irunlarrea s/n. E-31008 Pamplona, Spain
Received 22 November 1994;revision received 6 March 1995;accepted 6 March 1995
Abstract The effect of temporary drought on growth, carbon exchange and solute accumulation has been examined in alfalfa plants dependent on either N, or nitrate. Plants were subjected to cyclical moderate or severe drought (drought/recovery). Growth parameters, photosynthetic rate (P,), leaf conductance to water vapour (g,), chlorophyll content, and solute accumulation were determined. Growth decreased markedly under water deficit, but no significant differences between either groups of plants were found. Nitrogen-fixing plants developed higher root/shoot ratios maintaining larger leaves with increased specific leaf area and greater chlorophyll content than nitrate-fed ones. Leaf conductance and net photosynthetic rate declined simultaneously with the drought treatments in both groups of plants; however, Nr fling plants retained higher Pn and g, values than nitrate-fed plants at lower RWC. Upon rewatering, a considerable stomata1 closure remained in nitrate-fed plants. Drought treatment induced an increase in solute concentrations, mainly potassium, especially important in nitrate-fed plants. The interactions between the type of N nutrition and drought tolerance in alfalfa plants during temporary drought are discussed. Keywortis:
Alfalfa; Drought; Medicago
sativa cv.
Aragon; Nitrogen fixation; Nitrogen nutrition; Photosynthesis
1. Introduction
Alfalfa N nutrition is ensured both by atmospheric nitrogen fixation and mineral nitrogen assimilation, but the importance of each process on total plant production is largely influenced by environmental factors such as salinity [35] and water stress [6,33]. Several authors have addressed the inhibitory * Corresponding author, Tel.: (+34-48) 105600;Fax: (+3448) 105649. 0168-9452/95/$09.50 0 1995 SSDI 0168-9452(95)04108-7
effect of water stress on root nodule activity in legumes [7,9,31], including alfalfa [1,5]. However, some of these studies described interactions between water stress and N2 fixation without considering the effect of drought on plants depending on inorganic nitrogen. The particular way in which water stress is imposed might be of special importance in understanding the field response to drought and also in evaluating the plant’s capacity to acclimate to stress [14]. For short-term experiments withholding water is the most common method,
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but to simulate more realistic responses to drought, sustained or cyclic water stress is needed [19]. Previous reports showed that nodulated alfalfa plants responded to temporary drought by osmotic adjustment and enhanced drought tolerance [3]. The objective of this research was to examine growth, photosynthesis and solute accumulation in alfalfa plants grown under controlled environmental conditions in response to temporary drought, and relying on either nitrate or nitrogen fixation. 2. Materials and methods 2.1. Biological material and growth conditions Alfalfa (Medicago sativa cv. Aragon) seeds were surface sterilized and germinated on wet filter paper in Petri dishes. Seedlings were planted in 25 x 10 cm pots containing perlite (6 plants/pot). One set of plants (N2 plants) was inoculated at planting with Rhizobium meiiloti strain 102 F51 (Nitragin Co.) maintained on yeast extract manitol agar. This set received a N-free nutrient solution [8]. The other set (NO3 plants) received the same nutrient solution supplemented with 16 mM KNO,. Plants were grown in a greenhouse at 25/l 5°C 50/70% RH (day/night), and illuminated during 14h with natural daylight supplemented with fluorescent lamps (Sylvania DECOR 183, Germany), providing a Professional-58W, minimum photosynthetic photon flux density of around 300-400 pmol photons. rnm2.s-l. 2.2. Experimental design When 45 days old, plants were subjected to drought by withholding irrigation in a cyclic way as described previously [4]. Two degrees of stress (moderate, MS and severe, SS) were imposed. The moderate stress level was defined as the degree of stress that occurs when the plants show visible signs of wilting in the afternoon and recover turgor during the night (approximately 4 days/ cycle). The severe stress level was defined as the degree of stress that occurs when the plants show visible signs of wilting during the afternoon, and do not recover during the night (approximately 8 days/cycle). Predawn leaf water potential (q,,,) was measured before irrigation to determine the
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actual level of stress. Plants were subjected to three cycles of stress and recovery. Recovery was considered completed when predawn \k, of recovered plants reached the prestressed levels (RMS, RSS). Rewatering was performed with nutrient solution or deionized water in order to supply the different water treatments with the same amount of nutrients during drought. Plants were transferred prior the third cycle of stress to a controlled environment chamber with a day/night regime of 25/15”C and 60180% RH. A photosynthetic photon flux density of 310 prnol photons.m-2.s-’ (400-700 nm) on the upper leaves was provided by a fluorescent tube (Sylvania F 48T12 CW-WHO) and incandescent bulb mixture for a 15-h photoperiod. Measurements were made at the end of the third cycle and repeated 24h after rewatering. Midday leaf q,,, and photosynthesis were determined at the same time. Means f S.E. (n = 7-9) were calculated, and their differences tested for significance by using a LSD technique with the Student’s t-test. 2.3. Plant determinations Leaf water potential (q’,) was measured with a pressure chamber [25] and the relative water content (RWC) was calculated as follows: RWC = (fresh wt. - dry wt.)/(turgid wt. - dry wt.) x 100. CO2 exchange measurements were made on the youngest fully mature leaf using an infrared gas analyzer (Analytical Development Co., Hoddesdon, UK, LCA-2 model) operated in the differential mode as described by Sanchez-Diaz et al. [22]. Air of known COz concentration (400 prnol . mol-‘) and 60% RH was supplied at a constant flow (300 ml.min-‘) into the leaf chamber. Photosynthetic photon flux density was 1000 pmol photons.m-2.s- ’ during measurements. Chlorophyll content was determined on each leaf used in gas exchange measurements according to SCstak et al. [26] in acetone (80% v/v) extracts of fresh leaves. Plants were harvested for growth determinations and chemical analysis after rewatering. Leaf area was measured by using an automatic leaf area meter (Li-COR, LI-3000 model) and plant dry weight was determined after drying at 70°C for 2
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days. The total nitrogen was measured by Kjeldahl and plant phosphorus content was determined by the method of Allen et al. [2]. Calcium, potassium and magnesium were determined in acid solution of ashed samples obtained in an oven at 500°C. Potassium content was measured with a flame photometer, and calcium and magnesium were estimated by the EDTA titration method [2]. 3. Results and discussion Nitrate-fed alfalfa plants always showed higher RWC values than nitrogen-fixing ones for similar leaf qk, (Fig. 1). As reported by several authors in corn and sorghum [23], wheat [20] and in soybean [29], such results suggest that nitrate-fed leaves could be more resistant to dehydration than nitrogen-fixing leaves. However, differences in RWC values could also be a consequence of either different initial cell sap osmolarity, cell wall elasticity [ 131 or osmoregulation 1171,particularly when it is considered that nitrogen in nitrate-fed plants was supplied as KNOs. The rest of the results are expressed as a function of RWC since, according to some authors 113,281, a better relationship with growth and metabolism exists when expressing water status by using RWC rather than * W’ Net photosynthesis (P,) (Fig. 2A) and leaf conductance to water vapour (g,) (Fig. 3A) behaved similarly in both groups of plants during drought;
01 90
80
70
RWC
60
50
(%)
Fig. 2. Net photosynthetic rate (P,) in plants subjected to different nitrogen treatments (N,, 0; NO,, m during drought (A) and upon rewatering (B) as a function of relative water content (RWC). Recoveries are represented at the same RWC as in stressed plants to facilitate the comparison. Measurements were made in an external CO* concentration of 400 pmol . mol-‘, a photon flux density prnol. * and at leaf temof Otherwise, as Fig. I
0.3
-’
I _
;:;; 90g
8o: 70
2
-
60 50 2n
(Tyq - 1
-2
Leaf water potential
-4
-3
(MPa)
o.o+ 90
Fig. 1. Leaf relative water content (RWC) in plants subjected to different nitrogen treatments (N2, 0, NOs, 4 as a function of leaf water potential. Recoveries are represented by open (N$ and solid (NO,) circles. Each point is the mean of seven to nine plants. The bars indicate standard error (S.E.) of the mean. Standard errors lower than 10% were not represented.
,
,
80
70
RWC
, 60
50
40
(96)
Fig. 3. Total conductance to water vapour (g,J in plants subjected to different nitrogen treatments (N2, 0; NOs, m during drought (A) and upon rewatering (B) as a function of relative water content (RWC). Otherwise, as for Fig. 2.
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Table 1 Chlorophyll content (a and b) and chlorophyll a/b ratio of alfalfa plants subjected to different nitrogen and drought treatments Treatments
Chlorophyll (mgeg-’ dry wt.) a
b
ah
Nitrogen-fixing plants Control MS ss
8.51 l 0.73a 8.20 f 1.14a 5.18 f 0.75bc
2.42 f 0.22a 2.49 f 0.32ad 1.45 f 0.26bc
3.55 f 0.03a 3.63 * 0.09a 3.85 f 0.17ab
Nitrate-fed plants Control MS ss
9.17 l 0.85a 6.74 f 0.83b 3.58 zt 0.35~
2.63 f 0.22a 1.94 zt 0.25bd 0.93 l O.lck
3.46 f 0.05a 3.47 f 0.04a 3.97 zt 0.13b
Nitrogen treatments: Nz. NO?; drought treatments: control, moderate stress (MS), severe stress (SS). Recoveries are not significant (data not shown).
however, upon rewatering moderately stressed nitrogen-fixing plants recovered control g, levels whereas nitrate-fed plants did not (Fig. 3B). Ghashghaie and Saugier [lo] showed similar g, response to drought in high and low-N fertilized tall festue plants. In nitrogen-fixing plants, the partial recovery of P, (Fig. 2B), despite the almost complete recovery of g,, would implicate nonstomata1 effects on P, under cyclic drought conditions [4]. However, in nitrate-fed plants no recovery of & (Fig. 3B) and P, (Fig. 2B) were
observed, possibly indicating the involvement of other factors, such abscisic acid [ 12,361. Therefore, during drought nitrogen-fixing plants seemed to be less sensitive to temporary drought than nitratefed ones, retaining higher rates of P, at similar RWC values than nitrate-fed plants. Plants depending on nitrogen fixation maintained their chlorophyll contents at moderate water stress, whereas a significant decrease was found under severe stress (Table 1). Nitrate-fed plants showed a considerable reduction in chlorophyll
Table 2 Total, shoot and root dry weights, root-shoot dry weight ratio, leaf area and specific leaf area (SLA) of nitrogen-fixing and nitrate-fed Medicago sativa plants Nitrate-fed plants
Nitrogen-fixing plants Control Plant weight (g) Shoot weight (g) Root weight (g) Root/shoot (se g-‘) Leaf area (cm*) SLA (dm*. g-t)
2.07 l O.O8*a 0.92 * 0.02a 0.93 * O.O5*a 0.78 f O.l2*a 278.9 f 12.0a 2.82 f 0.08a
RMS 1.63 f 0.04b 0.67 * O.llb 0.92 f 0.02a 1.31 +z 0.09b 226.3 * 9.7b 3.17 * 0.20a
Control
RSS 1.60 f 0.65 + 0.89 f 1.32 f
0.26b 0.02b 0.14a 0.01b
202.3 f 13.0b 3.64 zt 0.31b
2.74 + 1.41 zt 1.41 l 1.05 f
RMS 0.14c 0.08~ 0.12b 0.07c
392.6 f 1.6c 2.96 * 0.02a
1.54 * 0.06b 0.79 f O.Olb 0.88 * 0.07a 1.26 zt 0.06bc 221.4 zk 6.4b 2.26 l 0.09~
RSS 1.53 * 0.79 l 0.75 * 1.29 +
0.07b 0.02b 0.04a 0.03bc
209.5 f 13.9b 2.19 zt 0.09~
Total, shoot and root dry weights, root-shoot dry weight ratio, leaf area and specific leaf area (SLA) of Medicago sat&a dependent on either N2 (nitrogen-fixing’plants) or 16 mM KNO, (nitrate-fed plants) under well watered conditions (control) and subjected to different drought treatmentsidata obtained upon rewatering) (RMS, moderate stress; RSS, severe stress). Values are means f S.E. (n = 7-9). Comparison between means were made with the Student’s t-test. Within each line, means followed by the same letter are not significantly different (P > 0.05). *Nodules are not included.
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Table 3 Leaf, stem and root nitrogen, phosphorus and cation concentrations in plants subjected to different nitrogen and water treatments Organ
Leaf
N treatment NZ
NO3
Stem
N2
NO3
Root
NZ
NO3
Water treatment Control RMS RSS Control RMS RSS Control RMS RSS Control RMS RSS Control RMS RSS Control RMS RSS
Concentration (mg-g-’ dry wt.) N
P
K
Ca
Mg
33.5 f 1.6a 30.6 f 0.8a 24.7 f l.lbc 26.8 f 0.5b 25.6 f 0.9bc 22.4 zt 1.6c 16.3 f 0.5a 16.5 A 0.6a 17.8 f 0.9a 12.2 f 0.7b 16.5 * 0.5a 17.4 * O.la 14.7 f 0.9a 14.7 l 1.5a 18.1 f l.lb 11.2 f 0.6c 16.2 f 0.7ab 23.8 f 0.4d
4.52 + 0.2a 4.19 f 0.2a 4.25 f 0.2a 2.38 + 0.2b 2.29 + O.lb 1.84 zt 0.5b 3.85 f 0.8a 3.63 * 0.9a 4.07 f 0.6a 2.19 l O.lb 2.35 f 0.3b 1.61 f 0.6b 3.96 f 0.3a 3.62 f 0.5ac 3.97 f 0.2a 1.99 f 0.6b 2.41 zt 0.6bc 2.14 f 0.6bc
34.7 * 0.9a 37.6 zt 0.8b 38.9 * 0.8b 60.4 zt 0.9~ 80.2 * 1.5d 91.1 * 3.le 30.2 f 0.8a 30.0 f 0.4a 28.7 f 0.2a 46.9 f 0.4b 53.3 f 0.4c 60.8 f 0.4d 13.3 * O.la 9.5 f 0.7b 8.2 zt 0.7b 14.6 + 1.9ac 16.3 + 0.4d 17.9 f 0.4d
13.1 f 0.2a 14.1 zt O.lb 18.7 f 0.1~ 8.9 f O.ld 7.4 f 0.2e 7.7 f O.le 5.2 f 0.2a 4.6 f 0.2b 3.4 f O.lc 3.8 f 0.1~ 3.4 f O.lc 2.9 f O.ld 1.9 l O.la 4.0 f O.lb 2.5 f 0.1~ 6.1 zt 0.2d 7.6 f O.le 10.3 f O.lf
6.6 f O.la 7.9 A O.Ob 8.7 f 0.1~ 4.4 f o.Od 4.5 * 0.2d 4.2 zt O.Od 3.8 f 0.2a 3.8 f O.la 3.3 * 0.2b 2.0 f o.Oc 1.9 f O.Ic 2.1 f O.lc 2.6 f O.la 2.3 f 0.3a 2.5 f O.la 2.8 f O.lab 3.1 f O.lb 3.0 f O.lb
Nitrogen treatments: Nz, NO,; data for water treatments data obtained upon rewatering. Otherwise as for Table 1
content at any drought level. The chlorophyll a/b ratio increased significantly only in severely stressed plants for both N treatments. These results suggest that nitrogen-fixing plants maintained a better leaf structure than nitrate-fed ones during dehydration. No decrease in chlorophyll content was also reported in drought tolerant genotypes of maize [21] and wheat [l 11. Growth parameters were only determined in rewatered plants because plant material was kept until the end of the third drought/recovery cycle for measurements. Under well watered conditions, nitrate-fed plants produced more shoot and root dry weights and leaf area than nitrogen-fixing plants; however after drought a marked decrease in these parameters was found (Table 2). Nitrogenfixing plants showed reduced shoot growth without changes in roots. Results agree with the fact that shoot growth is generally more affected by water stress than root growth, not only because the transpiring parts of the plant usually develop greater and longer water deficits [15,1X], but also because the shoot itself is usually more sensitive to water deficits [27]. In our experiments the root/
shoot ratio of nitrogen-fixing plants increased significantly (Table 2) [3]. Conversely, nitrate-fed plants showed decreased shoot and root growth and therefore the root/shoot ratio remained unchanged. These results contrast with those previously obtained in soybean [ 161and faba bean [24], where nitrate-fed plants had larger and more extensive root systems than nitrogen-fixing plants. On the other hand, increases in leaf thickness (low specific leaf area) of nitrate-fed plants (Table 2) could indicate that leaf expansion was more affected by drought in this type of plant. Table 3 shows that K, Ca and Mg leaf concentrations slightly increased in drought stressed nitrogen-fixing plants. However, nitrate-fed plants showed a very important increase of leaf K concentration whereas Ca and Mg remained almost unchanged. Increased leaf K concentration after drought both in nitrate-fed and nitrogen-fixing plants may contribute to osmotic adjustment [3]. The higher absolute values of K in nitrate-fed plants could be due to the extra potassium available in the nutrient solution as a consequence of the form of nitrogen supplied in nitrate-fed
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Table 4 Turgid weight/ dry weight ratio of alfalfa plants subjected to different nitrogen and water treatments Treatments
Turgid weight/dry weight (g*g_‘)
Science 107 (1995) 159-165
Acknowledgements This work was financed by the Asociacion de Amigos de la Universidad de Navarra and IEISA. References
Nitrogen-fixing plants Control RMS RSS
5.749 f 0.08a 5.335 f 0.13b 5.265 zt 0.12b
Nitrate-fed plants Control RMS RSS
6.546 zt 0.15~ 5.988 f 0.09a 5.950 f O.lOa
Nitrogen treatments: N,, NOs; data for water treatments obtained in rewatered leaves. Otherwise, as for Table 2.
plants. Nitrogen-fixing plants presented higher leaf N concentrations than nitrate-fed plants with a significant decline with severe stress, possibly due to decreased nitrogen metabolism [ 1,3,5]. However, the N concentration of stems and roots tended to increase in all plants suffering drought. In relation to phosphorus concentration, nitrogenfixing plants had significantly more P than nitratefed ones. The turgid weight/dry weight ratio (Table 4) has been linked to the degree of osmotic adjustment in some species [34,30,32]. Our results show higher turgid weight/dry weight in nitrate-fed than in nitrogen-fixing plants, suggesting that nitrate-fed plants had greater capacity for osmotic adjustment ~321. In summary, nitrogen-fixing plants increased root/shoot ratio absorbing more water from soil, and hence maintaining thinner leaves and higher chlorophyll content than did nitrate-fed plants. Photosynthetic rate was higher in nitrogen-fixing than in nitrate-fed plants for same RWC. Under well watered conditions nitrate-fed plants had higher total leaf area but under drought growth resulted more affected than in nitrogen-fixing plants. In addition, nitrate-fed plants showed strong stomata1 closure and developed thicker leaves. Results suggest that nitrogen-fixing alfalfa plants are more drought tolerant than nitrate-fed ones.
111J. Aguirreolea and M. Sanchez-Diax, CO, evolution by nodulated roots in Medicago sativa L. under water stress. J. Plant Physiol., 64 (1989) 476-480. 121SE. Allen, H.M. Grimmshaw, J.A. Parkinson, C. Quarmby and J.P. Roberts, Chemical Analysis, in: S.B. Chapman (Ed.), Methods in Plant Ecology. Blackwell Scientific Publishers, 1976, pp. 41 I-466. I31 M.C. Antolin and M. Sanchez-Diaz, Photosynthetic nutrient use efficiency, nodule activity and solute accumulation in drought stressed alfalfa plants. Photosynthetica, 27 (1992) 595-604. 141 M.C. Antolin and M. Sanchez-D@ Effects of temporary droughts in photosynthesis of alfalfa plants. J. Exp. Bot., 44 (1993) 1341-1349. 151 P.M. Aparicio-Tejo, M. Sanchez-D& and J.I. Pena, Nitrogen fixation, stomata1 response and transpiration in Medicago sativa, Trifolium repens and T. subterraneum under water stress and recovery. Physiol. Plant., 48 (1980) l-4. 161 P.R. Carter and C.C. Sheaffer, Alfalfa response to soil water deficits. III. Nodulation and N, fixation. Crop Sci., 23 (1983) 985-990. t71 M. Engin and J.I. Sprent, Effects of water stress on growth and nitrogen fixing activity of Trifolium repens. New Phytol., 72 (1973) 117-126. 181 H.J. Evans, Symbiotic nitrogen fixation in legume nodules, in: T.C. Moore (Ed.), Research Experiences in Plant Physiology, Berlin: Springer-Verlag, pp. 417-433. 191 G.A. Finn and W.A. Brun, Water stress effects on CO, assimilation, photosynthate partitioning, stomata] resistance and nodule activity in soybean. Crop Sci., 20 (1980) 43 1-434. 1101 J. Ghashghaie and B. Saugier, Effects of nitrogen deficiency on leaf photosynthetic response of tall festue to water deficit. Plant Cell Environ., I2 (1989) 261-271. [111 S. Gummuluru, S.L.A. Hobbs and S. Jana, Physiological responses of drought tolerant and drought susceptible durum wheat genotypes. Photosynthetica, 23 (1989) 479-485. 1121 J.E. Henson, CR. Jensen and NC. Turner, Leaf gas exchange and water relations of lupins and wheat. III. Abscisic acid and drought-induced stomata1 closure. Aust. J. Plant Physiol., 16 (1989) 429-442. 1131 W.M. Kaiser, Effects of water deficit on photosynthetic capacity. Physiol. Plant., 71 (1987) 142-149. [I41 W.M. Kaiser, Methods for studying the mechanism of water stress effects on photosynthesis, in: J.D.Tenhunen, F.M. Catarino, O.L. Lange and W.C. Oechel (Eds.),
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Plant Response to Stress. NATO AS1 Series, Vol. G15, Berlin: Springer-Verlag, 1987, pp. 77-93. (151 P.J. Kramer, Water Relations of Plants, London: Academic Press, 1983. (161 M.I. Minguez and F. Sau, Responses of nitrate-fed and nitrogen-fixing soybean to progressive water stress. J. Exp. Bot., 40 (1989) 497-502. 1171 J.A. Morgan, The effects of N nutrition on the water relations and gas exchange characteristics of wheat (Triticum aestivum L.). Plant Physiol., 80 (1986) 52-58. [I81 DC. Nielsen and A.D. Halvorson, Nitrogen fertility influence on water stress and yield of winter wheat. Agron. J., 83 (1991) 1065-1070. [I91 B.W. Pennypacker, K.T. Leath, W.L. Stout and R.R. Hill Jr., Technique for simulating field drought stress in the greenhouse. Agron. J., 82 (1990) 951-957. (20) SW. Ritchie, H.T. Inguyen and A.S. Holaday, Leaf water content and gas-exchange parameters of two wheat genotypes differing in drought resistance. Crop Sci., 30 (1990) 105-111. [21] R.A. Sanchez, A.J. Hall, N. Trapani and R.C. de Hunau, Effects of water stress on the chlorophyll content, nitrogen level and photosynthesis of leaves of two maize genotypes. Photosynth. Res., 4 (1983) 35-47. [22] M. Sanchez-Diaz, M. Pardo, M. Antolin, J. Peia and J. Aguirreolea, Effect of water stress on photosynthetic activity in the Medicago-Rhizobium-Glomus symbiosis. Plant Sci., 71 (1990) 215-221. [23] M. Sanchez-Diaz and P.J. Kramer, Behavior of corn and sorghum under water stress and during recovery. Plant Physiol., 48 (1971) 613-616. 1241 F. Sau and MI. Minguez, Response to water stress and recovery of nitrate-fed and nitrogen-fixing faba bean. J. Exp. Bot., 41 (1990) 1207-1211. [25] P.F. Scholander, H.T. Hammel, E.D. Badstreet and E.A. Hemmingsen, Sap pressure in vascular plants. Science, 148 (1965) 339-346. [26] Z. Se&k, J. Catsky and P. Jarvis, Plant Photosynthetic Production. Manual of Pethods. The Hague: Junk Publishers, 1971.
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1271 R.E. Sharp, W.K. Silk and T.C. Hsiao, Growth of the maize primary root at low water potentials. I. Spatial distribution of expansive growth. Plant Physiol., 87 (1988) 50-57. P-Y T.R. Sinclair and M.M. Ludlow, Who taught plant thermodynamics? The unfulfilled potential of plant water potential. Aust. J. Plant Physiol., 12 (1985) 213-217. 1291 R.J. Sloane, R.P. Patterson and J.E. Carter Jr.. Field drought tolerance of a soybean plant introduction. Crop Sci., 30 (1990) 118-123. 1301 M.A. Sobrado and N.C. Turner, Comparison of the water relations characteristics of Helianthus annuus and Helianthus petiolaris when subjected to water deficits. Oecologia 58 (1983) 309-313. 1311 J.I. Sprent, Water deficits and nitrogen fixing root nodules, in: T.R. Kozlowski (ed.), Water Deficits and Plant Growth, Vol. IV, New York: Academic Press, 1976, pp. 291-315. 1321 N.C. Turner, W.R. Stern and P. Evans,, Water relations and osmotic adjustment of leaves and roots of lupins in response to water deficits. Crop Sci., 27 (1987) 977-983. 1331 J. Wery, Contribution a I’itude de la nutrition azotee dune Legumineuse fourragere (Medicago sativa L.) et de Legumineuses a graines. These Docteur-Ingtnieur. E.N. S.A., Montpellier, 1983. 1341 J.R. Wilson, M.M. Ludlow, M.J. Fisher and E.-D. Schulze, Adaptation to water stress of the leaf water relations of four tropical forage legumes. Aust. J. Plant Physiol., 7 (1980) 207-220. 1351 A.N. Yousef and J.I. Sprent, Effects of NaCl on growth, nitrogen incorporation and chemical composition of inoculated and NH4N0, fertilized Vicia faba (L.) plants. J. Exp. Bot., 34 (1983) 941-950. 1361 J. Zhang, V. Schurr and W.J. Davies,. Control of stomata] behaviour by abscisic acid which apparently originates in the roots. J. Exp. Bot., 38 (1987) I 174-I I8 1.
s
CiENCE
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F:LSEVIER
Effects of temporary drought on nitrate-fed and nitrogen-fixing alfalfa plants M.C. Antolin*, J. Yoller, M. Sinchez-Diaz Departamento de Fisiologia Vegetal, Universidnd de Navarra. Irunlarrea s/n. E-31008 Pamplona, Spain
Received 22 November 1994;revision received 6 March 1995;accepted 6 March 1995
Abstract The effect of temporary drought on growth, carbon exchange and solute accumulation has been examined in alfalfa plants dependent on either N, or nitrate. Plants were subjected to cyclical moderate or severe drought (drought/recovery). Growth parameters, photosynthetic rate (P,), leaf conductance to water vapour (g,), chlorophyll content, and solute accumulation were determined. Growth decreased markedly under water deficit, but no significant differences between either groups of plants were found. Nitrogen-fixing plants developed higher root/shoot ratios maintaining larger leaves with increased specific leaf area and greater chlorophyll content than nitrate-fed ones. Leaf conductance and net photosynthetic rate declined simultaneously with the drought treatments in both groups of plants; however, Nr fling plants retained higher Pn and g, values than nitrate-fed plants at lower RWC. Upon rewatering, a considerable stomata1 closure remained in nitrate-fed plants. Drought treatment induced an increase in solute concentrations, mainly potassium, especially important in nitrate-fed plants. The interactions between the type of N nutrition and drought tolerance in alfalfa plants during temporary drought are discussed. Keywortis:
Alfalfa; Drought; Medicago
sativa cv.
Aragon; Nitrogen fixation; Nitrogen nutrition; Photosynthesis
1. Introduction
Alfalfa N nutrition is ensured both by atmospheric nitrogen fixation and mineral nitrogen assimilation, but the importance of each process on total plant production is largely influenced by environmental factors such as salinity [35] and water stress [6,33]. Several authors have addressed the inhibitory * Corresponding author, Tel.: (+34-48) 105600;Fax: (+3448) 105649. 0168-9452/95/$09.50 0 1995 SSDI 0168-9452(95)04108-7
effect of water stress on root nodule activity in legumes [7,9,31], including alfalfa [1,5]. However, some of these studies described interactions between water stress and N2 fixation without considering the effect of drought on plants depending on inorganic nitrogen. The particular way in which water stress is imposed might be of special importance in understanding the field response to drought and also in evaluating the plant’s capacity to acclimate to stress [14]. For short-term experiments withholding water is the most common method,
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but to simulate more realistic responses to drought, sustained or cyclic water stress is needed [19]. Previous reports showed that nodulated alfalfa plants responded to temporary drought by osmotic adjustment and enhanced drought tolerance [3]. The objective of this research was to examine growth, photosynthesis and solute accumulation in alfalfa plants grown under controlled environmental conditions in response to temporary drought, and relying on either nitrate or nitrogen fixation. 2. Materials and methods 2.1. Biological material and growth conditions Alfalfa (Medicago sativa cv. Aragon) seeds were surface sterilized and germinated on wet filter paper in Petri dishes. Seedlings were planted in 25 x 10 cm pots containing perlite (6 plants/pot). One set of plants (N2 plants) was inoculated at planting with Rhizobium meiiloti strain 102 F51 (Nitragin Co.) maintained on yeast extract manitol agar. This set received a N-free nutrient solution [8]. The other set (NO3 plants) received the same nutrient solution supplemented with 16 mM KNO,. Plants were grown in a greenhouse at 25/l 5°C 50/70% RH (day/night), and illuminated during 14h with natural daylight supplemented with fluorescent lamps (Sylvania DECOR 183, Germany), providing a Professional-58W, minimum photosynthetic photon flux density of around 300-400 pmol photons. rnm2.s-l. 2.2. Experimental design When 45 days old, plants were subjected to drought by withholding irrigation in a cyclic way as described previously [4]. Two degrees of stress (moderate, MS and severe, SS) were imposed. The moderate stress level was defined as the degree of stress that occurs when the plants show visible signs of wilting in the afternoon and recover turgor during the night (approximately 4 days/ cycle). The severe stress level was defined as the degree of stress that occurs when the plants show visible signs of wilting during the afternoon, and do not recover during the night (approximately 8 days/cycle). Predawn leaf water potential (q,,,) was measured before irrigation to determine the
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actual level of stress. Plants were subjected to three cycles of stress and recovery. Recovery was considered completed when predawn \k, of recovered plants reached the prestressed levels (RMS, RSS). Rewatering was performed with nutrient solution or deionized water in order to supply the different water treatments with the same amount of nutrients during drought. Plants were transferred prior the third cycle of stress to a controlled environment chamber with a day/night regime of 25/15”C and 60180% RH. A photosynthetic photon flux density of 310 prnol photons.m-2.s-’ (400-700 nm) on the upper leaves was provided by a fluorescent tube (Sylvania F 48T12 CW-WHO) and incandescent bulb mixture for a 15-h photoperiod. Measurements were made at the end of the third cycle and repeated 24h after rewatering. Midday leaf q,,, and photosynthesis were determined at the same time. Means f S.E. (n = 7-9) were calculated, and their differences tested for significance by using a LSD technique with the Student’s t-test. 2.3. Plant determinations Leaf water potential (q’,) was measured with a pressure chamber [25] and the relative water content (RWC) was calculated as follows: RWC = (fresh wt. - dry wt.)/(turgid wt. - dry wt.) x 100. CO2 exchange measurements were made on the youngest fully mature leaf using an infrared gas analyzer (Analytical Development Co., Hoddesdon, UK, LCA-2 model) operated in the differential mode as described by Sanchez-Diaz et al. [22]. Air of known COz concentration (400 prnol . mol-‘) and 60% RH was supplied at a constant flow (300 ml.min-‘) into the leaf chamber. Photosynthetic photon flux density was 1000 pmol photons.m-2.s- ’ during measurements. Chlorophyll content was determined on each leaf used in gas exchange measurements according to SCstak et al. [26] in acetone (80% v/v) extracts of fresh leaves. Plants were harvested for growth determinations and chemical analysis after rewatering. Leaf area was measured by using an automatic leaf area meter (Li-COR, LI-3000 model) and plant dry weight was determined after drying at 70°C for 2
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days. The total nitrogen was measured by Kjeldahl and plant phosphorus content was determined by the method of Allen et al. [2]. Calcium, potassium and magnesium were determined in acid solution of ashed samples obtained in an oven at 500°C. Potassium content was measured with a flame photometer, and calcium and magnesium were estimated by the EDTA titration method [2]. 3. Results and discussion Nitrate-fed alfalfa plants always showed higher RWC values than nitrogen-fixing ones for similar leaf qk, (Fig. 1). As reported by several authors in corn and sorghum [23], wheat [20] and in soybean [29], such results suggest that nitrate-fed leaves could be more resistant to dehydration than nitrogen-fixing leaves. However, differences in RWC values could also be a consequence of either different initial cell sap osmolarity, cell wall elasticity [ 131 or osmoregulation 1171,particularly when it is considered that nitrogen in nitrate-fed plants was supplied as KNOs. The rest of the results are expressed as a function of RWC since, according to some authors 113,281, a better relationship with growth and metabolism exists when expressing water status by using RWC rather than * W’ Net photosynthesis (P,) (Fig. 2A) and leaf conductance to water vapour (g,) (Fig. 3A) behaved similarly in both groups of plants during drought;
01 90
80
70
RWC
60
50
(%)
Fig. 2. Net photosynthetic rate (P,) in plants subjected to different nitrogen treatments (N,, 0; NO,, m during drought (A) and upon rewatering (B) as a function of relative water content (RWC). Recoveries are represented at the same RWC as in stressed plants to facilitate the comparison. Measurements were made in an external CO* concentration of 400 pmol . mol-‘, a photon flux density prnol. * and at leaf temof Otherwise, as Fig. I
0.3
-’
I _
;:;; 90g
8o: 70
2
-
60 50 2n
(Tyq - 1
-2
Leaf water potential
-4
-3
(MPa)
o.o+ 90
Fig. 1. Leaf relative water content (RWC) in plants subjected to different nitrogen treatments (N2, 0, NOs, 4 as a function of leaf water potential. Recoveries are represented by open (N$ and solid (NO,) circles. Each point is the mean of seven to nine plants. The bars indicate standard error (S.E.) of the mean. Standard errors lower than 10% were not represented.
,
,
80
70
RWC
, 60
50
40
(96)
Fig. 3. Total conductance to water vapour (g,J in plants subjected to different nitrogen treatments (N2, 0; NOs, m during drought (A) and upon rewatering (B) as a function of relative water content (RWC). Otherwise, as for Fig. 2.
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Table 1 Chlorophyll content (a and b) and chlorophyll a/b ratio of alfalfa plants subjected to different nitrogen and drought treatments Treatments
Chlorophyll (mgeg-’ dry wt.) a
b
ah
Nitrogen-fixing plants Control MS ss
8.51 l 0.73a 8.20 f 1.14a 5.18 f 0.75bc
2.42 f 0.22a 2.49 f 0.32ad 1.45 f 0.26bc
3.55 f 0.03a 3.63 * 0.09a 3.85 f 0.17ab
Nitrate-fed plants Control MS ss
9.17 l 0.85a 6.74 f 0.83b 3.58 zt 0.35~
2.63 f 0.22a 1.94 zt 0.25bd 0.93 l O.lck
3.46 f 0.05a 3.47 f 0.04a 3.97 zt 0.13b
Nitrogen treatments: Nz. NO?; drought treatments: control, moderate stress (MS), severe stress (SS). Recoveries are not significant (data not shown).
however, upon rewatering moderately stressed nitrogen-fixing plants recovered control g, levels whereas nitrate-fed plants did not (Fig. 3B). Ghashghaie and Saugier [lo] showed similar g, response to drought in high and low-N fertilized tall festue plants. In nitrogen-fixing plants, the partial recovery of P, (Fig. 2B), despite the almost complete recovery of g,, would implicate nonstomata1 effects on P, under cyclic drought conditions [4]. However, in nitrate-fed plants no recovery of & (Fig. 3B) and P, (Fig. 2B) were
observed, possibly indicating the involvement of other factors, such abscisic acid [ 12,361. Therefore, during drought nitrogen-fixing plants seemed to be less sensitive to temporary drought than nitratefed ones, retaining higher rates of P, at similar RWC values than nitrate-fed plants. Plants depending on nitrogen fixation maintained their chlorophyll contents at moderate water stress, whereas a significant decrease was found under severe stress (Table 1). Nitrate-fed plants showed a considerable reduction in chlorophyll
Table 2 Total, shoot and root dry weights, root-shoot dry weight ratio, leaf area and specific leaf area (SLA) of nitrogen-fixing and nitrate-fed Medicago sativa plants Nitrate-fed plants
Nitrogen-fixing plants Control Plant weight (g) Shoot weight (g) Root weight (g) Root/shoot (se g-‘) Leaf area (cm*) SLA (dm*. g-t)
2.07 l O.O8*a 0.92 * 0.02a 0.93 * O.O5*a 0.78 f O.l2*a 278.9 f 12.0a 2.82 f 0.08a
RMS 1.63 f 0.04b 0.67 * O.llb 0.92 f 0.02a 1.31 +z 0.09b 226.3 * 9.7b 3.17 * 0.20a
Control
RSS 1.60 f 0.65 + 0.89 f 1.32 f
0.26b 0.02b 0.14a 0.01b
202.3 f 13.0b 3.64 zt 0.31b
2.74 + 1.41 zt 1.41 l 1.05 f
RMS 0.14c 0.08~ 0.12b 0.07c
392.6 f 1.6c 2.96 * 0.02a
1.54 * 0.06b 0.79 f O.Olb 0.88 * 0.07a 1.26 zt 0.06bc 221.4 zk 6.4b 2.26 l 0.09~
RSS 1.53 * 0.79 l 0.75 * 1.29 +
0.07b 0.02b 0.04a 0.03bc
209.5 f 13.9b 2.19 zt 0.09~
Total, shoot and root dry weights, root-shoot dry weight ratio, leaf area and specific leaf area (SLA) of Medicago sat&a dependent on either N2 (nitrogen-fixing’plants) or 16 mM KNO, (nitrate-fed plants) under well watered conditions (control) and subjected to different drought treatmentsidata obtained upon rewatering) (RMS, moderate stress; RSS, severe stress). Values are means f S.E. (n = 7-9). Comparison between means were made with the Student’s t-test. Within each line, means followed by the same letter are not significantly different (P > 0.05). *Nodules are not included.
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Table 3 Leaf, stem and root nitrogen, phosphorus and cation concentrations in plants subjected to different nitrogen and water treatments Organ
Leaf
N treatment NZ
NO3
Stem
N2
NO3
Root
NZ
NO3
Water treatment Control RMS RSS Control RMS RSS Control RMS RSS Control RMS RSS Control RMS RSS Control RMS RSS
Concentration (mg-g-’ dry wt.) N
P
K
Ca
Mg
33.5 f 1.6a 30.6 f 0.8a 24.7 f l.lbc 26.8 f 0.5b 25.6 f 0.9bc 22.4 zt 1.6c 16.3 f 0.5a 16.5 A 0.6a 17.8 f 0.9a 12.2 f 0.7b 16.5 * 0.5a 17.4 * O.la 14.7 f 0.9a 14.7 l 1.5a 18.1 f l.lb 11.2 f 0.6c 16.2 f 0.7ab 23.8 f 0.4d
4.52 + 0.2a 4.19 f 0.2a 4.25 f 0.2a 2.38 + 0.2b 2.29 + O.lb 1.84 zt 0.5b 3.85 f 0.8a 3.63 * 0.9a 4.07 f 0.6a 2.19 l O.lb 2.35 f 0.3b 1.61 f 0.6b 3.96 f 0.3a 3.62 f 0.5ac 3.97 f 0.2a 1.99 f 0.6b 2.41 zt 0.6bc 2.14 f 0.6bc
34.7 * 0.9a 37.6 zt 0.8b 38.9 * 0.8b 60.4 zt 0.9~ 80.2 * 1.5d 91.1 * 3.le 30.2 f 0.8a 30.0 f 0.4a 28.7 f 0.2a 46.9 f 0.4b 53.3 f 0.4c 60.8 f 0.4d 13.3 * O.la 9.5 f 0.7b 8.2 zt 0.7b 14.6 + 1.9ac 16.3 + 0.4d 17.9 f 0.4d
13.1 f 0.2a 14.1 zt O.lb 18.7 f 0.1~ 8.9 f O.ld 7.4 f 0.2e 7.7 f O.le 5.2 f 0.2a 4.6 f 0.2b 3.4 f O.lc 3.8 f 0.1~ 3.4 f O.lc 2.9 f O.ld 1.9 l O.la 4.0 f O.lb 2.5 f 0.1~ 6.1 zt 0.2d 7.6 f O.le 10.3 f O.lf
6.6 f O.la 7.9 A O.Ob 8.7 f 0.1~ 4.4 f o.Od 4.5 * 0.2d 4.2 zt O.Od 3.8 f 0.2a 3.8 f O.la 3.3 * 0.2b 2.0 f o.Oc 1.9 f O.Ic 2.1 f O.lc 2.6 f O.la 2.3 f 0.3a 2.5 f O.la 2.8 f O.lab 3.1 f O.lb 3.0 f O.lb
Nitrogen treatments: Nz, NO,; data for water treatments data obtained upon rewatering. Otherwise as for Table 1
content at any drought level. The chlorophyll a/b ratio increased significantly only in severely stressed plants for both N treatments. These results suggest that nitrogen-fixing plants maintained a better leaf structure than nitrate-fed ones during dehydration. No decrease in chlorophyll content was also reported in drought tolerant genotypes of maize [21] and wheat [l 11. Growth parameters were only determined in rewatered plants because plant material was kept until the end of the third drought/recovery cycle for measurements. Under well watered conditions, nitrate-fed plants produced more shoot and root dry weights and leaf area than nitrogen-fixing plants; however after drought a marked decrease in these parameters was found (Table 2). Nitrogenfixing plants showed reduced shoot growth without changes in roots. Results agree with the fact that shoot growth is generally more affected by water stress than root growth, not only because the transpiring parts of the plant usually develop greater and longer water deficits [15,1X], but also because the shoot itself is usually more sensitive to water deficits [27]. In our experiments the root/
shoot ratio of nitrogen-fixing plants increased significantly (Table 2) [3]. Conversely, nitrate-fed plants showed decreased shoot and root growth and therefore the root/shoot ratio remained unchanged. These results contrast with those previously obtained in soybean [ 161and faba bean [24], where nitrate-fed plants had larger and more extensive root systems than nitrogen-fixing plants. On the other hand, increases in leaf thickness (low specific leaf area) of nitrate-fed plants (Table 2) could indicate that leaf expansion was more affected by drought in this type of plant. Table 3 shows that K, Ca and Mg leaf concentrations slightly increased in drought stressed nitrogen-fixing plants. However, nitrate-fed plants showed a very important increase of leaf K concentration whereas Ca and Mg remained almost unchanged. Increased leaf K concentration after drought both in nitrate-fed and nitrogen-fixing plants may contribute to osmotic adjustment [3]. The higher absolute values of K in nitrate-fed plants could be due to the extra potassium available in the nutrient solution as a consequence of the form of nitrogen supplied in nitrate-fed
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Table 4 Turgid weight/ dry weight ratio of alfalfa plants subjected to different nitrogen and water treatments Treatments
Turgid weight/dry weight (g*g_‘)
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Acknowledgements This work was financed by the Asociacion de Amigos de la Universidad de Navarra and IEISA. References
Nitrogen-fixing plants Control RMS RSS
5.749 f 0.08a 5.335 f 0.13b 5.265 zt 0.12b
Nitrate-fed plants Control RMS RSS
6.546 zt 0.15~ 5.988 f 0.09a 5.950 f O.lOa
Nitrogen treatments: N,, NOs; data for water treatments obtained in rewatered leaves. Otherwise, as for Table 2.
plants. Nitrogen-fixing plants presented higher leaf N concentrations than nitrate-fed plants with a significant decline with severe stress, possibly due to decreased nitrogen metabolism [ 1,3,5]. However, the N concentration of stems and roots tended to increase in all plants suffering drought. In relation to phosphorus concentration, nitrogenfixing plants had significantly more P than nitratefed ones. The turgid weight/dry weight ratio (Table 4) has been linked to the degree of osmotic adjustment in some species [34,30,32]. Our results show higher turgid weight/dry weight in nitrate-fed than in nitrogen-fixing plants, suggesting that nitrate-fed plants had greater capacity for osmotic adjustment ~321. In summary, nitrogen-fixing plants increased root/shoot ratio absorbing more water from soil, and hence maintaining thinner leaves and higher chlorophyll content than did nitrate-fed plants. Photosynthetic rate was higher in nitrogen-fixing than in nitrate-fed plants for same RWC. Under well watered conditions nitrate-fed plants had higher total leaf area but under drought growth resulted more affected than in nitrogen-fixing plants. In addition, nitrate-fed plants showed strong stomata1 closure and developed thicker leaves. Results suggest that nitrogen-fixing alfalfa plants are more drought tolerant than nitrate-fed ones.
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