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Effect of foliar spray of nutrient solutions on photosynthesis, dry matter accumulation and yield in seawater-stressed rice
Environmental and Experimental Botany 46 (2001) 129– 140 www.elsevier.com/locate/envexpbot
Effect of foliar spray of nutrient solutions on photosynthesis, dry matter accumulation and yield in seawater-stressed rice N. Sultana a,1, T. Ikeda b,*, M.A. Kashem a,1 a
Graduate School of Science and Technology, Niigata Uni6ersity, Niigata 950 -2181, Japan b Faculty of Agriculture, Niigata Uni6ersity, Niigata 950 -2181, Japan
Received 17 March 2000; received in revised form 10 April 2001; accepted 10 April 2001
Abstract The effects of seawater salinity and foliar application of nutrient solutions on rice in the early tillering stage and early reproductive phase of growth were investigated in a glasshouse. During early tillering stage, from 10 to 35 days after transplanting (DAT) and the early reproductive phase, from 75 to 100 DAT, potted rice plants were irrigated with Japan seawater of 0, 8.8, 17.5 and 35% (equivalent to an EC of 0.9, 5.7, 11.5 and 21.5 ms cm − 1, respectively). The nutrient solution of 1 mM Ca(NO3)2, MnSO4 or K2HPO4 was sprayed twice a week until the solution ran off the leaves. Photosynthesis and its related parameters were measured at 30 and 95 DAT in the early tillering stage and in the reproductive growth phase, respectively. Seawater salinity diminished photosynthesis rate and photosynthesisrelated parameters, such as stomatal conductance, intercellular CO2 concentration, leaf water and osmotic potential and relative leaf water content in both growth stages and have reduced tiller number, leaf area and top dry matter content in tillering stage. We have also studied the effect of salt-stress on the mineral content at 35 DAT. Na+ concentration increased, whereas Ca2 + , Mn2 + and K+ concentration were decreased with increasing stress. Seawater decreased fertile spikelets in the panicle, decreased accumulation of dry matter in the grain and concomitantly decreased grain yield. Foliar spray of Ca(NO3)2, MnSO4 or K2HPO4 partially minimized the salt-induced nutrient deficiency, increased photosynthesis, dry matter accumulation, number of fertile spikelet in the panicle and grain yield. Among the nutrient solutions, Ca(NO3)2 seemed to be the most effective, followed by MnSO4 and K2HPO4. These results suggested that foliar application of nutrient solutions partially alleviates the adverse effects of salinity on photosynthesis and photosynthesis-related parameters, yield and yield components through mitigating the nutrient demands of salt-stressed plants. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Growth stages; Mineral elements; Oryza sati6a; Photosynthesis; Seawater salinity
Abbre6iations: A, photosynthesis rate; Ci, internal CO2 partial pressure; DAT, days after transplanting; EC, electrical conductivity; gs, stomatal conductance; RLWC, relative leaf water content; s, osmotic potential; w, leaf water potential. * Corresponding author. Tel.: + 81-25-2626610; fax: +81-25-2626854. E-mail addresses: [email protected] (N. Sultana), [email protected] (T. Ikeda). 1 Present address: Plant Breeding Institute, The University of Sydney, P.O. Box 219, Narrabri, NSW 2390, Australia. S0098-8472/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 9 8 - 8 4 7 2 ( 0 1 ) 0 0 0 9 0 - 9
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1. Introduction Soil salinity is a major environmental stress that adversely affects plant growth and metabolism. Coastal rice crops in Asia are frequently affected by exposure to seawater brought in by cyclones around the Indian Ocean. This incidental exposure to salt occurs either during the vegetative or reproductive or both phases, presenting an increasing threat to rice productivity. Salt salinity affects plant physiology through changes of the water and ionic status in the cells (Sultana et al., 1999; Kashem et al., 2000b; Hasegawa et al., 2000). Ionic imbalance occurs in the cells due to excessive accumulation of Na+ and Cl− and reduces the uptake of other mineral nutrients, such as K+, Ca2 + and Mn2 + (Cramer and Nowak, 1992; Khan et al., 1997; Lutts et al., 1999). The function of Mn2 + at the cellular level of plant is to bind firmly to lamellae of chloroplasts, possibly to the outer surface of thylakoid membranes, affecting the chloroplast structure and photosynthesis (Lidon and Teixerira, 2000). External application of Mn2 + increased photosynthesis, net assimilation and relative growth in barley under salinity (Cramer and Nowak, 1992). Cytosolic Ca2 + is not only involved in biosynthesis and intracellular transport of proteins, but also in signaling components in rice plant (Kashem et al., 2000a). External supplied Ca2 + has been shown to ameliorate the adverse effect of salinity in plants, presumably by facilitating higher K+/ Na+ selectivity (Hasegawa et al., 2000). The role of K+ is vital for osmoregulation and protein synthesis, maintaining cell turgor and stimulating photosynthesis (Peoples and Koch, 1979). Higher levels of K+ in young expanding tissue is associated with salt tolerance in many plants (Gorham, 1993; Khatun and Flowers, 1995). NaCl also changes the anion concentrations in plants. A lowered supply of nitrate to growing leaves may be responsible for the inhibition of growth under saline conditions (Hu and Schmidhalter, 1998). Salinity can affect growth, dry matter accumulation and yield of rice (Sultana et al., 1999; Asch et al., 2000). The reduction in growth of salinized plants may result from the effect of salt on water status, ionic imbalance, nutrient and phytohor-
monal status, physiological processes, biochemical reactions or a combination of such factors (Volkmar et al., 1998; Hasegawa et al., 2000; Kashem et al., 2000b) and accompanied by a reduction of photosynthesis (Sultana et al., 1999). Low yield of grain under salinity might result from a number of causes, such as loss of photosynthetic capacity (Sultana et al., 1999; Horton, 2000), decreased the assimilates accumulation in the grain (Sultana et al., 1999; Asch et al., 2000) and reduction of seed setting in the panicles (Khatun and Flowers, 1995). Evidence accumulated that rice is a salt sensitive crop with peak sensitivity in the early seedling and early reproductive stages (Yeo et al., 1990; Lutts et al., 1995). Though a wide range of investigation has been carried out on the effects of salinization in a number of crops, the problem of seawater exposure and how to overcome this problem have not yet been sufficiently investigated in rice. To overcome the problem, plant breeders and physiologists have directed considerable efforts toward developing cultivars and agromanagement techniques to improve the physico-chemical properties of rice plants grown under saline conditions. Fertilizer application is now being tried to alleviate or neutralize growth inhibition due to salinization. Many physiological and chemical studies have also been conducted on the addition of nutrients to the soil medium in response to conditions of salt stress (Song and Fujiyama, 1996a,b; Abdel-Rahman, 1999). However, no studies have been undertaken concerning the ameliorative effects of foliar sprays of fertilizer solutions containing Ca, Mn or K applied during the early tillering and early reproductive stages of rice crops grown under seawater stress. The aim of this study was to test the hypothesis that foliar sprays of fertilizer solutions containing Ca, Mn or K could ameliorate the stress in rice plants caused by sea water salinity. For this purpose, rice plants at the tillering and early reproductive stages were sprayed with solutions of Ca(NO3)2, MnSO4 or K2HPO4 and we studied the effects of salt exposure on photosynthesis and its related parameters, mineral concentration, dry matter accumulation and yield and yield contributing characters of the rice plant.
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2. Materials and methods A moderately salt resistant, improved, semidwarf japonica rice variety (Oryza sati6a L. cv. Kosihikari) was germinated in a tray filled with silty clay-loam soil in a glasshouse at Niigata University, Japan. In this study, we conducted two separate experiments from April to September 1999. Each experiment was arranged as a two-way factorial experiment in a randomized block design with three replications. Each replication contained ten pots and each pot contained three seedlings.
2.1. Tillering stage (Experiment 1) Seedlings (25-day-old) were transplanted (May 15) to 3-l plastic pots filled with fertilized sandy loam soil having pH 7.4. The pots were kept in the well-ventilated glasshouse during the whole growing season. The air temperature and relative humidity in the glasshouse was in the range from 22 to 32°C and 60– 80%, respectively. Fertilizers were applied at 12 g N m − 2 (as NH4Cl), 8 g P m − 2 (as superphosphate) and 10 g K m − 2 (as KCl). Half of the N was applied as a basal dose in both the experiments, a quarter was applied at tillering stage and the rest was applied at panicle initiation stage. Seawater was collected from Japan sea (near Niigata University) and its electrical conductivity (EC) was 61 ms cm − 1. After transplanting, the base of each plant was covered with a plastic sheet to prevent the contamination of sprayed nutrients and also to minimize the evaporation. From May 25 (10 DAT) to June 20 (35 DAT), each pot was watered with a 0, 8.8, 17.5 or 35% of sea water (equivalent to EC about 0.9, 5.7, 11.5 or 21.5 ms cm − 1, respectively). These solutions were prepared by diluting seawater with deionized water and were applied far in excess of the saturation capacity of the soil. To maintain the required soil medium salt levels, the EC of the soil medium was measured periodically by portable EC meter and the required amount of treated water was added. From 10 to 35 DAT, each pot was sprayed twice a week with 1 mM
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solutions of Ca(NO3)2·4H2O, K2HPO4·12H2O or MnSO4·4H2O. Deionized water was sprayed as a control. Tween-20 (0.1%) was used as a wetting agent for each treatment, including the water as control. We sprayed the plants with solution until the leaves were completely wet and the solution ran off the leaves.
2.2. Reproducti6e phase (Experiment 2) The experiment was conducted from the end of flag leaf stage (30 July, 75 DAT) to milking stage (25 August, 100 DAT). The experimental conditions, such as sowing and cultural practices, were the same as in Experiment 1 and all the treatments were applied as described above.
2.3. Measurement of photosynthesis, photosynthetic parameters, leaf water and osmotic potential Photosynthetic rate (A), stomatal conductance (gs), intercellular CO2 concentration (Ci), water (w) and osmotic (s) potentials and relative leaf water content (RLWC) of the young expanded leaves (Experiment 1) and flag leaves (Experiment 2) were measured at tillering stage (30 DAT, June 15) and milking stage (95 DAT, August 20), respectively, according to the method previously described by Sultana et al. (1999).
2.4. Measurements of growth characteristics For measuring the tiller number and the leaf area, five plants were cut randomly at soil surface level at 35 DAT and then counted tiller number. Leaf area was measured with a leaf area meter (Automatic Area Meter, Model AAM-7, Hayashi Denko Co. Ltd.).
2.5. Estimation of mineral concentrations Oven dried samples of 35 DAT old plants were digested by HNO3 –HClO4, and Na+, Ca2 + , Mn2 + and K+ were analyzed by atomic absorption spectrophotometry (Mitsui et al., 1999).
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2.6. Measurement of dry weight, yield and yield components In both experiments, randomly five plants were cut at soil level at 35 DAT and after harvest, respectively. At tillering stage, dry weight of stem with leaf (top dry weight) was measured. After harvesting (30 September) five panicles form each treatment were taken randomly for counting the number of fertile spikelet/panicle. After counting the number of fertile spikelets, the same panicle was used for the measuring of panicle dry weight. The samples were oven-dried at 60°C for 72 h and the dry weight was recorded. For the dry weight of the developing grain, 20 ovaries (dehulled grain) were collected at dough stage (105 DAT, 30 August) for each treatment and were dried at 60°C for 48 h. Yield/plant also measured from the collected five plant for each replication.
2.7. Statistical analysis The MSTAT Development Team (1989) (MStat-C) software program was used for data analysis. The data were analyzed and the mean differences were adjudged as per LSD test.
3. Results Except slight yellowing of the leaves, no visible symptoms of salt injury were observed on the plants at 8.8 and 17.5% of seawater salinity, but at 35% seawater salinity, severe necrotic and chlorotic symptoms were present on leaves both at the tillering stage and early reproductive phases of growth (data not shown).
3.1. Effect of seawater and foliar spray on mineral content Mineral contents in the seawater treated plant are presented in Fig. 1. Elementary analysis of plant materials revealed that the concentrations of Na+ increased by 1.57, 2.14 and 2.86-fold in 8.8, 17.5 and 35% seawater treated plants, respectively (Fig. 1a). Seawater salinity decreased Ca2 + by 46%, Mn2 + by 36% and K+ by 37% at 35%
seawater treated plants compared to the control (Fig. 1b–d). Foliar application of nutrient solutions increased the mineral content up to 4% in the control plant (0% of seawater), whereas it was increased for Ca2 + by 25%, Mn2 + by 18% and K+ by 21% in 35% seawater treated plant (Fig. 1b–d).
3.2. Effect of seawater and foliar spray on photosynthesis and its related parameters We have studied the effects of seawater salinity and foliar spray of nutrients on photosynthesis (A) and its related parameters at 30 and 95 DAT (Tables 1 and 2). Salt stress led to a significant inhibition of leaf A (Table 1). The rate of photosynthesis was decreased when concentration of seawater increased from 8.8 to 35%. Salinity-induced low A was partially overcome when we applied nutrient solutions to the plants. For example, A was increased by 22% an application of Ca(NO3)2 in the 35% seawater treated plant at 30 DAT (Table 1). The increase in the salinity significantly decreased the gs of leaves (Table 1); however, a foliar spray of nutrients increased the gs compared to the control treatment. The tendency of increased A and gs were highest with the Ca(NO3)2 treatment; compared to other treatments. The Ci was not greatly affected in this stage of growth, but external application of nutrient solution improved the stress response of Ci (Table 1). Leaf water potential, osmotic potential and relative leaf water content (RLWC) significantly decreased with increasing concentrations of salinity at both growth stages (Table 2). Foliar application of nutrient solutions significantly reduced the decreasing tendency of these parameters; the Ca(NO3)2 treatment was most effective, followed by MnSO4 and K2HPO4.
3.3. Effect of seawater and foliar spray on growth characteristics at tillering stage The effects of seawater salinity and foliar application of nutrient solutions on growth characteristics of rice plant were studied at 35 DAT (Fig. 2) Seawater salinity significantly decreased the tiller number, leaf area and top dry weight (Fig. 2).
Sea water conc. (%)
Spray treatments
30 DAT
95 DAT
A (mmol CO2 m−2 s−1)
gs (mol CO2 m−2 s−1)
Ci (ppm)
A (mmol CO2 m−2 s−1)
gs (mol CO2 m−2 s−1)
Ci (ppm)
0
Water Ca(NO3)2 MnSO4 K2HPO4
20.3 90.47a 21.8 9 0.55a 21.4 9 0.23a 20.9 9 0.51a
0.86 9 0.02b 0.82 90.02b 0.85 90.02ab 0.87 90.01a
221 9 1.7b 224 92.3a 223 91.1ab 221 92.1b
24.9 9 1.15bc 28.3 9 1.73a 27.5 9 0.23ab 27.2 9 1.15ab
1.4 9 0.23a–c 1.6 90.11a 1.5 90.17ab 1.5 90.28ab
3029 5.7ab 308 9 4.6a 303 9 4.4ab 304 9 5.1ab
8.8
Water Ca(NO3)2 MnSO4 K2HPO4
16.3 90.51c 18.5 9 0.54b 17.8 9 0.46bc 17.0 9 0.26bc
0.45 9 0.03ef 0.57 90.03c 0.54 90.02c 0.52 90.02cd
196 9 3.7e 207 94.3c 204 92.1d 200 91.1de
20.5 9 0.51ab 23.0 9 1.73cd 22.7 9 0.57cd 21.9 9 0.57cd
0.75 90.05b–d 0.88 90.046a–d 0.84 90.023a–d 0.80 90.058a–d
2929 6.7c 3009 8.6a–c 2979 4.1bc 2959 2.9bc
17.5
Water Ca(NO3)2 MnSO4 K2HPO4
12.1 9 0.15e 13.6 9 0.4d 12.9 9 0.57de 12.5 9 0.26de
0.33 90.02g 0.48 90.01de 0.43 90.01f 0.40 90.02f
180 9 2.7h 188 92.8f 186 92.3fg 185 92.9g
14.7 90.4e 16.8 9 1.73e 15.9 9 1.15e 15.3 9 0.58e
0.54 9 0.023d 0.65 90.028de 0.61 90.020cd 0.60 90.028cd
270 9 5.8d 272 9 4.6d 269 9 5.2d 265 9 2.9d
35
Water Ca(NO3)2 MnSO4 K2HPO4
7.4 90.23g 9.6 90.23f 8.8 90.34fg 8.4 90.15fg
0.15 9 0.01i 0.25 90.02h 0.23 90.01h 0.22 90.01h
170 91.1j 175 9 2.3i 172 9 1.1j 171 9 1.7j
9.1 9 0.57f 11.1 9 0.26f 9.9 9 0.51f 9.5 9 0.57f
0.24 90.023d 0.32 9 0.011d 0.30 9 0.028d 0.27 9 0.028d
207 9 4.1e 215 92.9e 211 96.4e 208 94.6e
a
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Table 1 Effects of foliar spray of nutrient solutions on photosynthetic rate (A), stomatal conductance (gs), internal CO2 partial pressure (Ci) of rice grown under seawater salinity at 30 and 95 DATa
The values are the mean 9 S.E. (n =3); different letters indicate significant differences among the treatments at 1% level of significance in LSD test.
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However, the application of nutrient solution partially mitigated the adverse effects of salinity on these parameters.
Ca(NO3)2, MnSO4 or K2HPO4 seems to be increased the dry matter accumulation in the grain (Fig. 3a).
3.4. Effect of seawater and foliar spray on dry matter accumulation in de6eloping grains
3.5. Effect of seawater and foliar spray on yield and important yield components
The effect of seawater salinity and foliar spray of Ca(NO3)2, MnSO4, or K2HPO4 on dry matter accumulation of developing grains were examined (Fig. 3a). Salinity had a slight effect on grain dry matter at lower concentration and initial stages of growth (data not shown), but the effect was aggravated by the high concentration and long duration of salinity (Fig. 3a). Foliar spray of
Panicle dry weight, number of fertile spikelets/ panicle and yield/plant were measured after harvesting (Fig. 3b–d). There were significant differences (PB 0.01) for salinity and sprayed treatments for these parameters, but the interaction effect between salinity and spray was not significant for these parameters (data not shown). All these parameters decreased along with the
Fig. 1. Effect of seawater salinity and foliar spray of nutrient solutions either calcium, manganese or potassium on Ca, Mn and K concentration in rice leaves at 35 DAT. Vertical bars indicate mean 9 S.E. (n =3); different letters indicate significant differences among the treatment at the 0.01 probability level based on an LSD test.
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Table 2 Effects of foliar spray of nutrient solutions on leaf water potential (w), osmotic potential (s) and relative leaf water content (RLWC) of rice grown under seawater salinity at 30 and 95 DATa Seawater conc. (%)
Spray treatments
30 DAT
95 DAT
w (MPa)
s (MPa)
RLWC (%)
w (MPa)
s (MPa)
RLWC (%)
0
Water Ca(NO3)2 MnSO4 K2HPO4
−0.669 0.03cd −0.629 0.01d −0.639 0.02d −0.659 0.03cd
−0.8390.01g −0.8090.01g −0.8090.02g −0.929 0.01fg
69 9 2.9ab 70 9 2.3a 68 9 2.9a–c 69 9 1.2ab
−0.869 0.03fg −0.829 0.01g −0.839 0.02fg −0.859 0.03fg
−1.39 0.17e–g −1.290.11fg −1.290.06fg −1.190.06g
669 1.2ab 67 9 2.3a 67 9 1.2a 65 91.7bc
8.8
Water Ca(NO3)2 MnSO4 K2HPO4
−0.939 0.01b −0.889 0.02b–d −0.919 0.02bc −0.909 0.03bc
−1.190.06ef −0.949 0.02fg −0.959 0.02fg −0.9690.02bc
66 9 2.3a–d 68 92.3a–c 67 91.2a–d 67 9 0.6a–d
−1.039 0.06dfg −0.9990.05e–g −1.0190.02e–g −1.09 0.06e–g
−1.690.11dfg −1.390.17e–g −1.490.11e–g −1.590.11deg
62 91.2e 6592.3bc 649 1.7cd 63 91.7de
17.5
Water Ca(NO3)2 MnSO4 K2HPO4
−1.190.03b −0.999 0.01b −1.09 0.02b −1.049 0.03b
−1.690.11b −1.29 0.01de −1.490.15cd −1.590.11c
62 9 0.33d–f 65 90.6b–d 64 90.6c–e 64 9 1.1c–e
−1.49 0.23cd −1.190.10d–f −1.290.15c–e − 1.3 90.17cd
−2.09 0.28b–d −1.790.28d–f −1.890.15c–e −1.9790.11cd
55 9 0.57g 59 9 1.6f 58 9 2.3f 56 91.23g
35
Water Ca(NO3)2 MnSO4 K2HPO4
−1.99 0.11a −1.59 0.14a −1.69 0.17a −1.79 0.11a
−2.59 0.15a −2.190.10b −2.290.11b −2.390.10ab
56 91.1g 60 91.7fg 59 9 1.1fg 57 9 0.6g
−2.090.28a −1.790.17b −1.89 0.34ab −1.89 0.17ab
−2.790.17a −2.390.17a–c −2.59 0.28ab −2.69 0.12a
50 91.2j 5391.7a–c 52 9 1.2hi 51 9 1.73ij
a The values are the mean 9 S.E. (n=3); different letters indicate significant differences among the treatments at 1% level of significance in LSD test.
increasing concentrations of salinity. However, seed setting (number of fertile spikelets) and subsequently, grain yield were very sensitive to seawater salinity. Foliar spray of Ca(NO3)2, MnSO4 or K2HPO4 increased the number of panicles, seed setting in the panicle and grain dry weight, finally resulted in increased grain yield (Fig. 3c). Panicle dry weight, number of fertile spikelet/panicle and yield/plant were higher in Ca(NO3)2, followed by MnSO4 or K2HPO4 (Fig. 3b– d).
4. Discussion Seawater contains high concentration of Na salts and salinity reduced availability of nutrients in the soil (Muhammed et al., 1987). We have analyzed some cations, such as Na+, Ca2 + , Mn2 + and K+ in seawater-stressed rice (Fig. 1). At 35% of seawater, treated rice plant accumulated 2.9-fold higher Na+ and decreased Ca2 + by
46%, Mn2 + by 36% and K+ by 37% compared to the control. Salt induced nutrient deficiency has been reported by many researchers (Cramer et al., 1991; Khan et al., 1997; Lutts et al., 1999). The molecular mechanism of the discrimination of nutrient uptake under salinity is still unclear. However, the possible causes for discrimination of nutrient uptake under seawater salinity in the rice plant are: (a) ions in the high concentration in the external solution (i.e. Na+ or Cl−) are taken up at high rate, which may lead to excessive accumulation in the tissue. These ions may inhibit the uptake of other ions into the root (i.e. K+ or Ca2 + ) and their transport into the shoot through the xylem, eventually leading to deficiency in the tissue. (b) Na+ and Cl− transport across the plasma membrane in a saline environment must be considered in two cellular contexts, after salt stress shock and after re-establishment of ionic homeostasis. Immediately after salt stress, the H+ electrochemical gradient is altered. Influx of Na+
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dissipates the membrane potential, thereby facilitating the uptake of Cl− down the chemical gradient. An anion channel has been implicated in this passive flux. On the other hand, Na+ competes with K+ for intracellular influx because these cations are transported by common protein (Hasegawa et al., 2000). However, our important target is how to minimise the salt-induced nutrient deficiency in the rice plant. To overcome salt-induced nutrient defi-
ciency, we have sprayed respective nutrient solutions combined with surfactant in the shoot. At 35% of seawater, treated leaves contained :20% higher element concentration compared to unsprayed leaves. Our results are in agreement with the findings of many workers in different plant species (Smith et al., 1991; Abdel-Rahman, 1999) who found that nutrients were absorbed by the leaves when applied onto the shoot.
Fig. 2. Effect of nutrient solutions either Ca, Mn or K on tiller number, leaf area and top dry weight of rice plant grown under seawater salinity at 35 DAT. Vertical bars indicate mean 9S.E. (n =3); different letters indicate significant differences among the treatment at the 0.01 probability level based on an LSD test.
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Fig. 3. Effect of nutrient solutions containing either Ca, Mn or K on grain, panicle dry weight, fertile spikelets and yield of rice grown under seawater salinity. Vertical bars indicate mean 9S.E. (n =3); different letters indicate significant differences among the treatment at the 0.01 probability level based on an LSD test.
In the present study, we observed that seawater reduced the rate of photosynthesis and its related parameters (Tables 1 and 2), decreased growth characteristic such as tiller number, leaf area and
dry matter accumulation (Fig. 2) and decreased yield contributing characters, such as panicle number, fertile spikelets, grain dry matter accumulation and yield (Fig. 3). Foliar application of
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nutrient solutions partially overcame salt-induced detrimental effects in rice (Tables 1 and 2; Figs. 2 and 3). Understanding the regulatory mechanisms of photosynthesis in response to environmental changes is therefore important for plant productivity. Seawater salinity significantly reduced the rate of photosynthesis (Table 1). The rate of photosynthesis depends on stomatal and nonstomatal components (Bethke and Drew, 1992) and each of the components has a unique response to an environmental variable. Stomatal conductance is related to turgor pressure of cells. The turgor pressure is controlled by solute regulation within the guard cell protoplast and the relative water content of epidermal tissues. Accumulation of K+ and other organic ion increased the osmotic activity, causing a reduction in water potential and an influx of water from the surrounding cells. In addition, phytohormones and Ca2 + play important signaling role on the regulation of stomata (Sage and Reid, 1994) and Mn2 + bind firmly to the lamellae of chloroplasts, possibly to the outer surface of thylakoid membranes, affecting the chloroplast structure and photosynthesis (Lidon and Teixerira, 2000). Optimum balance of these physico-chemical components in the cells is directly or indirectly related to photosynthesis. Seawater salinity increased the water deficit and decreased the mineral nutrients that activate the ionic imbalance in the cytosol, leading to creating an unfavorable environment, therefore, reduced the rate of photosynthesis. The minerals Na+, Ca2 + , K+ and Mn2 + are not only involved in the regulation of photosynthesis but have other cellular regulatory function, which are directly or indirectly involved in growth and physiology of the plant. K+ plays a predominantly osmotic role in plants (Marshall and Porter, 1991). NaCl inhibits Ca2 + transport from roots to shoot by interfering with the release of Ca2 + into the root xylem, possibly via an effect on the active loading of Ca2 + into xylem vessels and salt stress inhibits the supply of Ca2 + to the shoot in several species (Lynch and La¨ uchli, 1985). However, supplementation of Ca2 + , Mn2 + and K+ through the nutrient solution increased the rate of photosynthesis and growth of salt-stressed plants by increasing
the concentration of Ca2 + and K+ (Colmer et al., 1996; Liu and Zhu, 1997) and Mn2 + (Cramer and Nowak, 1992) in shoots and leaves. Although Sohan et al. (1999) reported that Ca2 + supplements did not significantly ameliorate the negative effects of NaCl, our results showed that a nutrient solution providing supplemental calcium or potassium ameliorated the effects of seawater exposure on photosynthesis and photosynthesis-related parameters. The increased salt tolerance in rice by the addition of Ca(NO3)2 and K2HPO4 reported in our study is in agreement with other findings, though those researchers did not apply the nutrient solution as a foliar spray (Lopez and Satti, 1996; Song and Fujiyama 1996a,b; Cuartero and Ferna´ ndez-Mun˜ oz, 1999). We did not check whether the acclimation was due to the effect of the cation or anion of the foliar applications of Ca(NO3)2 and K2HPO4. We suggested that the − anion (NO− 3 or HPO4 ) may also be responsible for amelioration of photosynthesis and its related parameters because these two anions are important for the growth and development of plants, but Cramer and Nowak (1992) suggested that Mn and not SO4 was responsible for increasing the photosynthesis in salt-stressed barley plant when MnSO4 was sprayed. The deleterious effects of seawater on growth is partly due to the inhibition of photosynthesis, partly due to the induction of growth inhibitor (abscisic acid) which cause premature senescence of leaves and reduced leaf area (Arif and Tomas, 1993; Kashem et al., 2000b), partly due to decreased leaf protein content resulting from higher accumulations of Na+ and Cl− concentrations (Sultana et al., 1999), partly due to reduced ability to produce and utilize assimilates/photosynthates, reducing the supply of assimilates to the growing regions (Munns et al., 1995; Kashem et al., 2000b) and partly due to a nutrient imbalance caused mainly by lower absorption rates of K and lower utilization rates of K, Ca and P (Romero and Maran˜ o´ n, 1994). In agreement with this report, we found that seawater salinity reduced photosynthesis, reduced leaf area, imbalanced the mineral contents and as a result, decreased the growth of plants. We also found, however, that these detrimental effects of seawater on growth could be
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partially alleviated by application of nutrient solutions through decreasing the nutrient demand in salt-affected plants. Seed setting (fertile spikelet) is one of the major contributors to grain yield. In this study, we observed that seed is far more sensitive to salinity than other characteristics that contribute to yield and thus, grain yield was very sensitive to salinity. A lower rate of assimilate translocation from shoots to panicles and reduced pollen viability due to the effect of ionic toxicity may cause a reduction of flowering percentages, increase of floret sterility (Khatun and Flowers, 1995), lower the fertile spikelet/panicle, decrease panicle dry weight and reduce dry matter accumulation in grain and may finally lead to decreased yields, as we found in our study. Reduction in panicle growth by exposure to salt may be due to the strong inhibition of panicle exertion in rice by low hydrolic potentials induced by salt-induced water stress (Ekanayake et al., 1989). Salinity-induced reduction of dry matter accumulation in grains may be due to the reduced rate of photosynthesis and the reduced ability to utilize photosynthates for growth. The reduction in dry matter at the grain filling stage might be through inhibition of photoassimilation at an earlier stage, because high salinity reduces the contents of photosynthetic pigments and soluble proteins in the leaves/ ovaries; this change might cause the decline in leaves/ovary photosynthesis leading to the poor sugar accumulation in the ovaries (Sultana et al., 1999). However, when we applied spray treatments of Ca(NO3)2, MnSO4 and K2HPO4, the accumulation of dry matter increased in contrast to the control treatments, indicating that toxic ions, such as Na+ in the leaves, may interfere with phloem loading, restricting the uptake of nutrients from roots to shoot. Thus, when nutrients are applied to the leaves, the nutrient elements might penetrate into the leaves and restrict the inhibition due to toxic effects of Na+ and Cl− or minimizes the salinity induced nutrient deficiency.
Acknowledgements We are grateful to Dr R. Itoh, Associate Profes-
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sor, Laboratory of Crop Science, for his kind help. We also thank Dr Nilanta, CSIRO, Australia for critical editing and valuable suggestions. This study was financially supported by the Grant of the Ijiima Food Science Foundation, Tokyo, Japan.
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Kashem, M.A., Itoh, K., Iwabuchi, S., Hori, H., Mitsui, T., 2000a. Possible involvement of phosphoinositide-Ca + 2 signaling in the regulation of a-amylase expression and germination of rice seed (Oryza sati6a L.). Plant Cell Physiol. 41, 399 – 407. Kashem, M.A., Sultana, N., Ikeda, T., Hori, H., Loboda, T., Mitsui, T., 2000b. Alteration of starch –sucrose transition in germinating wheat seed under sodium chloride salinity. J. Plant Biol. 43, 121 –127. Khan, M.S.A., Hamid, A., Salahuddin, A.B.M., Quasem, A., Karim, M.A., 1997. Effect of sodium chloride on growth, photosynthesis and mineral ions accumulation of different types of rice (Oryza sati6a L.). J. Agron. Crop Sci. 179, 149 – 161. Khatun, S., Flowers, T.J., 1995. Effects of salinity on seed set in rice. Plant Cell Environ. 18, 61 –67. Lidon, F.C., Teixerira, M.G., 2000. Rice tolerance to excess Mn: Implication in the chloroplast lamellae and synthesis of a novel Mn protein. Plant Physiol. Biochem. 38, 969 – 978. Liu, J., Zhu, J., 1997. An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance. Proc. Natl. Acad. Sci. USA 94, 14960 –14964. Lopez, M.V., Satti, S.M.E., 1996. Calcium and potassium-enhanced growth and yield of tomato under sodium chloride stress. Plant Sci. 114, 19 –27. Lutts, S., Kinet, J.-M., Bouharmont, J., 1995. Changes in plant response to NaCl during development of rice (Oryza sati6a L.) varieties differing in salinity resistance. J. Exp. Bot. 46, 1843 –1852. Lutts, S., Bouharmont, J., Kinet, J.-M., 1999. Physiological characterisation of salt-resistant rice (Oryza sati6a L.) somaclones. Aust. J. Bot. 47, 835 –849. Lynch, J., La¨ uchli, A., 1985. Salt stress disturbs the calcium nutrition of barley (Hordeum 6ulgare L.). New Phytol. 99, 345 – 354. Marshall, B., Porter, J.R., 1991. Concepts of nutritional and environmental interactions determining plant productivity. In: Porter, J.R., Lawlor, D.W. (Eds.), Plant Growth: Interactions with Nutrition and Environment. Cambridge University Press, Cambridge, pp. 99 –124. Mitsui, T., Loboda, T., Itoh, A., Ikarashi, T., 1999. Sugar controlled Ca2 + uptake and a-amylase secretion in cultured cells of rice (Oryza sati6a L.). Plant Cell Physiol. 40, 884 –893.
Muhammed, S., Akbar, M., Neue, H.U., 1987. Effect of Na/Ca and Na/K ratios in saline culture solution on the growth and mineral nutrition of rice (Oryza sati6a L.). Plant Soil 104, 57 – 62. Munns, R., Schachtman, D.P., Condon, A.G., 1995. The significance of a two-phase growth response to salinity in wheat and barley. Aust. J. Plant Physiol. 22, 561 – 569. MSTAT Development Team, 1989. MSTAT User’s guide: A Microcomputer Program for the Design, Management, Analysis of Agronomic Research Experiments. Michigan State University, East Lansing, MI. Peoples, T.R., Koch, D.W., 1979. Role of potassium in carbon dioxide assimilation in Medicago sati6a L. Plant Physiol. 63, 878 – 881. Romero, J.M., Maran˜ o´ n, T., 1994. Long-term responses of Melilotus segetalis to salinity. 1. Growth and partitioning. Plant Cell Environ. 17, 1243 – 1248. Sage, R.F., Reid, C.D., 1994. Photosynthetic response mechanism to environmental change in C3 plants. In: Wilkinson, R.E. (Ed.), Plant – Environmental Interactions. Marcel Dekker, New York, pp. 413 – 499. Smith, C., Freney, J.R., Sherlock, R.R., Galbally, I.E., 1991. The fate of urea nitrogen applied in a foliar spray of wheat at heading. Fert. Res. 28, 129 – 138. Sohan, D., Jasoni, R., Zajicek, J., 1999. Plant – water relations of NaCl and calcium-treated sunflower plants. Environ. Exp. Bot. 42, 105 – 111. Song, J.Q., Fujiyama, H., 1996a. Ameliorative effect of potassium on rice and tomato subjected to sodium salinization. Soil Sci. Plant Nutr. 42, 493 – 501. Song, J.Q., Fujiyama, H., 1996b. Difference in response of rice and tomato subjected to sodium salinization to the addition of calcium. Soil Sci. Plant Nutr. 42, 503 – 510. Sultana, N., Ikeda, T., Itoh, R., 1999. Effect of NaCl salinity on photosynthesis and dry matter accumulation in developing rice grains. Environ. Exp. Bot. 42, 211 – 220. Volkmar, K.M., Hu, Y., Steppuhn, H., 1998. Physiological responses of plants to salinity: A review. Can. J. Plant Sci. 78, 19 – 27. Yeo, A.R., Yeo, M.E., Flowers, T.J., Flowers, S.A., 1990. Screening of rice (Oryza sati6a L.) genotypes for physiological characters contributing to salinity resistance, and their relationship to overall performance. Theor. Appl. Genet. 79, 377 – 384.
Effect of foliar spray of nutrient solutions on photosynthesis, dry matter accumulation and yield in seawater-stressed rice N. Sultana a,1, T. Ikeda b,*, M.A. Kashem a,1 a
Graduate School of Science and Technology, Niigata Uni6ersity, Niigata 950 -2181, Japan b Faculty of Agriculture, Niigata Uni6ersity, Niigata 950 -2181, Japan
Received 17 March 2000; received in revised form 10 April 2001; accepted 10 April 2001
Abstract The effects of seawater salinity and foliar application of nutrient solutions on rice in the early tillering stage and early reproductive phase of growth were investigated in a glasshouse. During early tillering stage, from 10 to 35 days after transplanting (DAT) and the early reproductive phase, from 75 to 100 DAT, potted rice plants were irrigated with Japan seawater of 0, 8.8, 17.5 and 35% (equivalent to an EC of 0.9, 5.7, 11.5 and 21.5 ms cm − 1, respectively). The nutrient solution of 1 mM Ca(NO3)2, MnSO4 or K2HPO4 was sprayed twice a week until the solution ran off the leaves. Photosynthesis and its related parameters were measured at 30 and 95 DAT in the early tillering stage and in the reproductive growth phase, respectively. Seawater salinity diminished photosynthesis rate and photosynthesisrelated parameters, such as stomatal conductance, intercellular CO2 concentration, leaf water and osmotic potential and relative leaf water content in both growth stages and have reduced tiller number, leaf area and top dry matter content in tillering stage. We have also studied the effect of salt-stress on the mineral content at 35 DAT. Na+ concentration increased, whereas Ca2 + , Mn2 + and K+ concentration were decreased with increasing stress. Seawater decreased fertile spikelets in the panicle, decreased accumulation of dry matter in the grain and concomitantly decreased grain yield. Foliar spray of Ca(NO3)2, MnSO4 or K2HPO4 partially minimized the salt-induced nutrient deficiency, increased photosynthesis, dry matter accumulation, number of fertile spikelet in the panicle and grain yield. Among the nutrient solutions, Ca(NO3)2 seemed to be the most effective, followed by MnSO4 and K2HPO4. These results suggested that foliar application of nutrient solutions partially alleviates the adverse effects of salinity on photosynthesis and photosynthesis-related parameters, yield and yield components through mitigating the nutrient demands of salt-stressed plants. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Growth stages; Mineral elements; Oryza sati6a; Photosynthesis; Seawater salinity
Abbre6iations: A, photosynthesis rate; Ci, internal CO2 partial pressure; DAT, days after transplanting; EC, electrical conductivity; gs, stomatal conductance; RLWC, relative leaf water content; s, osmotic potential; w, leaf water potential. * Corresponding author. Tel.: + 81-25-2626610; fax: +81-25-2626854. E-mail addresses: [email protected] (N. Sultana), [email protected] (T. Ikeda). 1 Present address: Plant Breeding Institute, The University of Sydney, P.O. Box 219, Narrabri, NSW 2390, Australia. S0098-8472/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 9 8 - 8 4 7 2 ( 0 1 ) 0 0 0 9 0 - 9
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1. Introduction Soil salinity is a major environmental stress that adversely affects plant growth and metabolism. Coastal rice crops in Asia are frequently affected by exposure to seawater brought in by cyclones around the Indian Ocean. This incidental exposure to salt occurs either during the vegetative or reproductive or both phases, presenting an increasing threat to rice productivity. Salt salinity affects plant physiology through changes of the water and ionic status in the cells (Sultana et al., 1999; Kashem et al., 2000b; Hasegawa et al., 2000). Ionic imbalance occurs in the cells due to excessive accumulation of Na+ and Cl− and reduces the uptake of other mineral nutrients, such as K+, Ca2 + and Mn2 + (Cramer and Nowak, 1992; Khan et al., 1997; Lutts et al., 1999). The function of Mn2 + at the cellular level of plant is to bind firmly to lamellae of chloroplasts, possibly to the outer surface of thylakoid membranes, affecting the chloroplast structure and photosynthesis (Lidon and Teixerira, 2000). External application of Mn2 + increased photosynthesis, net assimilation and relative growth in barley under salinity (Cramer and Nowak, 1992). Cytosolic Ca2 + is not only involved in biosynthesis and intracellular transport of proteins, but also in signaling components in rice plant (Kashem et al., 2000a). External supplied Ca2 + has been shown to ameliorate the adverse effect of salinity in plants, presumably by facilitating higher K+/ Na+ selectivity (Hasegawa et al., 2000). The role of K+ is vital for osmoregulation and protein synthesis, maintaining cell turgor and stimulating photosynthesis (Peoples and Koch, 1979). Higher levels of K+ in young expanding tissue is associated with salt tolerance in many plants (Gorham, 1993; Khatun and Flowers, 1995). NaCl also changes the anion concentrations in plants. A lowered supply of nitrate to growing leaves may be responsible for the inhibition of growth under saline conditions (Hu and Schmidhalter, 1998). Salinity can affect growth, dry matter accumulation and yield of rice (Sultana et al., 1999; Asch et al., 2000). The reduction in growth of salinized plants may result from the effect of salt on water status, ionic imbalance, nutrient and phytohor-
monal status, physiological processes, biochemical reactions or a combination of such factors (Volkmar et al., 1998; Hasegawa et al., 2000; Kashem et al., 2000b) and accompanied by a reduction of photosynthesis (Sultana et al., 1999). Low yield of grain under salinity might result from a number of causes, such as loss of photosynthetic capacity (Sultana et al., 1999; Horton, 2000), decreased the assimilates accumulation in the grain (Sultana et al., 1999; Asch et al., 2000) and reduction of seed setting in the panicles (Khatun and Flowers, 1995). Evidence accumulated that rice is a salt sensitive crop with peak sensitivity in the early seedling and early reproductive stages (Yeo et al., 1990; Lutts et al., 1995). Though a wide range of investigation has been carried out on the effects of salinization in a number of crops, the problem of seawater exposure and how to overcome this problem have not yet been sufficiently investigated in rice. To overcome the problem, plant breeders and physiologists have directed considerable efforts toward developing cultivars and agromanagement techniques to improve the physico-chemical properties of rice plants grown under saline conditions. Fertilizer application is now being tried to alleviate or neutralize growth inhibition due to salinization. Many physiological and chemical studies have also been conducted on the addition of nutrients to the soil medium in response to conditions of salt stress (Song and Fujiyama, 1996a,b; Abdel-Rahman, 1999). However, no studies have been undertaken concerning the ameliorative effects of foliar sprays of fertilizer solutions containing Ca, Mn or K applied during the early tillering and early reproductive stages of rice crops grown under seawater stress. The aim of this study was to test the hypothesis that foliar sprays of fertilizer solutions containing Ca, Mn or K could ameliorate the stress in rice plants caused by sea water salinity. For this purpose, rice plants at the tillering and early reproductive stages were sprayed with solutions of Ca(NO3)2, MnSO4 or K2HPO4 and we studied the effects of salt exposure on photosynthesis and its related parameters, mineral concentration, dry matter accumulation and yield and yield contributing characters of the rice plant.
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2. Materials and methods A moderately salt resistant, improved, semidwarf japonica rice variety (Oryza sati6a L. cv. Kosihikari) was germinated in a tray filled with silty clay-loam soil in a glasshouse at Niigata University, Japan. In this study, we conducted two separate experiments from April to September 1999. Each experiment was arranged as a two-way factorial experiment in a randomized block design with three replications. Each replication contained ten pots and each pot contained three seedlings.
2.1. Tillering stage (Experiment 1) Seedlings (25-day-old) were transplanted (May 15) to 3-l plastic pots filled with fertilized sandy loam soil having pH 7.4. The pots were kept in the well-ventilated glasshouse during the whole growing season. The air temperature and relative humidity in the glasshouse was in the range from 22 to 32°C and 60– 80%, respectively. Fertilizers were applied at 12 g N m − 2 (as NH4Cl), 8 g P m − 2 (as superphosphate) and 10 g K m − 2 (as KCl). Half of the N was applied as a basal dose in both the experiments, a quarter was applied at tillering stage and the rest was applied at panicle initiation stage. Seawater was collected from Japan sea (near Niigata University) and its electrical conductivity (EC) was 61 ms cm − 1. After transplanting, the base of each plant was covered with a plastic sheet to prevent the contamination of sprayed nutrients and also to minimize the evaporation. From May 25 (10 DAT) to June 20 (35 DAT), each pot was watered with a 0, 8.8, 17.5 or 35% of sea water (equivalent to EC about 0.9, 5.7, 11.5 or 21.5 ms cm − 1, respectively). These solutions were prepared by diluting seawater with deionized water and were applied far in excess of the saturation capacity of the soil. To maintain the required soil medium salt levels, the EC of the soil medium was measured periodically by portable EC meter and the required amount of treated water was added. From 10 to 35 DAT, each pot was sprayed twice a week with 1 mM
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solutions of Ca(NO3)2·4H2O, K2HPO4·12H2O or MnSO4·4H2O. Deionized water was sprayed as a control. Tween-20 (0.1%) was used as a wetting agent for each treatment, including the water as control. We sprayed the plants with solution until the leaves were completely wet and the solution ran off the leaves.
2.2. Reproducti6e phase (Experiment 2) The experiment was conducted from the end of flag leaf stage (30 July, 75 DAT) to milking stage (25 August, 100 DAT). The experimental conditions, such as sowing and cultural practices, were the same as in Experiment 1 and all the treatments were applied as described above.
2.3. Measurement of photosynthesis, photosynthetic parameters, leaf water and osmotic potential Photosynthetic rate (A), stomatal conductance (gs), intercellular CO2 concentration (Ci), water (w) and osmotic (s) potentials and relative leaf water content (RLWC) of the young expanded leaves (Experiment 1) and flag leaves (Experiment 2) were measured at tillering stage (30 DAT, June 15) and milking stage (95 DAT, August 20), respectively, according to the method previously described by Sultana et al. (1999).
2.4. Measurements of growth characteristics For measuring the tiller number and the leaf area, five plants were cut randomly at soil surface level at 35 DAT and then counted tiller number. Leaf area was measured with a leaf area meter (Automatic Area Meter, Model AAM-7, Hayashi Denko Co. Ltd.).
2.5. Estimation of mineral concentrations Oven dried samples of 35 DAT old plants were digested by HNO3 –HClO4, and Na+, Ca2 + , Mn2 + and K+ were analyzed by atomic absorption spectrophotometry (Mitsui et al., 1999).
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2.6. Measurement of dry weight, yield and yield components In both experiments, randomly five plants were cut at soil level at 35 DAT and after harvest, respectively. At tillering stage, dry weight of stem with leaf (top dry weight) was measured. After harvesting (30 September) five panicles form each treatment were taken randomly for counting the number of fertile spikelet/panicle. After counting the number of fertile spikelets, the same panicle was used for the measuring of panicle dry weight. The samples were oven-dried at 60°C for 72 h and the dry weight was recorded. For the dry weight of the developing grain, 20 ovaries (dehulled grain) were collected at dough stage (105 DAT, 30 August) for each treatment and were dried at 60°C for 48 h. Yield/plant also measured from the collected five plant for each replication.
2.7. Statistical analysis The MSTAT Development Team (1989) (MStat-C) software program was used for data analysis. The data were analyzed and the mean differences were adjudged as per LSD test.
3. Results Except slight yellowing of the leaves, no visible symptoms of salt injury were observed on the plants at 8.8 and 17.5% of seawater salinity, but at 35% seawater salinity, severe necrotic and chlorotic symptoms were present on leaves both at the tillering stage and early reproductive phases of growth (data not shown).
3.1. Effect of seawater and foliar spray on mineral content Mineral contents in the seawater treated plant are presented in Fig. 1. Elementary analysis of plant materials revealed that the concentrations of Na+ increased by 1.57, 2.14 and 2.86-fold in 8.8, 17.5 and 35% seawater treated plants, respectively (Fig. 1a). Seawater salinity decreased Ca2 + by 46%, Mn2 + by 36% and K+ by 37% at 35%
seawater treated plants compared to the control (Fig. 1b–d). Foliar application of nutrient solutions increased the mineral content up to 4% in the control plant (0% of seawater), whereas it was increased for Ca2 + by 25%, Mn2 + by 18% and K+ by 21% in 35% seawater treated plant (Fig. 1b–d).
3.2. Effect of seawater and foliar spray on photosynthesis and its related parameters We have studied the effects of seawater salinity and foliar spray of nutrients on photosynthesis (A) and its related parameters at 30 and 95 DAT (Tables 1 and 2). Salt stress led to a significant inhibition of leaf A (Table 1). The rate of photosynthesis was decreased when concentration of seawater increased from 8.8 to 35%. Salinity-induced low A was partially overcome when we applied nutrient solutions to the plants. For example, A was increased by 22% an application of Ca(NO3)2 in the 35% seawater treated plant at 30 DAT (Table 1). The increase in the salinity significantly decreased the gs of leaves (Table 1); however, a foliar spray of nutrients increased the gs compared to the control treatment. The tendency of increased A and gs were highest with the Ca(NO3)2 treatment; compared to other treatments. The Ci was not greatly affected in this stage of growth, but external application of nutrient solution improved the stress response of Ci (Table 1). Leaf water potential, osmotic potential and relative leaf water content (RLWC) significantly decreased with increasing concentrations of salinity at both growth stages (Table 2). Foliar application of nutrient solutions significantly reduced the decreasing tendency of these parameters; the Ca(NO3)2 treatment was most effective, followed by MnSO4 and K2HPO4.
3.3. Effect of seawater and foliar spray on growth characteristics at tillering stage The effects of seawater salinity and foliar application of nutrient solutions on growth characteristics of rice plant were studied at 35 DAT (Fig. 2) Seawater salinity significantly decreased the tiller number, leaf area and top dry weight (Fig. 2).
Sea water conc. (%)
Spray treatments
30 DAT
95 DAT
A (mmol CO2 m−2 s−1)
gs (mol CO2 m−2 s−1)
Ci (ppm)
A (mmol CO2 m−2 s−1)
gs (mol CO2 m−2 s−1)
Ci (ppm)
0
Water Ca(NO3)2 MnSO4 K2HPO4
20.3 90.47a 21.8 9 0.55a 21.4 9 0.23a 20.9 9 0.51a
0.86 9 0.02b 0.82 90.02b 0.85 90.02ab 0.87 90.01a
221 9 1.7b 224 92.3a 223 91.1ab 221 92.1b
24.9 9 1.15bc 28.3 9 1.73a 27.5 9 0.23ab 27.2 9 1.15ab
1.4 9 0.23a–c 1.6 90.11a 1.5 90.17ab 1.5 90.28ab
3029 5.7ab 308 9 4.6a 303 9 4.4ab 304 9 5.1ab
8.8
Water Ca(NO3)2 MnSO4 K2HPO4
16.3 90.51c 18.5 9 0.54b 17.8 9 0.46bc 17.0 9 0.26bc
0.45 9 0.03ef 0.57 90.03c 0.54 90.02c 0.52 90.02cd
196 9 3.7e 207 94.3c 204 92.1d 200 91.1de
20.5 9 0.51ab 23.0 9 1.73cd 22.7 9 0.57cd 21.9 9 0.57cd
0.75 90.05b–d 0.88 90.046a–d 0.84 90.023a–d 0.80 90.058a–d
2929 6.7c 3009 8.6a–c 2979 4.1bc 2959 2.9bc
17.5
Water Ca(NO3)2 MnSO4 K2HPO4
12.1 9 0.15e 13.6 9 0.4d 12.9 9 0.57de 12.5 9 0.26de
0.33 90.02g 0.48 90.01de 0.43 90.01f 0.40 90.02f
180 9 2.7h 188 92.8f 186 92.3fg 185 92.9g
14.7 90.4e 16.8 9 1.73e 15.9 9 1.15e 15.3 9 0.58e
0.54 9 0.023d 0.65 90.028de 0.61 90.020cd 0.60 90.028cd
270 9 5.8d 272 9 4.6d 269 9 5.2d 265 9 2.9d
35
Water Ca(NO3)2 MnSO4 K2HPO4
7.4 90.23g 9.6 90.23f 8.8 90.34fg 8.4 90.15fg
0.15 9 0.01i 0.25 90.02h 0.23 90.01h 0.22 90.01h
170 91.1j 175 9 2.3i 172 9 1.1j 171 9 1.7j
9.1 9 0.57f 11.1 9 0.26f 9.9 9 0.51f 9.5 9 0.57f
0.24 90.023d 0.32 9 0.011d 0.30 9 0.028d 0.27 9 0.028d
207 9 4.1e 215 92.9e 211 96.4e 208 94.6e
a
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Table 1 Effects of foliar spray of nutrient solutions on photosynthetic rate (A), stomatal conductance (gs), internal CO2 partial pressure (Ci) of rice grown under seawater salinity at 30 and 95 DATa
The values are the mean 9 S.E. (n =3); different letters indicate significant differences among the treatments at 1% level of significance in LSD test.
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However, the application of nutrient solution partially mitigated the adverse effects of salinity on these parameters.
Ca(NO3)2, MnSO4 or K2HPO4 seems to be increased the dry matter accumulation in the grain (Fig. 3a).
3.4. Effect of seawater and foliar spray on dry matter accumulation in de6eloping grains
3.5. Effect of seawater and foliar spray on yield and important yield components
The effect of seawater salinity and foliar spray of Ca(NO3)2, MnSO4, or K2HPO4 on dry matter accumulation of developing grains were examined (Fig. 3a). Salinity had a slight effect on grain dry matter at lower concentration and initial stages of growth (data not shown), but the effect was aggravated by the high concentration and long duration of salinity (Fig. 3a). Foliar spray of
Panicle dry weight, number of fertile spikelets/ panicle and yield/plant were measured after harvesting (Fig. 3b–d). There were significant differences (PB 0.01) for salinity and sprayed treatments for these parameters, but the interaction effect between salinity and spray was not significant for these parameters (data not shown). All these parameters decreased along with the
Fig. 1. Effect of seawater salinity and foliar spray of nutrient solutions either calcium, manganese or potassium on Ca, Mn and K concentration in rice leaves at 35 DAT. Vertical bars indicate mean 9 S.E. (n =3); different letters indicate significant differences among the treatment at the 0.01 probability level based on an LSD test.
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Table 2 Effects of foliar spray of nutrient solutions on leaf water potential (w), osmotic potential (s) and relative leaf water content (RLWC) of rice grown under seawater salinity at 30 and 95 DATa Seawater conc. (%)
Spray treatments
30 DAT
95 DAT
w (MPa)
s (MPa)
RLWC (%)
w (MPa)
s (MPa)
RLWC (%)
0
Water Ca(NO3)2 MnSO4 K2HPO4
−0.669 0.03cd −0.629 0.01d −0.639 0.02d −0.659 0.03cd
−0.8390.01g −0.8090.01g −0.8090.02g −0.929 0.01fg
69 9 2.9ab 70 9 2.3a 68 9 2.9a–c 69 9 1.2ab
−0.869 0.03fg −0.829 0.01g −0.839 0.02fg −0.859 0.03fg
−1.39 0.17e–g −1.290.11fg −1.290.06fg −1.190.06g
669 1.2ab 67 9 2.3a 67 9 1.2a 65 91.7bc
8.8
Water Ca(NO3)2 MnSO4 K2HPO4
−0.939 0.01b −0.889 0.02b–d −0.919 0.02bc −0.909 0.03bc
−1.190.06ef −0.949 0.02fg −0.959 0.02fg −0.9690.02bc
66 9 2.3a–d 68 92.3a–c 67 91.2a–d 67 9 0.6a–d
−1.039 0.06dfg −0.9990.05e–g −1.0190.02e–g −1.09 0.06e–g
−1.690.11dfg −1.390.17e–g −1.490.11e–g −1.590.11deg
62 91.2e 6592.3bc 649 1.7cd 63 91.7de
17.5
Water Ca(NO3)2 MnSO4 K2HPO4
−1.190.03b −0.999 0.01b −1.09 0.02b −1.049 0.03b
−1.690.11b −1.29 0.01de −1.490.15cd −1.590.11c
62 9 0.33d–f 65 90.6b–d 64 90.6c–e 64 9 1.1c–e
−1.49 0.23cd −1.190.10d–f −1.290.15c–e − 1.3 90.17cd
−2.09 0.28b–d −1.790.28d–f −1.890.15c–e −1.9790.11cd
55 9 0.57g 59 9 1.6f 58 9 2.3f 56 91.23g
35
Water Ca(NO3)2 MnSO4 K2HPO4
−1.99 0.11a −1.59 0.14a −1.69 0.17a −1.79 0.11a
−2.59 0.15a −2.190.10b −2.290.11b −2.390.10ab
56 91.1g 60 91.7fg 59 9 1.1fg 57 9 0.6g
−2.090.28a −1.790.17b −1.89 0.34ab −1.89 0.17ab
−2.790.17a −2.390.17a–c −2.59 0.28ab −2.69 0.12a
50 91.2j 5391.7a–c 52 9 1.2hi 51 9 1.73ij
a The values are the mean 9 S.E. (n=3); different letters indicate significant differences among the treatments at 1% level of significance in LSD test.
increasing concentrations of salinity. However, seed setting (number of fertile spikelets) and subsequently, grain yield were very sensitive to seawater salinity. Foliar spray of Ca(NO3)2, MnSO4 or K2HPO4 increased the number of panicles, seed setting in the panicle and grain dry weight, finally resulted in increased grain yield (Fig. 3c). Panicle dry weight, number of fertile spikelet/panicle and yield/plant were higher in Ca(NO3)2, followed by MnSO4 or K2HPO4 (Fig. 3b– d).
4. Discussion Seawater contains high concentration of Na salts and salinity reduced availability of nutrients in the soil (Muhammed et al., 1987). We have analyzed some cations, such as Na+, Ca2 + , Mn2 + and K+ in seawater-stressed rice (Fig. 1). At 35% of seawater, treated rice plant accumulated 2.9-fold higher Na+ and decreased Ca2 + by
46%, Mn2 + by 36% and K+ by 37% compared to the control. Salt induced nutrient deficiency has been reported by many researchers (Cramer et al., 1991; Khan et al., 1997; Lutts et al., 1999). The molecular mechanism of the discrimination of nutrient uptake under salinity is still unclear. However, the possible causes for discrimination of nutrient uptake under seawater salinity in the rice plant are: (a) ions in the high concentration in the external solution (i.e. Na+ or Cl−) are taken up at high rate, which may lead to excessive accumulation in the tissue. These ions may inhibit the uptake of other ions into the root (i.e. K+ or Ca2 + ) and their transport into the shoot through the xylem, eventually leading to deficiency in the tissue. (b) Na+ and Cl− transport across the plasma membrane in a saline environment must be considered in two cellular contexts, after salt stress shock and after re-establishment of ionic homeostasis. Immediately after salt stress, the H+ electrochemical gradient is altered. Influx of Na+
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dissipates the membrane potential, thereby facilitating the uptake of Cl− down the chemical gradient. An anion channel has been implicated in this passive flux. On the other hand, Na+ competes with K+ for intracellular influx because these cations are transported by common protein (Hasegawa et al., 2000). However, our important target is how to minimise the salt-induced nutrient deficiency in the rice plant. To overcome salt-induced nutrient defi-
ciency, we have sprayed respective nutrient solutions combined with surfactant in the shoot. At 35% of seawater, treated leaves contained :20% higher element concentration compared to unsprayed leaves. Our results are in agreement with the findings of many workers in different plant species (Smith et al., 1991; Abdel-Rahman, 1999) who found that nutrients were absorbed by the leaves when applied onto the shoot.
Fig. 2. Effect of nutrient solutions either Ca, Mn or K on tiller number, leaf area and top dry weight of rice plant grown under seawater salinity at 35 DAT. Vertical bars indicate mean 9S.E. (n =3); different letters indicate significant differences among the treatment at the 0.01 probability level based on an LSD test.
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137
Fig. 3. Effect of nutrient solutions containing either Ca, Mn or K on grain, panicle dry weight, fertile spikelets and yield of rice grown under seawater salinity. Vertical bars indicate mean 9S.E. (n =3); different letters indicate significant differences among the treatment at the 0.01 probability level based on an LSD test.
In the present study, we observed that seawater reduced the rate of photosynthesis and its related parameters (Tables 1 and 2), decreased growth characteristic such as tiller number, leaf area and
dry matter accumulation (Fig. 2) and decreased yield contributing characters, such as panicle number, fertile spikelets, grain dry matter accumulation and yield (Fig. 3). Foliar application of
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nutrient solutions partially overcame salt-induced detrimental effects in rice (Tables 1 and 2; Figs. 2 and 3). Understanding the regulatory mechanisms of photosynthesis in response to environmental changes is therefore important for plant productivity. Seawater salinity significantly reduced the rate of photosynthesis (Table 1). The rate of photosynthesis depends on stomatal and nonstomatal components (Bethke and Drew, 1992) and each of the components has a unique response to an environmental variable. Stomatal conductance is related to turgor pressure of cells. The turgor pressure is controlled by solute regulation within the guard cell protoplast and the relative water content of epidermal tissues. Accumulation of K+ and other organic ion increased the osmotic activity, causing a reduction in water potential and an influx of water from the surrounding cells. In addition, phytohormones and Ca2 + play important signaling role on the regulation of stomata (Sage and Reid, 1994) and Mn2 + bind firmly to the lamellae of chloroplasts, possibly to the outer surface of thylakoid membranes, affecting the chloroplast structure and photosynthesis (Lidon and Teixerira, 2000). Optimum balance of these physico-chemical components in the cells is directly or indirectly related to photosynthesis. Seawater salinity increased the water deficit and decreased the mineral nutrients that activate the ionic imbalance in the cytosol, leading to creating an unfavorable environment, therefore, reduced the rate of photosynthesis. The minerals Na+, Ca2 + , K+ and Mn2 + are not only involved in the regulation of photosynthesis but have other cellular regulatory function, which are directly or indirectly involved in growth and physiology of the plant. K+ plays a predominantly osmotic role in plants (Marshall and Porter, 1991). NaCl inhibits Ca2 + transport from roots to shoot by interfering with the release of Ca2 + into the root xylem, possibly via an effect on the active loading of Ca2 + into xylem vessels and salt stress inhibits the supply of Ca2 + to the shoot in several species (Lynch and La¨ uchli, 1985). However, supplementation of Ca2 + , Mn2 + and K+ through the nutrient solution increased the rate of photosynthesis and growth of salt-stressed plants by increasing
the concentration of Ca2 + and K+ (Colmer et al., 1996; Liu and Zhu, 1997) and Mn2 + (Cramer and Nowak, 1992) in shoots and leaves. Although Sohan et al. (1999) reported that Ca2 + supplements did not significantly ameliorate the negative effects of NaCl, our results showed that a nutrient solution providing supplemental calcium or potassium ameliorated the effects of seawater exposure on photosynthesis and photosynthesis-related parameters. The increased salt tolerance in rice by the addition of Ca(NO3)2 and K2HPO4 reported in our study is in agreement with other findings, though those researchers did not apply the nutrient solution as a foliar spray (Lopez and Satti, 1996; Song and Fujiyama 1996a,b; Cuartero and Ferna´ ndez-Mun˜ oz, 1999). We did not check whether the acclimation was due to the effect of the cation or anion of the foliar applications of Ca(NO3)2 and K2HPO4. We suggested that the − anion (NO− 3 or HPO4 ) may also be responsible for amelioration of photosynthesis and its related parameters because these two anions are important for the growth and development of plants, but Cramer and Nowak (1992) suggested that Mn and not SO4 was responsible for increasing the photosynthesis in salt-stressed barley plant when MnSO4 was sprayed. The deleterious effects of seawater on growth is partly due to the inhibition of photosynthesis, partly due to the induction of growth inhibitor (abscisic acid) which cause premature senescence of leaves and reduced leaf area (Arif and Tomas, 1993; Kashem et al., 2000b), partly due to decreased leaf protein content resulting from higher accumulations of Na+ and Cl− concentrations (Sultana et al., 1999), partly due to reduced ability to produce and utilize assimilates/photosynthates, reducing the supply of assimilates to the growing regions (Munns et al., 1995; Kashem et al., 2000b) and partly due to a nutrient imbalance caused mainly by lower absorption rates of K and lower utilization rates of K, Ca and P (Romero and Maran˜ o´ n, 1994). In agreement with this report, we found that seawater salinity reduced photosynthesis, reduced leaf area, imbalanced the mineral contents and as a result, decreased the growth of plants. We also found, however, that these detrimental effects of seawater on growth could be
N. Sultana et al. / En6ironmental and Experimental Botany 46 (2001) 129–140
partially alleviated by application of nutrient solutions through decreasing the nutrient demand in salt-affected plants. Seed setting (fertile spikelet) is one of the major contributors to grain yield. In this study, we observed that seed is far more sensitive to salinity than other characteristics that contribute to yield and thus, grain yield was very sensitive to salinity. A lower rate of assimilate translocation from shoots to panicles and reduced pollen viability due to the effect of ionic toxicity may cause a reduction of flowering percentages, increase of floret sterility (Khatun and Flowers, 1995), lower the fertile spikelet/panicle, decrease panicle dry weight and reduce dry matter accumulation in grain and may finally lead to decreased yields, as we found in our study. Reduction in panicle growth by exposure to salt may be due to the strong inhibition of panicle exertion in rice by low hydrolic potentials induced by salt-induced water stress (Ekanayake et al., 1989). Salinity-induced reduction of dry matter accumulation in grains may be due to the reduced rate of photosynthesis and the reduced ability to utilize photosynthates for growth. The reduction in dry matter at the grain filling stage might be through inhibition of photoassimilation at an earlier stage, because high salinity reduces the contents of photosynthetic pigments and soluble proteins in the leaves/ ovaries; this change might cause the decline in leaves/ovary photosynthesis leading to the poor sugar accumulation in the ovaries (Sultana et al., 1999). However, when we applied spray treatments of Ca(NO3)2, MnSO4 and K2HPO4, the accumulation of dry matter increased in contrast to the control treatments, indicating that toxic ions, such as Na+ in the leaves, may interfere with phloem loading, restricting the uptake of nutrients from roots to shoot. Thus, when nutrients are applied to the leaves, the nutrient elements might penetrate into the leaves and restrict the inhibition due to toxic effects of Na+ and Cl− or minimizes the salinity induced nutrient deficiency.
Acknowledgements We are grateful to Dr R. Itoh, Associate Profes-
139
sor, Laboratory of Crop Science, for his kind help. We also thank Dr Nilanta, CSIRO, Australia for critical editing and valuable suggestions. This study was financially supported by the Grant of the Ijiima Food Science Foundation, Tokyo, Japan.
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