OPCE-STR.vp Acta Bot. Croat. 71 (1), 13–29, 2012 CODEN: ABCRA 25 ISSN 0365–0588 eISSN 1847-8476 Effect of supplemental Ca 2+ on NaCl-stressed castor plants (Ricinus communis L.) SEEMA V. JOSHI1, NEHA T. PATEL1, INDU B. PANDEY2, AMAR NATH PANDEY1* 1Department of Biosciences, Saurashtra University, Rajkot-360005, India 2Department of Agronomy, Rajendra Agriculture University, Samastipur-848125, India Abstract – Greenhouse experiments were conducted to assess the effects of supplemental Ca2+ in salinised soil on germination and plant growth response of castor plant (Ricinus communis L. Var. Avani-31, Euphorbiaceae). NaCl amounting to 390 g was thoroughly mixed with soil of seven lots, of 100 kg each, to give electrical conductivity of 4.1 dS m–1. Further, Ca(NO3)2 × 4H20 to the quantity of 97.5, 195, 292.5, 390, 487.5, and 585 g was separately mixed with soil of six lots to give 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25, and 1:1.50 Na+/Ca2+ ratios, respectively. The soil of the seventh lot contained only NaCl and its Na+/Ca2+ ratio was 1:0. Soil without addition of NaCl and Ca (NO3)2 × 4H20 served as control, with a 0:0 Na+/Ca2+ ratio. Salinity significantly retarded seed germination and plant growth, but the deleterious effects of NaCl on seed germination were ameliorated and plant growth was restored with Ca2+ supply at the critical level (1:0.25 Na+/Ca2+ ratio) to salinised soil. Supply of Ca2+ above the critical level further retarded seed germination and plant growth due to the increased soil salinity. Salt stress reduced N, P, K+ and Ca2+ content in plant tissues, but these nutrients were restored by addition of Ca2+ at the critical level to saline soil. In contrast, Na+ content in plant tissues significantly increased in re- sponse to salinity, but significantly decreased with increasing Ca2+ supply to saline soil. The results are discussed in terms of the beneficial effects of Ca2+ supply on the plant growth of Ricinus communis grown under saline conditions. Keywords: Na+/Ca2+ ratio, Ricinus communis, seedling growth, salt tolerance, salt stress Introduction Soil salinity is a major abiotic stress to plant growth and development (SLATER et al. 2003). A high salt content lowers the osmotic potential of soil solution that reduces the soil water potential. Plants can absorb water only as long as the water potential of roots is lower (more negative) than that of the soil solution. In saline soils, plant cells have to decrease their water potential below that of the soil solution by lowering their solute potential ACTA BOT. CROAT. 71 (1), 2012 13 * Corresponding author, e-mail: anpandey2001@gmail.com Copyright® 2012 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved. U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:02 Color profile: Disabled Composite 150 lpi at 45 degrees through accumulation of solutes. This osmotic adjustment causes water stress to plants. In addition, ionic toxicity and many nutrient interactions in salt-stressed plants can reduce plant growth or damage the plants (MARSCHNER 1995, TAIZ and ZEIGER 2006). Salt toleran- ce of plants requires compartmentalization of potentially toxic ions in the vacuole and ac- cumulation of compatible solutes, (organic solutes) in the cytosol where they function in osmotic adjustment and osmoprotection. The osmoprotectants that accumulate most com- monly are proline and glycine betaine, although other molecules can accumulate to high concentrations in certain species (HASEGAWA et al. 2000) Application of gypsum has long been considered a common practice in reclamation of saline-sodic and sodic soils (MARSCHNER 1995). The addition of Ca2+ to the soil (as gyp- sum, lime or other soluble calcium salts) displaces Na+ from clay particles. This prevents the clay from swelling and dispersing (SUMNER 1993) and also makes it possible for Na+ to be leached deeper into the soil. Thus exogenously supplied Ca2+ not only improves soil structure, but also alters soil properties in various ways (SHABALA et al. 2003) that benefit the plant growth. Moreover, an improved Ca2+/Na+ ratio in the soil solution enhances the capacity of roots to restrict Na+ influx (MARSCHNER 1995). The importance of interaction between Na+ and Ca2+ was recognized after LAHAYE and EPSTEIN (1969) reported that exog- enously supplied Ca2+ may significantly alleviate detrimental effects of Na+ on the physio- logical performance of hydroponically grown plants. Since that time, many investigators have become interested in understanding the effects of divalent cations, specifically the ef- fects of Ca2+ on various physiological processes in plants (CRAMER et al. 1985; LAUCHLI 1990; RENGEL 1992; SHABALA et al. 2003, 2006; CHEN et al. 2007; VAGHELA et al. 2009). The spectrum of Na+/Ca2+ interactions in plants seems to be extremely broad, ranging from those at the molecular level, such as reduced binding of Na+ to cell wall or plasma mem- brane, to those manifested at the whole-plant level, such as effects on root and shoot elon- gation growth, increased uptake and transport of K+ or reduced Na+ accumulation in plants (LAUCHLI 1990; RENGEL 1992). Despite the impressive bulk of literature, the interaction of Na+ with Ca2+ in plants still remains unclear. Castor plant (Ricinus communis L.), an oil yielding crop, is native to India, the South Eastern Mediterranean region and Eastern Africa. It is cultivated in tropical regions of In- dia. Moreover, it is extensively cultivated in the marginal saline area of Kutch (north – west saline desert) of Gujarat State of India. This plant is the source of castor beans (used in or- namentation) and castor oil, which is extracted from seeds. The seed cake, which is left over after pressing contains a protein toxin known as ricin. There is evidence that Na+ induces Ca2+ deficiency in plant tissues (CRAMER 1997; PATEL et al. 2010). Consequently, it is assumed that Ca2+ supply to saline soils may mitigate Na+ toxicity to plants. An understanding of how and how far Ca2+ supply modifies respon- ses of plant species to salinity may be of practical significance. In the present investigation calcium nitrate Ca(NO3)2 × 4H2O, which is a nitrogenous fertilizer, was supplied to saline soil and the remedial effects of Ca2+ on salt stressed plants of R. communis were determined by studying germination, growth, water status and acquisition of macro-nutrients. Thus, the present study was designed to improve understanding of Na+/Ca2+ interactions at the whole plant level for this crop species, as such studies are lacking. 14 ACTA BOT. CROAT. 71 (1), 2012 JOSHI S. V., PATEL N. T., PANDEY I. B., PANDEY A. N. U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:02 Color profile: Disabled Composite 150 lpi at 45 degrees Materials and methods Study site The present study was carried out in a greenhouse of the botanical garden of Saurashtra University at Rajkot (22°18’ N Latitude, 70°56’ E Longitude) in Gujarat. For seedling emergence and plant growth the top 15 cm of black-cotton soil, which is predominant in the Saurashtra region of Gujarat, was collected from an agricultural field. This soil is a clayey loam containing 19.6% sand, 20.3% silt and 60.1% clay. The available soil water between wilting coefficient and field capacity ranged from 18.3% to 35.0%, respectively. The total organic carbon content was 1.3% and pH was 7.2. The electrical conductivity of soil was 0.3 dS m–1. N, P, K, Ca and Na contents were 0.15%, 0.05%, 0.03%, 0.05%, and 0.002%, respectively. This soil is fertile and fit for intensive agriculture. Physical and chemical properties of soil are given earlier (PANDYA et al. 2004). Na + /Ca 2+ ratios Surface soil was collected, air dried and passed through a 2 mm mesh screen. Eight lots of soil, of 100 kg each, were separately spread, about 50 mm thick, over polyethylene sheets. Sodium chloride (NaCl) amounting to 390 g was thoroughly mixed with soil of 7 lots to give electrical conductivity of 4.1 dS m–1. The soil was salinised to this level because this plant is cultivated on marginal saline lands in Kutch. Further, calcium nitrate (Ca(NO3)2 × 4H2O) in quantities of 97.5, 195, 292.5, 390, 487.5 and 585 g was separately mixed with soil of six lots to give 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 Na+/Ca2+ ratios, respectively, and then soil salinity for the corresponding lots was 4.3, 4.6, 4.9, 5.0, 5.1 and 5.2 dS m–1. The soil of seventh lot containing only NaCl was considered saline soil and its Na+/Ca2+ ratio was 1:0. There was no addition of NaCl and Ca(NO3)2 × 4H2O to the eighth lot of soil, which served as control with 0:0 Na+/Ca2+ ratio. The electrical conductivity of control soil was 0.3 dS m–1 and this value was approximately equal to 3.0 mM salinity. A total of eight grades of soil, defined according to their Na+/Ca2+ ratios, were used in this study. For the measurement of electrical conductivity a soil suspension was prepared in distilled water at a ratio of 1:2 in terms of weight. The suspension was shaken and allowed to stand over- night. Thereafter, electrical conductivity was determined with a Systronics conductivity meter 304, India. Available Ca 2+ , K + , Na + and Mg 2+ in soil For all grades of soil, Ca2+, K+, Na+ and Mg2+ were extracted with 1N CH3COONH4 adjusted to pH 7.0 and measured by Shimadzu double beam atomic absorption spectropho- tometer AA-6800, Japan following JONES, JR. (2001). Seedling emergence Twenty polyethylene bags for each grade of soil were each filled with 5 kg of soil. Tap water was added to each bag to bring the soils to field capacity and soils were allowed to dry for 7 days. Soils were then raked using fingers and seeds were sown on 15 August 2008. Seeds of R. communis Var. Avani-31 were collected from the saline desert of Kutch. Bags were kept in an uncontrolled greenhouse under natural temperature and light. Ten seeds ACTA BOT. CROAT. 71 (1), 2012 15 EFFECT OF SUPPLEMENTAL Ca2+ ON NaCl-STRESSED CASTOR PLANTS U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:02 Color profile: Disabled Composite 150 lpi at 45 degrees were sown in each bag at a depth of 8–12 mm. Immediately after sowing soils were watered (300 mL water was added to raise the soil moisture to field capacity) and thereafter about 100–150 mL water was added to soil (just to wet the surface soil) on alternate days. Irriga- tion of soil with the required amount of water was taken as a measure to control the Na+/Ca2+ ratio. Emergence of seedlings was recorded daily over a period of 30 days and data of cumulative emergence of seedlings were analysed by t-test (compared 0:0 and 1:0 Na+/Ca2+ treatments) and one-way ANOVA (compared treatments ranging from 1:0 to 1:1.50 Na+/Ca2+). Plant growth For the growth studies, the two seedlings that emerged first were left in each of the 20 bags for each grade of soil and the others were uprooted. Seedlings grown in soils at 0:0 (control), 1:0 (saline), 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 Na+/Ca2+ ratios exhibit- ed emergence of the second leaf after 7, 9, 8, 8, 8, 9, 9 and 9 days, respectively. Emergence of the second leaf confirmed the establishment of seedlings. Following the emergence of the second leaf, the more vigorous of the two seedlings was allowed to grow in each bag and the other was uprooted. Thus twenty replicates for each of eight grades of soil (0:0, 1:0, 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 Na+/Ca2+ ratios) were prepared. This gave a total of 160 bags, which were arranged in twenty randomized blocks. Plants were watered (to raise the soil moisture to field capacity) on alternate days and allowed to grow for 4 months. The experiment was terminated on 15 December 2008. The mean maximum temperature of the greenhouse increased from 31.7 ± 0.6 °C in August to 35.9 ± 0.8 °C in October and thereafter consistently decreased to 30.5 ± 0.6 °C in December 2008. Plants contained in 20 bags at each grade of soil were washed to remove soil particles adhered to roots. Morpho- logical characteristics of each plant were recorded. Shoot height and root length (tap root) were measured. Leaf area was marked out on graph paper. Fresh and dry weights of leaves, stems, tap roots and lateral roots were determined. Water content (g g–1 dry weight) in plant tissues (leaves, stems, tap roots and lateral roots) was calculated using fresh and dry weight values. Data recorded for morphological characteristics, dry weight and water content of tissues were analyzed by t-test to assess the effect of salinity on plant growth and by one-way ANOVA to assess the effect of calcium nitrate treatment on the growth of salinised plants. Determination of water potential and proline content Ten additional plants grown in soil at each grade of soil were used for the measurement of water potential and proline determination in plant tissues. Water potential of leaves, stems, tap roots and lateral root tissues was measured by Dewpoint Potential Meter WP4 (Decagon Devices, Inc.Pullman, WA, USA) following PATEL et al. (2010). All the mea- surements were taken between 8 to 10.30 a.m. Concentration of proline in plant tissues was determined following BATES et al. (1973). Extract of 0.5g fresh plant material with aqueous sulphosalicylic acid was prepared. The extracted proline was made to react with ninhydrin to form chromophore. Toluene was added to terminate the reaction. Optical density of chromophores was measured at 520 nm by a Systronics UV-VIS spectrophotometer 118, India. A stock solution of proline was used to prepare a standard curve for proline concentration and optical density. Data were analyzed by t-test and one-way ANOVA. 16 ACTA BOT. CROAT. 71 (1), 2012 JOSHI S. V., PATEL N. T., PANDEY I. B., PANDEY A. N. U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:02 Color profile: Disabled Composite 150 lpi at 45 degrees Mineral analyses of plant materials Mineral analyses were performed in triplicate on leaves, stems, tap roots and lateral root tissues of seedlings grown at each level of Na+/Ca2+ ratio. Total nitrogen was deter- mined by a micro-Kjeldahl method and phosphorus content was estimated by the chloro- stannous molybdophosphoric blue color method in sulphuric acid (PIPER 1944). Concen- trations of Ca2+, Mg2+, Na+ and K+ were determined by Shimadzu double beam atomic absorption spectrophotometer AA-6800, (Shimadzu Corporation, Kyoto, Japan) after tri- acid (HNO3: H2SO4: HClO4 in the ratio of 10: 1: 4) digestion. Data were analyzed by t-test and one-way ANOVA. Results The concentration of available Ca2+, K+, Mg2+ and Na+ in salinised soil increased with increasing calcium nitrate (Ca(NO3)2 × 4H2O) concentrations (Fig. 1). Salt stress signifi- cantly (p<0.01) reduced the percent emergence of seedlings (Tab. 1). Supply of external Ca2+ to the salinity treatment significantly enhanced the germination percentage (p<0.01) and the process was stimulated. These effects were evident until Na+/Ca2+ ratio in soil increased to 1:0.25 and 1:0.50. Seed germination again decreased with further supply of Ca2+ to salinised soil. ACTA BOT. CROAT. 71 (1), 2012 17 EFFECT OF SUPPLEMENTAL Ca2+ ON NaCl-STRESSED CASTOR PLANTS y = 47.080 + 0.006x (r=0.877, p<0.01) y = 2.960 + 0.0004x (r=0.963, p<0.01) y = 20.130 + 0.001x (r=0.971, p<0.01) y = 40.610 + 0.001x (r=0.956, p<0.01) 0 20 40 60 80 100 0 1000 2000 3000 4000 5000 6000 7000 Ca 2+ added (mg kg ) E x tr a c ta b le C a 2 + , M g 2 + , K + a n d N a + (m g k g ) –1 – 1 Fig. 1. Concentrations of available Ca2+ (�), Mg2+ (�), K+ (�) and Na+ (�)(mg kg–1) in salinised soil in relation to increasing supply of Ca(NO3) × 4H2O. Valus are mean ±SEM. The data points shown correspond to 1:0, 1:0.25, 1:0.50, 1:0.75, 1:1, 1:1.25 and 1:1.50 Na+/Ca2+ ratios respectively, on the X axis. U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:02 Color profile: Disabled Composite 150 lpi at 45 degrees 18 A C T A B O T .C R O A T .71 (1),2012 JO S H I S .V .,P A T E L N .T .,P A N D E Y I.B .,P A N D E Y A .N . Tab. 1. Effect of salinity and Ca2+ nutrition on leaf, stem, shoot and root characteristics of Ricinus communis seedlings as indicated by mean ± SEM. Na+/Ca2+ Total seedling Shoot Root Leaf Leaf Stem Shoot dry weight Tap root Lateral root Total root Root/Shoot ratio emergence height length area dry weight dry weight (leaf+stem) dry weight dry weight dry weight dry weight (%) (cm) (cm) (cm2) (mg) (mg) (mg) (mg) (mg) (mg) ratio 0:0 93 ± 2 42 ± 1 27 ± 1 198 ± 18 660 ± 64 758 ±39 1419 ± 92 132 ± 5 106 ± 12 238 ± 12 0.17 ± 0.1 1:0 84 ± 2 36 ± 1 19 ± 1 144 ± 3 546 ± 24 556 ± 36 1103 ± 51 97 ± 5 72 ± 6 170 ± 7 0.16 ± 0.1 1:0.25 88 ± 2 41 ± 1 25 ± 1 208 ± 9 651 ± 38 762 ± 21 1413 ± 43 134 ± 12 110± 11 244 ± 22 0.17 ± 0.1 1:0.50 92 ± 2 37 ± 0.4 18 ± 1 165 ± 8 616 ± 51 661 ± 43 1277 ± 72 127 ± 11 96 ± 11 223 ± 15 0.18 ± 0.1 1:0.75 80 ± 2 35 ± 1 16 ± 1 152 ± 5 564 ± 31 542 ± 27 1106 ± 29 112 ± 12 81 ± 12 193 ± 18 0.17 ± 0.1 1:1 77 ± 2 31 ± 1 14 ± 1 137 ± 4 520 ± 23 525 ± 35 1045 ± 52 96 ± 5 70 ± 8 167 ± 8 0.16 ± 0.1 1:1.25 67 ± 2 29 ± 1 13 ± 1 128 ± 3 498 ± 20 476 ± 35 974 ± 47 89 ±7 56 ± 6 146 ± 11 0.15 ± 0.1 1:1.50 52 ± 1 27 ± 1 11 ± 0.3 114 ± 4 402 ± 36 404 ± 29 806 ± 50 68± 8 46 ± 6 114 ± 14 0.14 ± 0.1 t – values 3.48** 4.59** 7.29** 3.07** 3.11** 4.06** 4.52** 3.54** 3.12** 4.07** NS F – values 29.82** 39.45** 36.89** 28.16** 5.05** 12.80** 13.17** 4.48** 5.67** 8.00** NS LSD0.05 8.3 3.2 2.0 16.0 106.4 91.5 151.8 30.9 26.2 44.9 NS Results of 1:0 and 0:0 Na+/Ca2+ treatments were compared by t-test. Results of treatments ranging from 1:0 to 1:1.50 were compared by F-test. ** Values are significant at p<0.01, N.S. = Non significant. U : \ A C T A B O T A N I C A \ A c t a - B o t a n 1 - 1 2 \ 4 6 7 J o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 2 C o l o r p r o f i l e : D i s a b l e d C o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s Salinity significantly retarded (p<0.01) elongation of stems and roots (Tab. 1). Supply of Ca2+ to salinity treatment reversed the negative effect of NaCl. For example, stem height and root length of plants grown in soil at 1:0.25 Na+/Ca2+ ratio were almost equal to those of plants grown under control conditions. A further increase in supply of external Ca2+ where Na+/Ca2+ exceeded the 1:0.25 ratio caused reduction in stem height and root length. In addition, salinity significantly reduced (p<0.01) the expansion of leaves. There was recovery in leaf expansion with increase of Ca2+ supply to salinised soil until 1:0.25 Na+/Ca2+ ratio. Following this Na+/Ca2+ ratio in soil, leaf expansion exhibited a decreasing trend. The dry weight of leaves, stems, shoots (leaves + stems), and roots significantly de- creased (p<0.01) in response to salinity (Tab. 1). When compared with the control, the reduction of dry matter caused by salinity was 17.2%, 26.6%, 26.3% and 31.4% for leaves, stems, tap roots and lateral roots, respectively. However, dry weight of tissues exhibited either a complete or a significant recovery (p<0.01) in the plants grown with 1:0.25 Na+/Ca2+ ratio. Ca2+ supplies to the saline soil exceeding 1:0.25 Na+/Ca2+ ratio caused significant decreases in the dry weight of all tissues. Root/shoot dry weight ratio of plants did not change with salinity and Ca2+ treatments. Salt stress significantly reduced (p<0.01) the water content in leaves, stems, tap roots and lateral roots (Tab. 2). Supply of Ca2+ to salinity treatment resulted in a significant reco- very (p<0.01) of water content in tissues. The results suggested that water content in the tis- sues of seedlings increased up to 1:0.25 Na+/Ca2+ ratio and was almost equal to that in con- trol plant tissues. Moreover, water content in tissues exhibited a decreasing trend when Na+/Ca2+ exceeded the 1:0.25 ratio. Tissues according to their water content can be arrang- ed in the decreasing order of lateral roots, tap roots, leaves and stems. Water potential of leaves, stems, tap roots and lateral roots of plants grown in saline soil became significantly (p<0.05) more negative than that in tissues of control plants. It is evident that water potential of tissues of plants grown in soil at 1:0.25 Na+/Ca2+ ratio was significantly (p<0.01) restored. Further increase in the supply of external Ca2+ to salinity treatment again reduced water potential of tissues. According to their water potential (low to high negative values), tissues can be arranged in decreasing order of lateral roots, tap roots, leaves and stems. Proline content significantly increased (p<0.05) in leaves, stems, tap roots and lateral root tissues in response to salinity (Tab. 2). Results suggested that proline content in tissues decreased to minimum level with 1:0.25 Na+/Ca2+ treatments, but it further increased as the external supply of Ca2+ to saline soil increased. According to their proline content tissues can be arranged in decreasing order of leaves, stems, tap roots and lateral roots. Na+ content in the leaf, stem and root tissues of plants significantly increased (p<0.05) in response to salinity (Tab. 3), but increasing the Ca2+ in saline soil significantly reduced (p<0.01) the Na+ content in the tissues. Salinity significantly reduced (p<0.05) K+ content in the tissues. There was a complete recovery in K+ content of plants grown under the 1:0.25 Na+/Ca2+ ratio. Reduction in K+ content in tissues was again recorded when Na+/Ca2+ in soil exceeded the 1:0.25 ratio. The K+/Na+ ratio of tissues significantly decreased (P<0.05) in response to salinity, but increasing supply of Ca2+ to salinity treatment significantly increased (p<0.01) their K+/Na+ ratio. Concentrations of N, P and Ca2+ in the tissue of plants significantly decreased (p<0.05) in response to salinity. It is evident that concentrations of these nutrients were completely restored in tissues of plants ACTA BOT. CROAT. 71 (1), 2012 19 EFFECT OF SUPPLEMENTAL Ca2+ ON NaCl-STRESSED CASTOR PLANTS U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:02 Color profile: Disabled Composite 150 lpi at 45 degrees 20 A C T A B O T .C R O A T .71 (1),2012 JO S H I S .V .,P A T E L N .T .,P A N D E Y I.B .,P A N D E Y A .N . Tab. 2. Effect of salinity and Ca2+ nutrition on water content, water potential and proline content in tissues of Ricinus communis seedlings as indicated by mean ± SEM. Na+/Ca2+ Water Content (g g–1 DW) Water Potential (-MPa) Proline Content (µ mol g–1 FW) ratio Leaves Stems Tap Roots Lateral Roots Leaves Stems Tap Roots Lateral Roots Leaves Stems Tap Roots Lateral Roots 0:0 3.4 ± 0.1 2.6 ± 0.1 3.8 ± 0.1 4.3 ± 0.1 2.9 ± 0.3 4.1 ± 0.1 2.2 ± 0.1 1.6 ± 0.2 26 ± 2 26 ± 1 22 ± 1 18 ± 1 1:0 2.9 ± 0.1 2.0 ± 0.1 3.4 ± 0.0 3.9 ± 0.1 3.8 ± 0.2 4.7 ± 0.1 2.9 ± 0.1 2.4 ± 0.1 34 ± 1 29 ± 1 26 ± 1 22 ± 1 1:0.25 3.3 ± 0.1 2.6 ± 0.1 3.8 ± 0.1 4.1 ± 0.1 3.1 ± 0.1 4.5 ± 0.1 2.4 ± 0.1 1.9 ± 0.2 27 ± 2 25 ± 1 23 ± 1 19 ± 1 1:0.50 3.1 ± 0.1 2.3 ± 0.1 3.6 ± 0.1 3.9 ± 0.1 3.5 ± 0.1 4.6 ± 0.1 2.5 ± 0.1 2.1 ± 0.2 29 ± 1 26 ± 1 24 ± 1 20 ± 1 1:0.75 3.0 ± 0.1 2.1 ± 0.1 3.4 ± 0.1 3.8 ± 0.1 3.7 ± 0.1 4.6 ± 0.0 2.6 ± 0.0 2.3 ± 0.2 32 ± 1 28 ± 2 25 ± 1 21 ± 1 1:1 2.8 ± 0.0 2.0 ± 0.1 3.2 ± 0.1 3.7± 0.0 3.9 ± 0.2 4.7 ± 0.2 2.8 ± 0.3 2.4 ± 0.1 34 ± 0.4 29 ± 1 26 ± 1 21 ± 0 1:1.25 2.7 ± 0.0 1.9 ± 0.1 3.0 ± 0.1 3.5 ± 0.1 4.1 ± 0.1 4.9 ± 0.1 3.0 ± 0.1 2.7 ± 0.2 35 ± 1 30 ± 2 27± 1 22 ± 1 1:1.50 2.6 ± 0.0 1.7 ± 0.1 2.9 ± 0.0 3.3 ± 0.1 4.5 ± 0.1 5.3 ± 0.2 3.9 ± 0.2 3.1 ± 0.1 38 ± 0.3 31 ± 1 27 ± 1 22 ± 2 t - values 4.311** 3.795** 6.165** 4.701** 6.584* 7.235* 6.913* 6.235* 6.547* 6.333* 7.216* 6.621* F - values 9.572** 7.578** 22.605** 32.176** 8.473** 8.672** 10.752** 6.637** 12.060** 5.683** 6.265** 5.625** LSD 0.05 0.2 0.3 0.2 0.1 0.2 0.2 0.2 0.2 1.4 1.5 1.3 1.2 Results of 1:0 and 0:0 Na+/Ca2+ treatments were compared by t-test. Results of treatments ranging from 1:0 to 1:1.50 were compared by F-test. Values are significant at p<0.01 (**) and p<0.05 (*). U : \ A C T A B O T A N I C A \ A c t a - B o t a n 1 - 1 2 \ 4 6 7 J o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 2 C o l o r p r o f i l e : D i s a b l e d C o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s A C T A B O T .C R O A T .71 (1),2012 21 E F F E C T O F S U P P L E M E N T A L C a 2+ O N N aC l-S T R E S S E D C A S T O R P L A N T S Tab. 3. Effect of salinity and Ca2+ nutrition on nutrient content (mg g–1 DW) of tissues (leaf, stem, tap root and lateral root) of Ricinus communis seedlings as indicated by mean ± SEM. Tissue Na+/Ca2+ N K+ P Na+ Ca2+ Mg2+ K+/Na+ Ratio (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) ratio 0:0 23.0 ± 0.7 27.3 ± 0.7 2.4 ± 0.0 8.8 ± 0.1 12.6 ± 0.8 1.1 ± 0.3 3.1 ± 0.1 1:0 20.0 ± 1.2 23.4 ± 0.3 2.0 ± 0.1 10.0 ± 0.3 10.1 ± 0.4 0.9 ± 0.2 2.3 ± 0.1 1:0.25 23.0 ± 0.5 26.6± 0.4 2.5 ± 0.2 9.0 ± 0.3 13.6 ± 0.5 1.1 ± 0.3 3.0 ± 0.1 1:0.50 22.0 ± 0.3 26.1 ± 0.4 2.5 ± 0.2 8.6 ± 0.3 12.6 ± 0.5 1.1 ± 0.2 3.0 ± 0.1 1:0.75 21.0 ± 0.5 24.8 ± 0.8 2.4 ± 0.1 8.2 ± 0.3 12.1 ± 0.2 1.1 ± 0.1 3.0 ± 0.2 Leaf 1:1 21.0± 0.6 24.0 ± 0.3 1.9 ± 0.2 7.4 ± 0.3 11.8 ± 0.4 1.1 ± 0.2 3.3 ± 0.1 1:1.25 20.0 ± 0.2 22.3 ± 0.2 1.7 ± 0.1 6.9 ± 0.2 11.2 ± 0.6 1.0 ± 0.2 3.2 ± 0.1 1:1.50 19.0 ± 0.6 21.9 ± 0.3 1.5 ± 0.2 6.3 ± 0.3 10.9 ± 0.7 1.0 ± 0.2 3.5 ± 0.2 t – values 5.002* 7.986* 5.292* 6.928* 5.339* NS 11.003* F – values 4.679** 18.810** 6.369** 19.805** 5.581** NS 6.650** LSD 0.05 0.8 0.5 0.2 0.4 0.6 NS 0.1 0:0 21.0 ± 1.0 21.6± 0.3 2.2 ± 0.1 9.1 ± 0.1 12.5 ± 0.7 1.0 ± 0.3 2.4 ± 0.1 1:0 19.0 ± 1.2 18.5 ± 0.3 1.9± 0.0 10.8 ± 0.3 10.6± 0.4 0.8 ± 0.2 1.7 ± 0.1 1:0.25 22.0 ± 0.6 21.2 ± 0.8 2.2 ± 0.1 9.4 ± 0.3 13.4 ± 0.4 1.0 ± 0.3 2.3 ± 0.1 1:0.50 21.0 ± 0.5 19.5 ± 0.5 2.1 ± 0.1 8.6 ± 0.2 12.4 ± 0.3 1.0 ± 0.4 2.3 ± 0.1 1:0.75 20.0 ± 0.7 18.5 ± 0.8 2.0 ± 0.1 8.2 ± 0.4 11.8 ± 0.4 1.0 ± 0.2 2.3 ± 0.1 Stem 1:1 18.0 ± 0.7 16.3 ± 0.7 1.8 ± 0.1 7.2 ± 0.2 11.6 ± 0.3 0.9 ± 0.2 2.3 ± 0.1 1:1.25 18.0 ± 0.7 15.2 ± 0.7 1.7 ± 0.1 7.0 ± 0.4 11.1 ± 0.2 0.9 ± 0.1 2.2 ± 0.1 1:1.50 18.0 ± 0.4 14.1 ± 0.8 1.7 ± 0.1 6.7 ± 0.2 10.9 ± 0.7 0.9 ± 0.2 2.1 ± 0.1 t – values 5.774* 5.529* 5.196* 4.348* 7.208* NS 5.232* F – values 4.996** 13.976** 4.807** 24.982** 5.067** NS 5.677** LSD 0.05 0.9 0.8 0.1 0.4 0.5 NS 0.1 U : \ A C T A B O T A N I C A \ A c t a - B o t a n 1 - 1 2 \ 4 6 7 J o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 3 C o l o r p r o f i l e : D i s a b l e d C o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s 22 A C T A B O T .C R O A T .71 (1),2012 JO S H I S .V .,P A T E L N .T .,P A N D E Y I.B .,P A N D E Y A .N . Tab. 3. – continued Tissue Na+/Ca2+ N K+ P Na+ Ca2+ Mg2+ K+/Na+ Ratio (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) (mg g–1 DW) ratio 0:0 20.0 ± 1.0 18.4 ± 0.6 2.0 ± 0.1 9.7± 0.6 11.8 ± 0.1 0.9 ± 0.3 1.9 ± 0.1 1:0 16.0 ± 1.0 13.9 ± 0.4 1.7 ± 0.1 10.8 ± 0.8 10.0 ± 0.3 0.7 ± 0.3 1.3 ± 0.1 1:0.25 20.0 ± 0.4 18.2 ± 0.5 1.9 ± 0.1 9.9 ± 0.7 12.1 ± 0.3 0.9 ± 0.2 1.9 ± 0.2 1:0.50 19.0 ± 0.6 16.9 ± 0.7 1.9 ± 0.1 8.6 ± 0.4 12.1 ± 0.3 0.9 ± 0.2 2.0 ± 0.2 1:0.75 18.0 ± 0.6 16.7 ± 0.3 1.8 ± 0.1 8.4 ± 0.4 11.9 ± 0.3 0.8 ± 0.2 2.0 ± 0.1 Tap root 1:1 18.0 ± 0.5 15.2 ± 0.2 1.5 ± 0.1 7.6 ± 0.2 11.8 ± 0.5 0.8 ± 0.2 2.0 ± 0.0 1:1.25 17.0 ± 0.6 14.6 ± 0.6 1.4 ± 0.2 7.3 ± 0.2 11.7 ± 0.4 0.8 ± 0.2 2.0 ± 0.1 1:1.50 16.0 ± 0.9 13.9 ± 0.9 1.3 ± 0.2 7.0 ± 0.3 10.7 ± 0.3 0.8 ± 0.1 2.0 ± 0.1 t – values 4.703* 4.666* 4.645* 4.715* 4.754* NS 5.871* F – values 4.870** 8.897** 4.508** 8.520** 4.723** NS 4.798** LSD 0.05 0.8 0.7 0.1 0.6 0.5 NS 0.2 0:0 19.0 ± 1.3 13.2 ± 0.5 1.7 ± 0.0 10.2 ± 0.5 14.4 ± 0.6 0.8 ± 0.0 1.3 ± 0.1 1:0 14.0 ± 0.9 8.9 ± 0.4 1.4± 0.0 11.6 ± 0.5 12.6 ± 0.3 0.7 ± 0.1 0.8 ± 0.0 1:0.25 19.0 ± 0.5 13.6 ± 0.7 1.6 ± 0.1 10.5 ± 0.4 15 ± 0.5 1.0 ± 0.1 1.3 ± 0.1 1:0.50 19.0 ± 0.6 13.4 ± 0.5 1.6 ± 0.1 9.7 ± 0.3 14.8 ± 0.4 1.0 ± 0.2 1.4 ± 0.1 1:0.75 18.0 ± 1.0 13.1 ± 0.5 1.6 ± 0.1 8.8 ± 0.3 13.5 ± 0.7 1.0 ± 0.2 1.5 ± 0.0 Lateral root 1:1 18.0 ± 0.6 12.5 ± 0.4 1.4 ± 0.1 7.9 ± 0.5 13.1 ± 0.5 0.9 ± 0.0 1.6 ± 0.1 1:1.25 17.0 ± 1.0 11.7 ± 0.5 1.2 ± 0.0 7.7 ± 0.5 12.8 ± 0.3 0.9 ± 0.3 1.5 ± 0.1 1:1.50 16.0 ± 0.9 11.3 ± 0.6 1.2 ± 0.1 7.3 ± 0.7 12.1 ± 0.1 0.9 ± 0.3 1.6 ± 0.1 t – values 5.879* 5.000* 5.090* 11.094* 6.079* NS 5.120* F – values 4.948** 10.296** 4.963** 10.778** 6.063** NS 8.830** LSD 0.05 1.0 0.6 0.1 0.6 0.5 NS 0.1 Results of 1:0 and 0:0 Na+/Ca2+ treatments were compared by t-test. Results of treatments ranging from 1:0 to 1:1.50 were compared by F-test. Values are significant at p<0.01 (**), and p<0.05 (*), NS = Non significant. U : \ A C T A B O T A N I C A \ A c t a - B o t a n 1 - 1 2 \ 4 6 7 J o s h i . v p 2 6 . o u j a k 2 0 1 2 1 0 : 1 7 : 0 3 C o l o r p r o f i l e : D i s a b l e d C o m p o s i t e 1 5 0 l p i a t 4 5 d e g r e e s grown in soil with a 1:0.25 Na+/Ca2+ ratio. Moreover, high Ca2+ in saline soil reduced the concentration of these nutrients in the tissues. Concentrations of Mg2+ in plants were not significantly affected by Na+ and / or Ca2+ levels in the soil. Discussion The deleterious effects of NaCl on germination of R. communis were ameliorated by in- crease of Ca2+ to a critical level (1:0.25 Na+/Ca2+ ratio) in the salinised soil. The detrimen- tal effect of NaCl salinity on germination is associated with an accumulation of toxic ions (MOHAMMAD and SEN 1990), a decrease of available water to the seeds (PUJOL et al. 2000) or both. The beneficial effect of Ca2+ did not persist when Ca2+ supply exceeded the critical level. In the present study, the concentration of available Na+ and soil salinity increased with increase in the external supply of Ca2+ to the saline soil. Secondly, the water uptake by the germinated seeds decreased with both salinity (20.2 ± 0.5%) and increased Ca2+ levels (13.6 ± 0.6%). Therefore, the beneficial effect of Ca2+ on R. communis seed germination appears due to counteraction of the toxic effect of Na+. An insufficient level of Ca2+ in the germination medium could result in a general deterioration and loss of selectivity of the plasma membrane (WHITTINGTON and SMITH 1992). This aggravates salt effects, probably by increasing membrane permeability and leads to a higher accumulation of toxic ions and/or leakage of solutes (CRAMER et al. 1987, LAUCHLI 1990). A positive response to Ca2+ application on germination rate under saline conditions has also been reported in Phaseolus vulgaris (CACHORRO et al. 1994), in wimmera ryegrass (MARCAR 1986), in barley (BLISS et al. 1986), in Salvadora oleoides (VAGHELA et al. 2009). The detrimental effect of Ca2+, above 1:0.50 Na+/Ca2+ ratio, on seed germination might be due to the decreased osmotic potential of soil solution because soil salinity increased with increase in Ca2+ supply. A reduction in water content and water potential of leaves, stems, tap roots and lateral roots of plants grown in saline soil might have resulted in internal water deficit to plants, which in turn, reduced the elongation of stems and roots and dry matter accumulation in tis- sues. It is found that plants subjected to water stress show a general reduction in size and dry matter production (TAIZ and ZEIGER 2006). In general, salinity can reduce plant growth or damage to the plants through (i) osmotic effect (causing water deficit), (ii) toxic effect of ions and (iii) imbalance of the uptake of essential nutrients (RAMOLIYA et al. 2004). These modes of action may operate on the cellular as well as on higher organizational levels and influence all the aspects of plant metabolism (KRAMER 1983, GARG and GUPTA 1997). R. Communis exhibited a reduction in leaf area (photosynthetic area) in response to salinity treatment. GARG and GUPTA (1997) reported that salinity causes reduction in leaf area as well as in rate of photosynthesis, which together result in reduced crop growth and yield. Also, a high concentration of salt tends to slow down or stop root elongation (KRAMER 1983) and causes reduction in root production (GARG and GUPTA 1997). Supply of Ca2+ to the salinised soil ameliorated the harmful effects of NaCl on R. communis and plant growth was restored at the 1:0.25 Na+/Ca2+ ratio. It has been reported that supplemental Ca2+ in sa- linised growth media alleviated inhibition of barley root growth (SHABALA et al. 2003), shoot growth of Phaseolus vulgaris (CACHORRO et al. 1994), shoot and root growth both in Salva- dora oleoides (VAGHELA et al. 2009). In maize plants grown with a high Na+:Ca2+ ratio, the hydraulic conductance was reduced; supplemental Ca2+ (10 mM) improved growth by re- storing hydraulic conductance back to that of the control plants (CRAMER 1992). ACTA BOT. CROAT. 71 (1), 2012 23 EFFECT OF SUPPLEMENTAL Ca2+ ON NaCl-STRESSED CASTOR PLANTS U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:03 Color profile: Disabled Composite 150 lpi at 45 degrees The inhibiting effect of salinity on plant growth was lowest in leaves and highest for stems, tap roots and lateral roots. Consequently, leaves were more resistant and other tis- sues were sensitive to soil salinity. Likewise, the recovery of dry matter at 1:0.25 Na+/Ca2+ ratio was 98.6%, 100.4%, 101.5% and 103.8% for leaves, stems, tap roots and lateral roots, respectively. Results suggested that there was a resemblance in the shoot and root growth of plants and their root/shoot dry weight ratio did not change with salinity and Ca2+ treatments. Salt tolerance in plants is associated with the accumulation of organic solutes in cytoplasm to balance the osmotic pressure of ions in the vacuoles. Proline accumulates in the cyto- plasm without having any detrimental effects on cytosolic enzymes activities (STEWART and LEE 1974, HASEGAWA et al. 2000). In R. communis, osmotic adjustment was achieved by K+ (as evidenced by higher K+ than Na+ content in tissues) and increase in the quantity of proline in tissues when water content decreased because of salinity. In addition to its conventional osmoprotective role, proline prevents NaCl-induced K+ efflux from roots and may operate as ion channel regulators (CUIN and SHABALA 2005) or reactive oxygen species (ROS) scavengers (BOHNERT et al. 1995). Such a regulatory role does not require signifi- cant amounts of proline to be accumulated and is, therefore, of low carbon cost to the plant. Results further indicated that increase in water content and water potential of tissues with Ca2+ treatment was related to decrease in proline content. In the present study, there was a significant decrease of Ca2+ content in all the tissues with salinity treatment. As a result, Na+ induced Ca2+ deficiency in tissues. It is reported that uptake of Ca2+ from the soil solution may decrease because of ion interaction, precipi- tation and increase in ionic strength that reduce the activity of Ca2+ (JANZEN and CHANG 1987). It is found that salinity can alter Ca2+ uptake and transport leading to Ca2+ deficiency in plants (CRAMER et al. 1987). Consequently, addition of Ca2+ to salinised soil to the critical level resulted in recovery of shoot and root growth. Supply of Ca2+ exceeding the critical level again reduced the shoot and root growth. In the present study, increased nitrate content together with chloride content caused an increase in soil salinity with Ca2+ treat- ment. The increased soil salinity, in other words, the decreased osmotic potential, might be responsible for retardation of growth at high supply of Ca2+. K+ is a major osmoticum in plant cells (MARSCHNER 1995) and, therefore is essential for all extension growth. It is evidenced that in salt-stressed roots of cotton, Na+ displaced membrane-associated Ca2+, which was believed to be primarily located at the plasma mem- brane (CRAMER et al. 1985). In addition, NaCl-salinity displaced membrane-associated Ca2+ on protoplasts of corn (LYNCH and LAUCHLI 1988) and barley (BITTISNICH et al. 1989), and on plasma membrane vesicles of melon (YERMIYAHU et al. 1994). One consequence of the displacement of membrane-associated Ca2+ by Na+ is the immediate increase of K+ efflux across the plasma membrane of salt-stressed cotton roots (CRAMER et al. 1985). This effect may be related to the rapid depolarization of the membrane potential upon salinisa- tion (CRAMER 1997). In the present study, the increased efflux of K+ might be one of the rea- sons for the significant decrease of K+ content in tissues of R. communis in response to NaCl salinity. However, recovery of K+ content in tissues with external Ca2+ supply at the critical level (1:0.25 Na+/Ca2+ ratio) may be the result of repolarization of membrane. There is abundant evidence that salinity alters the ion transport and contents of plants (CRAMER 1997). In general, Na+ uptake and concentrations increase and Ca2+ uptake and 24 ACTA BOT. CROAT. 71 (1), 2012 JOSHI S. V., PATEL N. T., PANDEY I. B., PANDEY A. N. U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:03 Color profile: Disabled Composite 150 lpi at 45 degrees concentrations decrease in plant cells and tissues as the external Na+ concentration in- creases (RENGEL 1992, CRAMER 1997). Likewise, as external Ca2+ concentrations increase Na+ uptake and concentrations decrease and Ca2+ uptake and concentrations increase. One consequence of these Na+:Ca2+ interactions is the reduction of K+ content in salinised plants, which can be prevented with supplemental Ca2+. SHABALA et al. (2006) reported that supplemental Ca2+ may prevent K+ efflux from the cell by blocking the depolarization – ac- tivated outward – rectifying K+ channels. In addition, salinity generates reactive oxygen species (SLATER et al. 2003) which activates non-selective cation channels (NSCC) induc- ing further K+ leak (DEMIDCHIK et al. 2002). This leak is additional to one caused by mem- brane depolarization (CHEN et al. 2007). As a result supplemental Ca2+ may prevent such ROS – induced NSCC activation and associated K+ leak. However, increase in soil salinity with high Ca2+ supply caused a decrease in K+ content in tissues and it can be accounted for low osmotic potential of soil solution. Isosmotic concentrations of mannitol have similar effects as saline treatments with supplemental Ca2+ (10 mM) indicating that K+ efflux is af- fected by osmotic factors in these solutions and not associated with Na+-specific displace- ment of membrane-associated Ca2+ (CRAMER et al. 1985). Na+ content significantly increased in tissues of salt-stressed plants, but decreased with increase in Ca2+ supply to saline soil. It is reported that uptake mechanisms of both K+ and Na+ are similar (SCHROEDER et al. 1994). Na+ can not move through the plasma membrane lipid bilayer, but the ion is transported through both low- and high- affinity transport sys- tems, which are necessary for K+ acquisition. As a consequence, Na+ could enter the cell through high affinity K+ carriers or through the low affinity channels (NSCC) that are strongly influenced by Ca2+. These cation channels could allow entry of large amount of Na+ from a highly saline soil if not adequately regulated (AMTMANN and SANDERS 1999). Low affinity K+ uptake is not inhibited by Na+ but the high affinity process is restricted (SCHROEDER et al. 1994). Similarly Na+ toxicity in plants is correlated with two proposed Na+ uptake pathways (NIU et al. 1995). The K+ and Na+ profiles of R. communis suggest that a similar mechanism might operate in this species. It has been shown that Ca2+ is an ef- ficient blocker of NSCC, a major route for Na+ uptake into the cell (DEMIDCHIK and TESTER 2002, DEMIDCHIK and MAATHUIS 2007) and, thus, may directly reduce the amount of Na+ accumulation in plants. For R. communis, external supply of Ca2+ reduced Na+ content on the whole plant level. Further, the high K+ content and low Na+ content in leaves, stems and tap roots tissues suggest that this plant has the characteristic for rapid transport of K+ to shoot tissues. Intracellular K+/Na+ homeostasis is a key component of salinity tolerance in plants (TESTER and DAVENPORT 2003). In general, salinity reduces N accumulation in plants (FEIGIN 1985).This is due to the fact that an increase in chloride uptake and accumulation is mostly accompanied by a decrease in shoot nitrate concentration (TORRES and BINGHAM 1973, GARG and GUPTA 1997). The interaction between salinity and P is very complex and there is no clear cut mechanism for decreased, increased or unchanged P uptake in response to salinisation in different species (GRATTAN and GRIEVE 1992). However, it is known that P concentration is related to the rate of photosynthesis, but it decreases the conversion of fixed carbon into starch (OVERLACH et al. 1993) and therefore decrease of P in leaves will reduce shoot growth. Besides the role of Mg2+ in chlorophyll structure and as an enzyme cofactor, another important role of Mg2+ in plants is in the export of photosynthates (MARSCHNER ACTA BOT. CROAT. 71 (1), 2012 25 EFFECT OF SUPPLEMENTAL Ca2+ ON NaCl-STRESSED CASTOR PLANTS U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:03 Color profile: Disabled Composite 150 lpi at 45 degrees 1995). External Ca2+ supply reversed the effects of Na+ and concentrations of N and P were restored in tissues of seedlings grown at 1:0.25 Na+/Ca2+ ratio. The high influx or low efflux of nutrients might be responsible for restoration or recovery of nutrients. The increased salinity (low osmotic potential) can be accounted for decrease of nutrients when Ca2+ supply exceeded the critical level. In the present study, available Ca2+ in salinised soil with supplemental Ca2+ at the criti- cal level (1:0.25 Na+/Ca2+ ratio) was two times higher than that in non-saline control soil. Thus, it can be suggested that available Ca2+ in saline soil should be maintained nearly two times higher than that in normal soil in order to ameliorate the injurious effects of NaCl on seed germination and growth of Ricinus communis. Conclusions Results of the present investigation show that germination and growth of R. communis plants were dependent upon external supply of Ca2+ up to the critical level (1:0.25 Na+/Ca2+ ratio) to the salinised soil. Our results are in accordance with the assumption that external Ca2+ supply to the saline soil may alleviate Na+ toxicity to castor plants. The beneficial ef- fects of high Ca2+ concentration are reflected in: (a) the almost complete recovery in germi- nation percentage; (b) the negative effect of soil salinity on elongation of stems and roots, leaf area development and dry matter accumulation in tissues can be reduced by additional supply of Ca2+; (c) water content and water potential of leaves, stems, tap roots and lateral root tissues increased with increase in Ca2+ up to the critical level in salinised soil; (d) it seems that much of growth reduction associated with salinity is due to high Na+ and low Ca2+ levels in tissues, thus increasing Ca2+ concentration reduces the uptake of Na+ and in- creases Ca2+ uptake, consequently decreasing Na+ toxicity; (e) a decrease in the efflux of K+ and probably other mineral nutrients resulted in the restoration of nutrients. Moreover, the beneficial effects of Ca2+ did not persist when the external supply of this element ex- ceeded the critical level because further Ca2+ supply increased soil salinity. Acknowledgement This study was supported with funds from Departmental Special Assistance provided by the University Grants Commission, New Delhi, Government of India. References AMTMANN, A., SANDERS, D., 1999: Mechanisms of Na+ uptake by plant cells. Advances in Botanical Research 29, 76–112. BATES, L. S., WALDREN, R. P., TEARE, F. D., 1973: Rapid determination of free proline from water stress studies. Plant and Soil 39, 205–207. BITTISNICH, D., ROBINSON, D., WHITECROSS, M., 1989: Membrane-associated and intracel- lular free calcium levels in root cells under NaCl stress. In: DAINTY, J., de MICHELIS, M. J., MARRÉ, E. RASI-CALDOGNO, F. (eds.), Plant membrane transport: The current posi- tion. Proceedings 8 International Workshop on Plant Membrane Transport, Venice, 681–682. Elesevier Science Publishing Company, Inc., New York. 26 ACTA BOT. CROAT. 71 (1), 2012 JOSHI S. V., PATEL N. T., PANDEY I. B., PANDEY A. N. U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:03 Color profile: Disabled Composite 150 lpi at 45 degrees BLISS, R. D., PLATT-ALOIA, K. A., THOMSON, W. W., 1986: Osmotic sensitivity in relation to salt sensitivity in germinating barley seeds. Plant, Cell and Environment 9, 721–725. BOHNERT, H. I., NELSON, D. E., JENSEN, R. G., 1995: Adptations to environmental stresses. The Plant Cell 7, 1099–1111 CACHORRO, P., ORTIZ, A., CERDA, A., 1994: Implications of calcium nutrition on the re- sponse of Phaselous vulgaris L. to salinity. Plant and Soil 159, 205–212. CHEN, Z., POTTOSIN, I. I., CUIN, T. A., FUGALSANG, A. T., TESTER, M., JHA, D., ZEPEDA- -JAZO, I., ZHOU, M., PALMGREN, M. G., NEWMAN, I. A., SHABALA, S., 2007. Root plasma membrane transporters controlling K+ /Na+ homeostasis in salt- stressed barley. Plant Physiology 145, 1714–1725. CRAMER, G. R., LAUCHLI, A., POLITO, V. S., 1985: Displacement of Ca2+ by Na+ from the plasmalemma of root cells. A primary response to salt stress? Plant Physiology 79, 207–211. CRAMER, G. R., LYNCH, J., LAUCHLI, A., EPSTEIN, E., 1987: Influx of Na+, K+ and Ca2+ into roots of salt-stressed cotton seedlings. Effects of supplemental Ca2+. Plant Physiology 83, 510–516. CRAMER, G. R., 1992: Kinetics of maize leaf elongation. II. Response of a Na-excluding cultivar and Na-including cultivar to varying Na/Ca salinities. Journal of Experimental Botany 43, 857–864. CRAMER, G. R., 1997: Uptake and role of ions in salt tolerance. In: JAIWAL, P. K., SINGH, R. P., GULATI, A. (eds.), Strategies for improving salt tolerance in higher plants. 55–86. Oxford and IBH Publishing Co., Pvt. Ltd., New Delhi. CUIN, T. A., SHABALA, S., 2005: Exogenously supplied compatible solutes rapidly amelio- rate NaCl-induced potassium efflux from barley roots. Plant and Cell Physiology 46, 1924–1933. DEMIDCHIK, V., TESTER, M. A., 2002: Sodium fluxes through nonselective cation channels in the plant plasma membrane of protoplasts from Arabidopsis roots. Plant Physiology 128, 379–387. DEMIDCHIK, V., BOWEN, H. C., MAATHUIS, F. J. M., SHABALA, S. N., TESTER, M. A., WHITE, P. J., DAVIES, J. M., 2002: Arabidopsis thaliana root nonselective cation channels mediate calcium uptake and are involved in growth. Plant Journal 32, 799–808. DEMIDCHIK, V., MAATHUIS, F. J. M., 2007: Physiological roles of nonselective cation channels in plants: from salt stress to signaling and development. New Phytologist 175, 387–404. FEIGIN, A., 1985: Fertilization management of crops irrigated with saline water. Plant and Soil 89, 285–299. GARG, B. K., GUPTA, I. C., 1997: Saline Wastelands Environment and Plant Growth. Scientific Publishers, Jodhpur, India. GRATTAN, S. R., GRIEVE, C. M., 1992: Mineral element acquisition and growth response of plants grown in saline environments. Agriculture, Eco- systems and Environment 38, 5–300. ACTA BOT. CROAT. 71 (1), 2012 27 EFFECT OF SUPPLEMENTAL Ca2+ ON NaCl-STRESSED CASTOR PLANTS U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:03 Color profile: Disabled Composite 150 lpi at 45 degrees HASEGAWA, P. M., BRESSAN, R. A., ZHU, J. K., BOHNERT, H. J., 2000: Plant cellular and molecular responses to high salinity. Annual Review of Plant Physiology and Plant Molecular Biology 51, 463–499. JANZEN, H. H., CHANG, C., 1987: Cation nutrition of barley as influenced by soil solution composition in a saline soil. Canadian Journal of Soil Science 67, 619–629. JONES, JR., J. B., 2001: Laboratory guide for conducting soil tests and plant analysis. CRC Press LLC, New York. KRAMER, P. J., 1983: Water relations of plants. Academic Press, New York. LAHAYE, P. A., EPSTEIN, E., 1969: Salt toleration by plants: enhancement with calcium. Science 166, 395–396. LAUCHLI, A., 1990: Calcium, salinity and the plasma membrane. In: LEONARD, R. T., HEPLER, P. K. (eds.), Calcium in plant growth. The American Society of Plant Physiolo- gists. 26–35. Rockville MD. LYNCH, J., LAUCHLI, A., 1988: Salinity affects intracellular calcium in corn root proto- plasts. Plant Physiology 87, 351–356. MARCAR, N. E., 1986: Effect of the calcium on the salinity tolerance of Wimmera ryegrass (Lolium rigidum Gaud., cv. Wimmera) during germination. Plant and Soil 93, 129–132. MARSCHNER, H., 1995: Mineral nutrition of higher plants. Academic Press, London. MOHAMMAD, S., SEN, D. N., 1990: Germination behavior of some halophytes in Indian desert. Indian Journal of Experimental Biology 28, 545–549. NIU, X., BRESSAN, R. A., HASEGAWA, P. M., PARDO, J. M., 1995: Ion homeostasis in NaCl stress environments. Plant Physiology 109, 735–742. OVERLACH, S., DIEKMANN, W., RASCHKE, K., 1993: Phosphate translocator of isolated guard-cell chloroplasts from Pisum sativum L. transport glucose-6-phosphate. Plant Physiology 101, 1201–1207. PATEL, A. D., JADEJA, H. R., PANDEY, A. N., 2010: Effect of salinisation of soil on growth, water status and nutrient accumulation in seedlings of Acacia auriculiformis (Faba- ceae). Journal of Plant Nutrition 33, 914–932. PANDYA, D. H., MER, R. K., PRAJITH, P. K., PANDEY, A. N., 2004: Effect of salt stress and manganese supply on growth of barley seedlings. Journal of Plant Nutrition 27, 1361–1379. PIPER, C. S., 1944: Soil and plant analysis. Interscience, New York. PUJOL, J. A., CALVO, J. F., DAIZ, L. R., 2000: Recovery of germination from different osmo- tic conditions by four halophytes from southeastern Spain. Annals of Botany 85, 279–286. RAMOLIYA, P. J., PATEL, H. M., PANDEY, A. N., 2004: Effect of salinization of soil on growth and macro- and micro-nutrient accumulation in seedlings of Salvadora persica (Salva- doraceae). Forest Ecology and Management 202, 181–193. RENGEL, Z., 1992: The role of calcium in salt toxicity. Plant Cell and Environment 15, 625–632. 28 ACTA BOT. CROAT. 71 (1), 2012 JOSHI S. V., PATEL N. T., PANDEY I. B., PANDEY A. N. U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:03 Color profile: Disabled Composite 150 lpi at 45 degrees SCHROEDER, J. I., WARD, J. M., GASSMANN, W., 1994: Perspectives on the physiology and structure of inward-rectifying K channels in higher plants, biophysical implications for K uptake. Annual Review of Biophysics and Biomolecular Structure 23, 441–471. SHABALA, S., SHABALA, L., VOLKENBURGH, E. V., 2003: Effect of calcium on root develop- ment and root ion fluxes in salinised barley seedlings. Functional Plant Biology 30, 507–514. SHABALA, S., DEMIDCHIK, V., SHABALA, L., CUIN, T. A., SMITH, S. J., MILLER, A. J., DAVIES, J. M., NEWMAN, I. A., 2006: Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ – permeable channels. Plant Physiology 141, 1653–1665. SLATER, A., SCOTT, N. W., FOWLER, M. R., 2003: Plant biotechnology. The genetic manipu- lation of plants. Oxford University Press, New York. STEWART, G. R., LEE, J. A., 1974: The role of proline accumulation in halophytes. Planta 120, 279–289. SUMNER, M. E., 1993: Sodic soils: new perspectives. Australian Journal of Plant Physio- logy 31, 683–750. TAIZ, L., ZEIGER, E., 2006: Plant physiology. Sinauer Associates, Inc., Publishers, Sunder- land, USA. TESTER, M., DAVENPORT, R., 2003: Na+ tolerance and Na+ transport in higher plants. Annals of Botany 91, 503–527. TORRES, B. C., BINGHAM, F. T., 1973: Salt tolerance of Mexican wheat. I. Effect of NO3 and NaCl on mineral nutrition, growth and grain production of four wheats. Proceedings of the Soil Science Society of America 37, 711–715. VAGHELA, P. M., PATEL, A. D., PANDEY, I. B., PANDEY, A. N., 2009: Implications of calcium nutrition on the response of Salvadora oleoides (Salvadoraceae) to soil salinity. Arid Land Research and Management 23, 311–326. WHITTINGTON, J., SMITH, F. A., 1992: Calcium-salinity interactions affect ion transport in Chara corallina. Plant Cell and Environment 5, 727–733. YERMIYAHU, U., NIR, S., BEN-HAYYIM, G., KAFKAFI, U., 1994: Quantitative competition of calcium with sodium or magnesium for sorption sites on plasma membrane vesicles of melon (Cucumis melos L.) root cells. Journal of Membrane Biology 138, 55–63. ACTA BOT. CROAT. 71 (1), 2012 29 EFFECT OF SUPPLEMENTAL Ca2+ ON NaCl-STRESSED CASTOR PLANTS U:\ACTA BOTANICA\Acta-Botan 1-12\467 Joshi.vp 26. o ujak 2012 10:17:03 Color profile: Disabled Composite 150 lpi at 45 degrees