Effects of controlled irrigation on water potential, nitrogen uptake and biomass production in Dalbergia sissoo seedlings

Environmental and Experimental Botany 55 (2006) 209–219

Effects of controlled irrigation on water potential, nitrogen uptake and biomass production in Dalbergia sissoo seedlings Bilas Singh1 , Genda Singh∗ Division of Forest Ecology and Desert Development, Arid Forest Research Institute, New Pali Road, Jodhpur 342005, India Accepted 3 November 2004

Abstract Biomass production, pattern of nodulation, nutrient uptake, net photosynthetic rate (Pn ), leaf temperature (Tleaf ), leaf nitrate reductase (NR) activity and free proline of Dalbergia sissoo seedlings planted in containers with 120 kg soil were studied under different water stress levels to assess the productive potential of the species in dry areas. Seedlings were irrigated at 20 mm (W1 ), 14 mm (W2 ), 10 mm (W3 ), 8 mm (W4 ) throughout the experimental period to maintain the respective treatment up to the lowest soil water content of 7.43%, 5.64%, 4.30% and 3.23%, respectively. There was a treatment (W5 ) in which seedling were irrigated once to −0.03 MPa and left without re-irrigation. Decreased irrigation level resulted in lowering of leaf water potential (LWP), net photosynthetic rate (Pn ), total number of root nodules and nodule dry mass and nitrogen uptake in the seedling. Pn , leaf nitrate reductase (NR) activity and seedling biomass were highest in W1 indicating a positive relations of NR activity with CO2 assimilation and biomass production. The decrease in Pn , leaf NR activity and LWP was sharp at W3 onwards. Monthly changes in the values of Pn , Tleaf and NR activity indicate environmental effect on these physiological variables. Proline was detected only in the seedlings of W3 , W4 and W5 treatments after February and was highest in the seedlings of W5 treatment. The study suggests that severe water deficit adversely affect physiological and biochemical processes that resulted in reduced growth, nutrient uptake and biomass productivity in D. sissoo seedlings. Re-irrigation above W3 level is recommended for this species. © 2004 Published by Elsevier B.V. Keywords: Dalbergia sissoo; Leaf water potential; Nitrate reductase; Proline; Soil water stress

1. Introduction

∗

Corresponding author. Tel.: +91 291 742550. E-mail addresses: [email protected] (B. Singh), [email protected], g [email protected] (G. Singh). 1 Division of Forest Resource Management and Economics, Jodhpur, India. 0098-8472/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.envexpbot.2004.11.001

Leguminous species depend on the atmospheric nitrogen to fulfill their nitrogen requirement. They are used for soil N enrichment under wasteland development programmes (Pokhriyal et al., 1997; Singh et al., 2001). However, the process of symbiotic N2 -fixation in root and assimilation of nitrogen in leaves or root

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of higher plants requires both energy and carbohydrates produced during photosynthetic carbon assimilation (Niu et al., 2003). Therefore, carbon partitioning is coordinated by a sophisticated regulatory system to meet the growing demand of tree seedlings for both carbohydrates and the nitrogen (Huber et al., 1996). In this context, nitrogen metabolism appears to be tightly coupled with adequate supply of nitrogen and water. The availability of water seems to be the most important factor limiting plant growth and productivity in dry areas. A water deficit can slow seedling development and decrease biomass by reducing the carbohydrate supply for optimum growth. During water deficit, the decreases in water availability for transport associated processes lead to changes in the concentration of many metabolites, followed by disturbances in the carbohydrates and amino acid metabolism (Osbert et al., 1995). Increase in the synthesis of compatible solutes like amino acids (i.e., proline), sugars and sugar alcohols and Gly-betaine (Girousse et al., 1996) during water deficits are the best examples. Acclimation to low soil water availability allows essential reactions of the primary metabolism to continue and enables to plant to tolerate water deficits. The acquisition of nitrogen and phosphorus are generally affected under water deficit either due to drought or withdrawal of irrigation affecting overall productivity. Drought induced N deficiency was found to limit recovery of photosynthesis in prairie grasses (Heckathorn et al., 1997). Assessment of biochemical changes (Good and Zapalachinsky, 1994) may be an important tool to evaluate the performance of tree seedlings for management and productivity enhancement in dry areas. Dalbergia sissoo is a multipurpose tree of Indian subcontinent and valued for its fuel wood and timber uses and soil ameliorative characteristics (Rajbanshi and Gupta, 1985) through nitrogen fixation (Pokhriyal et al., 1997). Recently, a large-scale mortality in D. sissoo has been experienced in response to drought. It needs proper investigation about physiology of this species in relation to water stress for better management strategy and to increase the productivity. The study relating biochemical changes, particularly nitrogen metabolism under soil water stress could be one of the important aspects to assess the productive potential of D. sissoo in dry region. Therefore, the specific objective of this study was to investigate the influence of soil water deficit on bio-

chemical changes in D. sissoo seedlings in relation to nitrogen uptake and accumulation, CO2 assimilation and biomass production. Monitoring water stress tolerance and productivity of D. sissoo at varying levels of irrigation was the ultimate objective.

2. Materials and methods 2.1. Site characteristics Experiment was carried out at the experimental farm of Arid Forest Research Institute, Jodhpur (72.03 E, 26.45 N). The three prominent seasons in the year are summer, monsoon and winter. The soil of the container was loamy sand (coarse loamy, mixed, hyperthermic family of Typic camborthides according to US soil taxonomy) with water holding capacity of 10.67% (w/w) at −0.03 MPa. Soil water storage in 0–50 cm layer varies from 82 mm at −0.03 MPa to 24 mm at −1.5 MPa. Soil had pH 8.32 and EC 0.52 dS m−1 in 1:2 soil water suspension. Soil available nitrogen was 12.56 mg kg−1 , phosphorous was 10.01 mg kg−1 P2 O5 and potash was 106 mg kg−1 K. Cumulative rainfall was 237 mm and pan evaporation was 2109 mm during August 1998 to May 1999 showing high water deficit at the experimental site. However, only 9 mm of rain in two events (i.e., 6 mm on 27 January and 3 mm on 16 February 1999) was received during the treatment period of October 1998–May 1999. Environmental variables varied within months. Mean monthly minimum and maximum air temperatures were 10.0 and 25.0 ◦ C in January and increased gradually to 27.9 and 40.7 ◦ C, respectively, in May. Vapor pressure deficit (VPD) increased from 1286 Pa (389 in morning to 2183 Pa at midday) in January to 5115 Pa (2943 Pa in morning to 7753 Pa at midday) in May (Fig. 1). Potential evapotranspiration fluctuated between 2.47 mm day−1 in December to 8.54 mm day−1 in May (Rao et al., 1971). Photosynthetically active radiation (PAR) was highest at midday (13:00 h) and oscillated between 756 ␮mol m−2 s−1 in December 1998 to 1933 ␮mol m−2 s−1 in May 1999. 2.2. Planting material and experimental design Four-month-old single clone seedlings of D. sissoo were planted during first week of August 1998 in gal-

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Fig. 1. Temporal changes in photosynthetically active radiation (PAR) and vapor pressure deficit (VPD).

vanized iron containers of 45 cm diameter and 55 cm depth. One hundred and twenty kilograms of sandy soil was filled up to 50 cm depth of container (i.e., 0.08 m3 ) leaving 5 cm depth for irrigation. A drainage hole was present in the container. The experiment had five treatments comprising different irrigation levels depending on soil water content at different pressures determined with a pressure plate apparatus (soil water retention curve) (Table 1). The treatments were initiated in the second week of October 1998 after proper establishment of the seedlings, which attained average height and collar diameter of 52 and 0.4 cm, respectively. At the time of treatment initiation, soil of all the containers was fully saturated at field capacity by addition of water. Drainage of excess water was allowed till the soil water ceased to drain down. Different irrigation levels were: 20 mm (3.2 l), 14 mm (2.2 l), 10 mm (1.6 l) and 8 mm (1.3 l) per irrigation throughout the

experimental period and abbreviated as W1 , W2 , W3 and W4 , respectively. There was a treatment (W5 ) in which no further water was added to the containers after saturation at field capacity initially. Soil water content was monitored gravimetrically after oven drying of the soil samples at 105 ◦ C temperature to a constant weight. The seedlings were re-irrigated when the soil water content approached to 7.43% (−0.10 MPa), 5.64% (−0.50 MPa), 4.30% (−1.00 MPa) and 3.23% (−1.50 MPa) in W1 , W2 , W3 and W4 treatments, respectively. Total quantity of water added was 112.0 l in W1 in 35 irrigation events, 63.8 l in W2 in 29 irrigation events, 27.2 l in W3 in 17 irrigation events and 16.9 l in W4 in 13 irrigation events in 210 days. Eight replications were taken in each treatment and experiment was laid in Complete Randomized Block Design. The experiment was terminated in the first week of May 1999 when the seedlings of W5 treatments suffered of

Table 1 Soil water potential, corresponding soil water content and irrigation levels S. no.

(i) (ii) (iii) (iv) (v) (vi). a

Soil water potential (MPa)

0.03 0.05 0.10 0.50 1.00 1.50

Irrigation level (0–50 cm soil layer)a

Soil water Percent

Liter

Millimeter

Millimeter

Liter

10.67 9.88 7.43 5.64 4.30 3.23

13.04 11.90 8.91 6.77 5.16 3.88

82.0 74.8 56.0 42.6 32.4 24.4

(ii)–(iii) = 18.8 (W1 ) (iii)–(iv) = 13.4 (W2 ) (iv)–(v) = 9.88 (W3 ) (v)–(vi) = 8.00 (W4 ) (i) to till death (W5 )

3.2 2.2 1.6 1.3

Irrigation levels were 20, 14, 10 and 8 mm for W1 , W2 , W3 and W4 treatments, respectively.

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permanent wilting i.e., 9 months after planting of the seedlings. 2.3. Measurements Leaf water potential (LWP) was recorded monthly in triplicate for each treatment. LWP was measured on leaf discs in a leaf chamber (L-52; Wescor, Logan, UT, USA) connected to a Dew Point Micro-voltmeter (Wescor HR-33T) between 05:00 and 07:00 h from January 1999 to May 1999 before the re-irrigation of the seedlings in each treatment. Leaf disc of 0.5 cm diameter was punched out from the attached leaves (without leaf abrasion) and was transferred into a leaf chamber and after 15 min of equilibration the water potential was determined (Campbell et al., 1973). The discs were collected subsequently for each measurement and at the time of observation recording. Net photosynthetic rate (Pn ) and leaf temperature (Tleaf ) was recorded with open system of portable CO2 Gas Analyzer, Model CI-301 (CT-301 PS0), CID Inc., Vancouver, USA. Pn observations were recorded in triplicate fortnightly at 10:00 h and averaged to provide a mean value for each month. All these observations were recorded on leaves of middle canopy of the seedlings. Self shading within the cuvette was minimized by ensuring that the leaves did not overlap, particularly in the seedlings of W3 , W4 and W5 treatments, the leaves of which were comparatively smaller than that in W1 and W2 treatments. Nitrate reductase (NR) activity was measured in vitro (Wray and Filner, 1970). Two hundred and fifty milligrams of fresh leaves were cut into small pieces and homogenized in 5 ml of extraction media containing 0.1 M phosphate buffer of pH 7.5 and 1 mM cysteine. The reaction mixture containing 0.5 ml of enzyme extract, 0.01 ml of KNO3 (0.1 M), 0.5 ml of phosphate buffer (0.1 M, pH 7.5), 0.1 ml of NADH (1 mM) and 0.1 ml of double distilled water was incubated for 45 min and at 30 ◦ C. The reaction was terminated by adding of 1 ml of sulphanilamide (1% in 3N HCl) and 1 ml of 0.02% NEDD (N-1-naphthylethylene diamine dihydrochloride). The reaction mixture was centrifuged to discard proteins and absorbance was recorded at 540 nm against blank. The standard curve was prepared by using 1 ␮g/ml of sodium nitrite (NaNO2 ). Enzyme activity was calculated in terms of nitrite released per gram fresh weight leaf per hour. Free proline was estimated following the procedure of

Bates et al. (1973). Two hundred and fifty milligrams fresh leaf was homogenized in 3% sulphosalicylic acid, centrifuged and supernatant was used for estimation of proline content. One milliliter of aliquot, 2.0 ml of acetic acid and 2.0 ml of ninhydrin reagent (1.25 g of ninhydrin in 30 ml of glacial acetic acid and 12 ml of 6 M orthophosphoric acid) was mixed and the tubes containing the mixture were heated in boiling water bath for 1 h. The tubes were then cooled in ice bath. To this mixture, 4.0 ml of toluene was added and coloured compound was partitioned in separating funnel. Absorbance of pink coloured toluene layer was recorded at 520 nm against blank. Reference curve was prepared by using 0.01 mg/ml of proline. Seedlings were harvested when mortality occurred in W5 treatment (i.e., at 9 months of age after plantation) for estimation of biomass production. Shoot removed from the collar region and fresh weight of stem and leaves were recorded. Roots along with soil mass were taken out from the container by turning the container upside down and then roots were separated carefully and soil particles adhered to the root surface were removed. Root nodules were removed, counted and their fresh weight recorded. Fresh weight of root was recorded immediately after measurement. Dry weights of the stem, leaves and roots were recorded after oven drying of samples for 72 h at 80 ◦ C. Soil samples were collected before planting and after harvesting of the seedling. Soil samples were analyzed following the procedure of Jackson (1973). Soil pH and electrical conductivity (Ec ) were determined in 1:2 soil water ratio. Soil available nitrogen (NH4 -N and NO3 N) and phosphorous were determined after extracting the soil in 2 M KCl and Olson’s extraction solutions, respectively, and plant nitrogen was determined after sulphuric acid digestion using Fiastar autoanalyzer system (Model Enviroflow-5012, Tecator AB, Hoganas, Sweden). Potassium was determined after ammonium acetate extraction (Simard, 1993) using Perkin-Elmer Double Beam Atomic Absorption Spectrophotometer (Model-3110, Huenenberg, Sweden). 2.4. Statistical analysis of data Data were analyzed by one-way ANOVA using biomass, physiological and biochemical parameters as the dependent variables for each month. Further, since the data were repeatedly measured from December

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1998 to May 1999, the variation due to month was analyzed using repeated measure analyses. Pearson correlation was also computed. We used protected LSD comparisons at a threshold P value of 0.05 to test the differences between the treatments. All these analyses were carried out using SPSS statistical package.

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and their dry mass were lowest in the seedlings of W5 treatment. 3.3. Soil available nitrogen and plant nitrogen uptake Availability of NH4 -N and NO3 -N was low in May 1999 compared to the values of 2.17 ± 0.03 (mean ± S.D.) and 10.39 ± 0.18 mg kg−1 , respectively in July 1998 (Table 3). Both these soil nutrients differed significantly (P < 0.05) between the treatments. Available NH4 -N was highest in W1 and decreased with soil water deficit. Availability of NO3 -N was greater in the soil of W3 , W4 and W5 compared to the soils in W1 and W2 treatments. Total nitrogen uptake depended upon the seedling nitrogen concentration and the biomass. Nitrogen accumulation was greater (P < 0.001) in the seedlings of W1 (Table 3) and it decreased as a function of decreased irrigation level. In severely stressed seedlings of W5 treatment, total nitrogen uptake was 0.022 g seedling−1 that is one thirteenth of that in W1 treatment.

3. Results 3.1. Seedling growth and biomass Seedlings of W1 had significantly (P < 0.001) greater height and collar diameter and produced higher biomass compared to the seedlings of other treatments (Table 2). Irrigation level of W3 caused 66% reduction in the dry mass than in the treatment W1 . The seedlings of W5 produced one-tenth biomass compared to the seedlings in W1 treatment. Seedlings of W1 treatment produced highest (P < 0.01) root biomass as compared with the seedlings of other treatments, whereas lowest root biomass was in the seedlings of W5 treatment. Percent biomass allocation to the seedling root of W1 was 25%, which increased (P < 0.05) to 35–50% in the seedlings of W3 , W4 and W5 treatments. Percent leaf biomass reduction was greater than the percent stem biomass reduction.

3.4. Leaf water status, CO2 assimilation and leaf temperature Increasing water stress caused reduction in leaf water potential (LWP). LWP in the seedlings of W1 , W2 , W3 and W4 indicated variations due to month i.e., temporal shift whereas it showed a gradual decrease in the seedling in W5 treatment (Fig. 2). LWP did not differ between the seedlings of W1 and W2 but it was significantly low for the seedlings of W3 , W4 and W5 . Differences in LWP between the seedlings of W1 and W5 were 0.40 MPa in January, 1.16 MPa in March and

3.2. Nodulation and nodule weight Nodule number and total nodule dry mass were highest in the seedlings of W1 treatment. Both these variables decreased with decrease in irrigation level being significantly (P < 0.01) low in the seedlings of W3 and W4 treatments (Table 2). Number of nodules

Table 2 Influence of water stress on growth (cm), total dry biomass (g seedling−1 ), nodule number and nodule dry mass (g seedling−1 ) in 9-month-old planted D. sissoo seedlings Parameter

W1

Height Collar diameter Leaf dry mass Stem dry mass Root dry mass Nodule number Nodule dry mass

97.0 0.96 33.72 40.38 26.52 85 1.18

W2 ± ± ± ± ± ± ±

5.1 a 0.03 a 2.82 a 3.59 a 2.66 a 3.44 a 0.03 a

90.0 0.91 27.35 29.66 18.78 60 0.60

W3 ± ± ± ± ± ± ±

4.4 a 0.02 a 2.85 b 3.36 b 1.92 b 2.27 b 0.02 b

77.0 0.74 9.16 14.32 12.47 13 0.29

W4 ± ± ± ± ± ± ±

3.0 b 0.03 b 0.86 c 1.36 c 1.42 c 1.41 c 0.1 c

71 0.71 4.47 9.29 11.82 7 0.09

P

W5 ± ± ± ± ± ± ±

4.3 b 0.07 b 0.80 d 2.32 d 1.83 d 1.25 d 0.007 d

60.0 0.48 1.43 3.68 5.00 2 0.01

± ± ± ± ± ± ±

4.8 c 0.06 c 0.15 e 0.63 e 0.86 e 0.49 e 0.003 e

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

Irrigation levels of 20 mm (W1 ), 14 mm (W2 ), 10 mm (W3 ), 8 mm (W4 ) throughout the experiment and 82 mm only once in W5 . Values are mean ± S.D. of five replications. Similar alphabet in a row indicates non-significant (P < 0.05).

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Table 3 Influence of water stress on soil available NH4 -N and NO3 -N and nitrogen accumulation in 9-month-old planted D. sissoo seedlings Parameter

W1

W2

W3

W5

P

1.84 ± 0.05 b 6.34 ± 0.11 b

1.73 ± 0.08 c 6.16 ± 0.10 b

<0.01 <0.05

16.87 ± 3.05 d 21.41 ± 1.55 c 32.56 ± 0.59 c

3.95 ± 0.51 e 8.34 ± 0.88 d 9.34 ± 0.11 d

<0.001 <0.001 <0.001

W4

Soil available N (mg kg−1 soil) NH4 -N 2.10 ± 0.13 a NO3 -N 5.41 ± 0.41 a

1.88 ± 0.06 b 5.21 ± 0.26 a

1.93 ± 0.06 b 6.50 ± 0.24 b

Total N content (mg seedling−1 ) Leaf 75.71 ± 3.02 a Stem 77.89 ± 4.43 a Root 121.00 ± 1.64 a

63.82 ± 4.11 b 70.88 ± 7.89 a 93.75 ± 1.80 b

28.68 ± 2.72 c 29.28 ± 1.27 b 36.43 ± 0.54 c

Irrigation levels of 20 mm (W1 ), 14 mm (W2 ), 10 mm (W3 ), 8 mm (W4 ) throughout the experiment and 82 mm only once in W5 . Values are mean ± S.D. of three replications. Soil available NH4 -N and NO3 -N in July 1998 were 2.17 and 10.39 mg kg−1 soil, respectively. Similar alphabet in a row indicates non-significant (P < 0.05).

4.89 MPa in May when soil water content decreased to 2.56% in W5 treatment (−1.96 MPa). LWP was highest during winter whereas it decreased to lowest during May 1999 when leaf drying and leaf abscission occurred in the seedlings of W5 treatment. Net photosynthetic rate (Pn ) was highest for the seedlings of W1 treatment and was related with high LWP and soil water content. Compared to that in W1 , the reduction in Pn was significant (P < 0.05) in the seedlings at W3 , W4 , and W5 (Fig. 3). Net photosynthetic rate increased from January to March and decreased thereafter showing lowest value in May. Leaf temperature (Tleaf ) was lowest in the seedling of W1 and showed an increasing trend with decrease in irrigation level in all the months (Fig. 4). Tleaf was lowest

in January. It increased up to April and decreased in May 1999. 3.5. NR activities and proline accumulation Decrease in irrigation level reduced nitrate reductase (NR) activity (r = 0.62, P < 0.01) (Fig. 3). NR activity was highest (403 ␮mol g−1 fresh weight h−1 ) in the seedlings of W1 treatment in which Pn was highest (Fig. 3). NR activity increased from December to March and dropped to lowest in May through April for the seedlings of all the treatments (Fig. 3). NR activity in May was even belower than the value in December for the seedlings of W3 , W4 and W5 treatments. However, the decrease in NR activity in severely stressed

Fig. 2. Decrease in leaf water potential (05:00–07:00 h) with lowering of irrigation level in D. sissoo seedlings in different months. Error bars are ±S.D. Similar alphabet in same month indicates non-significant (P > 0.05) difference between treatments.

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Fig. 3. Effect of water deficit and environmental factors on net photosynthetic rate (Pn ) and nitrate reductase activity (NR) at irrigation level of 20 mm (W1 ), 14 mm (W2 ), 10 mm (W3 ), 8 mm (W4 ) and 82 mm only once (W5 ). Error bars are ±S.D. Difference due to treatments and months are significant (P < 0.05).

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Fig. 4. Effect of water stress and months on leaf temperature in D. sissoo seedlings. Each value is mean of three replications ± S.D. as the error bars. Similar alphabet in same month indicates non-significant (P > 0.05) difference between treatments.

seedlings of W5 (at a soil water potential of -1.96 MPa) caused one-eighth NR activity than the activity in the seedlings of W1 treatment. Proline was below the detection limit in the months of January and February in the seedlings of all the treatments (Fig. 5). However, proline was detected in the seedlings of W3 , W4 and W5 treatments in March, April and May. In April, the concentration of proline was 4.33 ␮g g−1 fresh weight in the seedlings of W3 treatment which increased further with the increase in soil water stress to W4 treatment. In severely water stressed W5 treatment, proline concentration was three-fold to that in W3 .

4. Discussion 4.1. Seedling growth and biomass Soil water deficits negatively affected seedling growth and total dry mass. When compared with that in W1 , the reduction in growth and biomass of the seedlings in W3 and W4 treatments was due to limited water availability that had affected leaf expansion and carbon assimilation (Table 2). Significantly low biomass in the seedlings of W5 treatment was due to continuous depletion in soil water leading to reduction

in leaf size or leaf area and total dry mass (Waldron and Terry, 1987). Greater root biomass in the seedlings W1 compared to the seedlings of other treatments was due to development and growth of the rootlets of secondary or tertiary orders for absorption of available soil water. However, relative increase in percent biomass allocation to root (from 25% in W1 to 35–50% in W3 , W4 and W5 seedlings) was due to stress stimulated root growth through greater allocation of photosynthates towards roots (Pallardy and Rhoods, 1993). Bongarten and Teskey (1987) found that the seedlings exposed to repeated drought cycles, increased dry matter allocation to the root at the expense of allocation to the stem. However, in the present study, the percent leaf biomass reduction was greater than the percent stem biomass reduction indicating that the allocation to root was at the expense of the leaves. Comparatively low root dry mass in the seedlings of W2 was due to photosynthates utilization in maximizing leaf biomass at mild water stress. 4.2. Nodulation and nodule weight Greater nodule number and total nodule dry mass in the seedlings of W1 and their reduction with decrease in irrigation level indicated a negative effect of water deficits on nodule formation. Exceptionally low num-

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Fig. 5. Effect of water stress and months on free proline concentration in D. sissoo seedlings. Each value is mean of three replications ± S.D. as the error bars. Similar alphabet in same month indicates non-significant (P > 0.05) difference between treatments.

ber of nodules and their low dry mass in the seedlings of W5 treatment suggested that severe soil water deficit adversely affected nodulation through the effect on carbohydrate transport and bacterial activity. Srivastava and Ambasht (1994) observed a reduction in N fixation under low soil moisture regime in Casuarina equisetifolia plantation. A decline in nodule number and nodule dry mass with decreasing soil water availability and LWP indicated negative correlation in nitrogen fixation activity with soil water stress similar to the observation of Huang et al. (1975). 4.3. Soil available nitrogen and plant nitrogen uptake Decrease in nitrogen availability in May 1999 as compared to than in July 1998 was due to utilization of nitrogen in growth and development of the seedlings and/or due to the leaching losses during irrigation. Highest availability of soil NH4 -N in W1 was appeared to be the result of nitrogen fixation as evidenced by greater number of root nodule and nodule dry mass. Availability of NO3 -N was greater in the soil of W3 , W4 and W5 treatments compared to those in the W1 and W2 suggesting its favourable absorption as evidenced by highest nitrogen uptake (P < 0.001) in the seedlings

of W1 treatment. Decrease in nitrogen uptake with decrease in irrigation level indicated that soil water deficit negatively affected nitrogen uptake and, therefore, nitrogen assimilation. This is in consistent with the result of Naik et al. (1982) who found a close relationship between NR activity and nitrogen assimilation with plant nitrogen uptake. 4.4. Leaf water status and CO2 assimilation Temporal changes in leaf water potential (LWP) in the seedlings of W1 , W2 , W3 and W4 treatments indicated significant relations between environmental factors like solar radiation and air temperature and seedling water status (Fig. 2). Higher LWP during winter was due to low VPD (Fig. 1). Monthly variations in LWP suggest that D. sissoo is very sensible to changes in soil water content thereby showing moderate drought tolerance. A greater difference in the LWP between the seedlings of W1 and W5 treatments suggests less recovery under severe soil water stress, particularly in the seedlings of W5 , where LWP decline was high. Significantly low LWP in W5 in April and May compared to that in January–March was probably due to fast depletion of soil water with increasing air temperature and VPD and irreversible damage of leaf tissue evidenced by leaf drying and abscission at soil water

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potential of <−2.02 MPa as reported for Quercus ilex (Kyriakopoulos and Larcher, 1976). Highest Pn for the seedlings of W1 treatment was related with highest LWP, soil water content and leaf nitrogen content and lowest leaf temperature. A direct relationship between nitrogen content and Pn has also been reported in other studies (Grassi et al., 2001; Medivilla et al., 2003). Greater Pn in W1 and W2 showed more CO2 fixation and greater biomass production compared to the seedlings of other treatments (Ni and Pallardy, 1991). Significant (P < 0.05) reduction in Pn in the seedlings at W3 , W4 , and W5 , suggests that water deficit reduces the rate of CO2 assimilation (Rhodenbaugh and Pallardy, 1993). An increase in Pn from January to March and decreased thereafter indicated that the environmental factors like air temperature, PAR, and VPD are favourable in March influencing CO2 assimilation (Fig. 3). However, increased air temperature, Tleaf , solar radiation and VPD in April and May adversely affected CO2 assimilation. Negative relations of Pn and LWP with soil water deficit reflected moderate drought tolerance behaviour in D. sissoo seedlings (Allen et al., 1999). 4.5. NR activities and proline accumulation Highest NR activity in the seedlings of W1 treatment was due to sufficient soil water availability and highest Pn discussed earlier (Fig. 3). It suggests that NR activity was positively related with photosynthetic CO2 assimilation. Soil water deficit reduced NO3 -N absorption and transport to the leaf, which in turn, decreased NR activity. Relatively less nitrogen availability in the soil of W1 and W2 compared to that in W3 , W4 and W5 treatments suggested lesser absorption of nitrogen from soil in the later treatments. A positive correlation between photosynthetic CO2 assimilation and NR activity supports the inference. Foyer et al. (1998) recorded similar observation for maize leaves. Lowest NR activity in the seedlings of W5 treatment indicated adverse effect of water stress on nitrate metabolism (Pokhriyal et al., 1991a). Favourable environmental conditions and higher rate of CO2 assimilation seemed to influence NR activity as evidenced by highest NR activity in March. However, increased temperature and evaporative water loss in April and May decreased NR activity like decline in photosynthetic CO2 assimilation discussed earlier (Fig. 3). This result is in consistent with the ob-

servation of Pokhriyal et al. (1991b) where high NR activity in the month of March and August and low activity in July and June was observed in D. sissoo nodules. Low air temperature and low solar radiations reduced physiological activities and soil water stress during winter affecting biosynthesis of proline (Fig. 5). This is evidenced by detection of proline in the seedlings of W3 , W4 and W5 treatments in March–May during which these environmental variables increased. It indicates that the enzyme (
 -pyrroline5-carboxylate synthatase, P5CS) involved in proline biosynthesis was regulated via proline feed back inhibition (Stewart and Larher, 1980). Increase in proline content as a result of water deficit (from W3 to W5 treatments) in the present study is in consistent with the observation of Hanson and Hits (1982). Newton et al. (1986) observed 40-fold higher proline at −1.6 MPa leaf water potential compared to that at −0.4 MPa leaf water potential in Pinus taeda seedling. A 3.0fold increase in proline concentration in severely water stressed W5 than that in W3 treatment showed that water stress leads to change in the concentration of proline (Garg et al., 1998). This study suggests that mild water stress had no significant effects on growth and biomass reduction and biomass allocation in leaf, stem and root. However, growth and biomass were severely affected at high soil water stress (low irrigation level). Nitrogen metabolism was affected at severe water stress as indicated by reduced NR activity and increased proline concentration in the seedlings of W3 , W4 and W5 treatments. Decline in Pn , NR activity, number of nodule and LWP with increasing water deficit suggests stress tolerant behaviour of D. sissoo seedlings. However, a drastic reduction in these variables and biomass for the seedlings of W5 demonstrated that D. sissoo is moderate drought tolerant species. W2 level of soil water availability is suitable for physiological activity and, therefore, for growth and biomass production of D. sissoo.

Acknowledgements Authors are thankful to the Director, Arid Forest Research Institute, Jodhpur for providing facilities and to Late Dr. G.N. Gupta, Scientist ‘SF’ for constructive

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criticisms and fruitful suggestions. Technical help rendered by Mr. T.R. Rathod is gratefully acknowledged.

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