Effect of soil salinity on proline content in tissues of Avicinnia officinalis Linn.

              

The dissertation submitted in partfulfilment for the

degree of

MASTER OF SCIENCE

IN

PLANT SCIENCE

BY

Vejlani Aliasgar A.

Department of Biosciences,

Saurashtra university,

Rajkot.

MARCH-2011

Summary

Greenhouse experiments were conducted to assess the effects of soil salinity on emergence, growth, water status, proline content and mineral accumulation of seedlings of Avicennia officinalis  Linnn.. seawater salt was added to the soil and salinity was maintained at 0.3, 2.5, 5.1, 7.7, 10.3, 12.6, 15.4, 17.9, 20.5, 23.0, 25.6,  28.2, 30, 32, 34, and 36 dsm^-1. This mangrove is highly salt tolerant during germination. Growth of seedlings was significantly promoted by low salinity and optimum growth was obtained at 17.9 dsm^-1. Higher salinities inhibited plant growth. Water potential of tissues became significantly more negative with increasing salinity, proline content in tissues significantly increased as salinity increased. The concentration of Na in tissues also increased significantly, whereas K, Ca, and N  content decreased with increase in salinity. Concentration of Mg did not change with increasing salinity.

CONTENTS

                                                                            

1.   INTRODUCTION

2.   MATERIAL AND METHODS

3.   RESULT

4.   DISSUSSION

5.   SUMMARY

6.   REFERENCES

1.     Introduction

Mangroves could tolerate a large range of salinities under natural conditions (Suarez et al., 1998). The growth and physiological mechanisms of mangroves differ in nature due to their complexity of structure and differences in flooding regime, tidal inundation, rapid influx of extra nutrients as well as type of soil (Clough, 1984; Naidoo, 1987). They adapt themselves to the fluctuating environment in several ways such as (i) salt exclusion by root ultra filtration  (Scholander, 1968), (ii) salt recretion via glands (Roth, 1992), (iii) ion accumulation in leaf cells (Popp, 1994), (iv) leaf succulence (Roth, 1992) and (v) accumulating organic acids as osmotica to counter toxic effects of salinity (Popp, 1984). Like other halophytes, mangroves decrease their water and osmotic potentials to maintain turgor at high salinity (Naidoo, 1987; Khan et al., 2000 a, b). Salinity required for optimal growth varies from 10% to 50% seawater (Downton, 1982; Clough, 1984; Naidoo, 1987; Lin and Sternberg, 1992, 1995) and a decline in their optimal growth is obtained with a further increase in salinity. Similarly, lower water potential and accumulation of inorganic ions are the results from extreme saline environments for most of the plants (Ball and Farquhar, 1984; Naidoo, 1987).

The mangroves are found in sandy and muddy intertidal zones of Arabian Sea along semi-arid region of Saurashtra and contiguous saline desert of Kutch in Gujarat State of India. Overexploitation, pollution and other biotic stresses have drastically reduced and fragmented the mangrove forests. Little information exists on the salt tolerance of mangroves of Saurashtra and Kutch and this information is crucial to the success of restoration effort in the region. Avicennia officinalis  Linn.  is a common species along the coasts of Saurashtra and Kutch. It can be planted for mangrove restoration, because it exhibits wide physiological tolerance and it creates an environment suitable for other mangrove species after it has become well established.  It is assumed that A. officinalis   growing along the coasts of Saurashtra and Kutch in Gujarat State of India is acclimated to hypersalinity that prevails in arid regions, and is able to tolerate high level of salinity. Thus, the present study was designed to investigate the tolerance of A. officinalis of Gujarat coasts in India to the multiple (sixteen) salinity levels and such studies are lacking for mangroves growing in arid.

2.      Materials and methods

Study area

The present study was carried out in a greenhouse of the botanical garden of Saurashtra University at Rajkot (22°18'N Lat, 70°56'E Long) in Gujarat. For the emergence and growth of seedlings, the top 15 cm black-cotton soil (Vestisol), which is predominant in 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. Nitrogen, phosphorus, potassium, calcium and sodium 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).

Salinisation of soil

Surface soil was collected, air dried and passed through a 2 mm mesh screen. Sixteen lots of soil, of 100 kg each, were separately spread, about 50 mm thick, over polyethylene sheets. Seawater salt amounting to 160, 400, 680,980, 1200, 1600, 1980, 2320, 2600, 2800, 3000, 3300, 3600, 3800 and 4000 g was then thoroughly mixed with soil of fifteen lots, respectively to give electrical conductivities of 2.5, 5.1, 7.7,10.3, 12.6, 15.4, 17.9,  20.3, 23.0, 25.6.0,  28.2, 30,  32, 34, 36 d Sm^-1. There was no addition of seawater salt to fifteenth lot of soil that served as control. Seawater salinity at Jamnagar coast in Saurashtra varies from 45 to 48 dS m^-1 during the rainy (monsoon) season. The electrical conductivity of control soil was 0.3 dS m^-1 and this value was approximately equal to 3mM salinity. For the measurement of electrical conductivity a soil suspension was prepared in distilled water at 1:2 soil  :water ratio. The suspension was shaken and allowed to stand overnight. Thereafter, electrical conductivity of the supernatant solution was determined with a conductivity meter.

Seedling emergence

Twenty polyethylene bags for each level of soil salinity were each filled with 5 kg of soil. Tap water was added to each bag until water level was 2 cm   above the soil surface. Propagules of A. officinalis were collected from Jamnagar coast. Bags were kept in an uncontrolled greenhouse under natural temperature and light. Ten propagules were sown (propagules were pressed completely into the soil) in each bag on 3 October 2010. Tap water was added daily to compensate evapotranspiration loss. Emergence of seedlings was recorded daily over a period of 82 days.

Seedling growth

For the growth studies, two seedlings that emerged first were left in each of 20 bags at each level of salinity and others were uprooted. Seedlings grown in soils at 0.3, 2.5, 5.1, 7.7, 10.3, 12.6, 15.4, 17.9, 20.3, 23, 25.6, 28.2, 30, 32, 34 and 36 dS m^-1 salinities exhibited emergence of the second leaf after 18, 18, 20, 20, 21, 21, 21, 22, 22, 21, 21,23, 22, 22, 22 and 22 days, respectively. Emergence of the second leaf confirmed the establishment of seedlings. Following emergence of the second leaf, one seedling having better vigor was allowed to grow in each bag and another seedling was further uprooted. Thus twenty replicates factorialzed with sixteen grades of soil (0.3 , 2.5, 5.1, 7.7, 10.3, 12.6, 15.4, 17.9, 20.3, 23.0, 25.6, 28.2, 30, 32, 34 and 36.0 dS m^-1) were prepared. This gave a total of 320 bags, which were arranged in   randomized blocks. Seedlings were watered daily to maintain water level above the soil surface and experiment was terminated after three months. Five seedlings died at   36 dS m^-1   salinity during  the course of experiment . Seedlings contained in 15 bags at each salinity level were washed with tap water to remove salt on leaves and soil particles adhered to roots. Roots of A. officinalis are adventitious. Roots, 4 to 5 in number, emerge from the root stock. Morphological characteristics of each seedling were recorded. Shoot height and   roots length   of the seedling were measured. Leaf area was marked out on graph paper. Fresh and dry weights of tissues (leaves, stems,  roots ) were determined. Water content (%) in plant tissues was calculated using following expression.

Water content (%)    =       Fresh weight – Dry weight × 100         

                                                    Fresh weight

Data recorded for morphological characteristics, dry weight and water content of different components were analyzed by one-way ANOVA to assess the effect of salinity on plant growth.

Determination of water potential and proline content

Ten additional plants grown in soil at each level of salinity were used for measurement of water potential and proline determination in plant tissues. Water potential of leaves, stems and  roots was measured by Dewpoint Potential Meter WP4 following Patel and Pandey (2010). All the measurements were taken between 8 to 10.30 A.M. Concentration of proline in plant tissues was estimated 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 and read at 520 nm. Data were analyzed by one-way ANOVA.  

Measurement of electrolyte leakage (membrane permeability)

Electrolyte leakage was determined in triplicate for each tissue of seedlings grown in soils at sixteen salinity levels. For measurement of electrolyte leakage 500mg fresh material of each tissue (leaf, stem, root) was cut in to small pieces and placed in 50ml glass vials, rinsed with distilled water to remove electrolyte released during leaf disc excision. Vials were then filled with 30ml of distilled water and allowed to stand in the dark for 24 h at room temperature. Electrical conductivity (EC1) of bathing solution was determined at the end of incubation period. Vials were heated in temperature - controlled water bath at 95^oC for 20 min. and then cooled to room temperature and electrical conductivity (EC2) was measured. Electrolyte leakage was calculated as percentage of EC1/ EC2 (Shi et al. 2006)

Mineral analyses of plant materials

Mineral analyses were performed on leaves, stems and root tissues. Plant parts of the seedlings grown in soil at same level of salinity were pooled separately. Plant samples were grind using mortar and pestle. Three subsamples of plant tissues were analyzed. Total nitrogen was determined by Kjeldahl method and (Piper, 1944). Concentrations of Ca, Mg, Na and K were determined by Shimadzu double beam atomic absorption spectrophotometer AA-6800 after triacid (HNO₃: H₂SO₄: HClO₄ in the ratio of 10: 1: 4) digestion. Mineral data were analyzed by one-way ANOVA.

3. Results

Effect of salinity on seedling emergence

Seedlings began to emerge 9 days after sowing and 45% propagule germinated over a period of 82 days under control (0.3 dS m^-1   salinity) condition (Fig. 1). Seedling emergence in saline soils was delayed and reduced by increasing concentration of salt .Propagule germination decreased from 45% at 0.3 dS m^-1   to  26  % at 36 dS m^-1    salinity.

Effect of salinity on stem and root elongation and leaf expansion

Salinity significantly stimulated (p < 0.01) stem and   root elongation until 17.9 dS m^-1, but extension growth declined with further increases in salinity (Fig 2 a,b,c).  However, root length was   almost similar to stem height under the control and saline conditions. Leaf area significantly increased (p < 0.01) until 17.9 dS m^-1 salinity, but decreased at higher salinities (Fig 3).

Effect of salinity on dry weight

Dry weight of leaves, stems, shoots (leaves + stems)   and   roots was significantly promoted (p < 0.01) by salinity until 17.9 dS m^-1, but it declined with further increases in salinity (Fig. 4 a,b,c,d). Root/shoot dry weight ratio (Fig.4 c), was 0.86 for plants grown in control soil and it significantly decreased (p < 0.01) with increasing soil salinity.

Effect of salinity on water content and water potential of tissues

Water content (%) significantly increased (p < 0.01) in leaves, stems   and  roots until 17.9 dS  m^-1 salinity, but declined with further increases in salinity(Fig.5 a,b,c) .Water content was maximum in stems and minimum in  roots. Tissues according to their water content can be arranged in the following decreasing order: Roots > Stems > Leaves. Water potential significantly became more negative in tissues (p < 0.01) as soil salinity increased (Fig. 6). Tissues according to their negative water potential values (low to high negative) can be arranged into the following decreasing order: Roots > Stems > Leaves.

Effect of salinity on proline content of tissues

Proline content (m mol / g FW material) significantly increased (p < 0.01) in leaves, stems, and root tissues with increase in soil salinity (Fig. 7c). Tissues according to their proline content can be arranged into the following decreasing order: stems > leaves > roots.

Effect of salinity on mineral accumulation

Sodium content significantly increased (p < 0.01) in leaves, stems and roots with increase in salinity. Potassium   content significantly decreased (p < 0.01) in leaves, stems and   roots  tissues, in response to increasing salinity (Fig.8 a,b,c). The Na/K ratio significantly increased (p < 0.01) in leaves, stems and root tissues in response to increasing soil salinity.

Concentration of Ca significantly decreased (p < 0.01) in leaves, stems and   roots in response to increasing salt concentration in soil. The concentration of Mg   did not change in   leaves, stems and root tissues with increase in soil salinity.(Fig. 9 a, b) Concentration of N significantly decreased   (p < 0.01)   in leaves, stems and root tissues in response to increasing soil salinity (Fig. 10).

Effect of salinity on membrane leakage

Electrolyte leakage significantly (P<0.01) increased in leaves, stems and roots with increase in salinity (Fig.11).The electrolyte leakage was maximum in roots followed by stems and leaves, sequentially. As a result , tissues can be arranged in the following decreasing order for electrolyte leakage :roots >stems >leaves. 

3.      Discussion

Optimum germination was obtained in non – saline control soil and increasing salinity delayed and reduced germination of A. officinalis. Similar results have been reported by others for seed germination of halophytes (Khan and Weber, 1986; Ungar, 1996; Katembe et al., 1998, Gulzar and Khan, 2001; Khan, 2002; Li et al., 2002). Salt can affect seed germination either by osmotic effect, restricting the supply of water to embryo, (Agboola, 1998; Pujol et al., 2000) or by ionic effect, causing specific injury through ions to the metabolic machinery, (Pollack and Waisel, 1972; Mohammed and Sen, 1990). A. officinalis is characterized by viviparous germination. Adaptation of viviparous propagules to saline environments actually starts when they are still attached to the mother tree by continuously absorbing salt from the tree or by a desalinating process (Joshi et al., 1972; Zheng et al., 1999).Under natural conditions, germination of propagules of A. officinalis occurs during the season with high precipitation when salinity level of seawater is usually reduced. It appears that high salt tolerance at germination and low salinity level of seawater during the germination period together enable A. officinalis to invade coastline along the Arabian Sea.

Growth of A. marina seedlings was stimulated by low salinity and their optimum growth was at 17.9 dS m^-1. Similar results have been reported for halophytes that have optimal growth in the presence of salt (Naidoo and Raghunanan, 1990; Ayala and O’Leary, 1995; Khan et al., 2000a; Patel and Pandey, 2007). Soil salinity at 17.9 dS m^-1 was equal to 38% seawater treatment because salinity of seawater during the rainy season at Jamnagar coast is about 48.0 dS m^-1.  Optimum growth at 50 % seawater was recorded for A. marina  from Sunderban (Karim and Karim, 1993) and Pakistan (Khan and Aziz, 2001). However, Naidoo (1987) obtained growth stimulation for A. marina at 25% seawater. High media salinity affects plant growth due to low water potential, ion toxicities, nutrient deficiencies or a combination of all these factors (Khan et al., 2000 a). Patel and Pandey (2007) reported that seedlings of Cassia montana, a halophyte tree in the coastal area of Saurashtra, exhibited optimum growth at 7.9 dS m^-1 salinity. Evidently,  A. marina can be grouped among highly salt tolerant plants.

Water content of tissues increased until 17.9 dS m^-1 and then declined with increased salinity. Similar result has been reported for S. fruticosa (Khan et al., 2000). Halophytes are characterized by their capacity to adjust tissue water potential to a level that is more negative than that of the soil water potential of the habitat in which they are growing (Ungar, 1991). Mangroves lower tissue osmotic potential through the net accumulation of solutes in response to salinity and water deficits (Turner and Jones, 1993; Suarez et al., 1998).

Avicennia officinalis can shed salts via leaf glands when supplied with salinity in the growth medium (Fitzgerald et al., 1992). On the contrary high Na concentration was maintained in tissues with increase in salinity. There is evidence that halophytes and glycophytes accumulate NaCl in vacuoles (Flowers et al., 1977). High internal salt concentrations provide potential benefits to plants growing under conditions where soil osmotic potential is more negative than that of seawater because of high soil salinity (Ungar, 1991). Increased salt content lowers the internal water potential which is required to permit water uptake. Na⁺ and Cl^- accumulated in leaf tissues provide osmotic adjustment and turgor to maintain growth (Yeo, 1983). The cation K⁺ is essential for cell expansion, osmo-regulation and cellular and whole-plant homeostasis (Schachtman et al., 1997). High stomatal K⁺ requirement is reported for photosynthesis (Chow et al., 1990). The role of K⁺ in response to salt stress is also well documented, where Na⁺ depresses K⁺ uptake (Fox and Guerinot, 1998). In the present study, significant decrease of K⁺ content in all the tissues of seedlings with increasing soil salinity suggests that Na⁺ inhibited K⁺ uptake. The Na/K ratio increased in leaves and stems with increase in salinity suggesting an increase in transportation of Na⁺ from root to shoot. Tattini (1995) reported that Na/K ratio increases in salt tolerant species with increasing salinity in the external medium because mass transport of sodium takes place from root to shoot via the transpiration stream.

The result on electrolyte leakage showed increasing trend with the increase in salt concentration. The presence of Nacl in the membrane permeability caused a disturbance in membrane permeability (Ghoulam et.al. 2002). The higher leakage of solutes was probably due to enhanced H₂O₂   accumulation   and lipid peroxidation under salt stress (Dionisio-sese and Tobita 1978). Our findings   are also in agreement with the results of Kaya et. al.(2003) in cucumber and Tiwari et.al. (2010).  

The accumulation of compatible solutes is a common response to salinity in higher plants (Stewart and Lee, 1974; Storey et al., 1977). These compounds are not toxic to cytoplasmic enzymes functions at high concentrations (Storey et al., 1977), thus help in osmotic adjustment. The increase of proline content in tissues with increase in Na content indicates that higher proline accumulation may contribute to the alleviation of NaCl stress in A. officinalis. Glycinebetaine is known to occur in different Avicennia species and it acts as a compatible osmoticum to overcome toxic effects of Na⁺ and Cl^- (Popp and Polania, 1989). Though the proline concentration increased, it might be insufficient to play a significant role in the osmotic balance of the cell. The proline accumulation was greater in shoot tissues than that in roots. Munns (2002) concluded that organic solutes are often lower in roots than shoots.

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). Calcium is important during salt stress, e.g., in preserving membrane integrity (Rengel, 1992), signaling in osmoregulation (Mansfield et al., 1990) and influencing  K⁺/Na⁺ selectivity (Cramer et al., 1987). In the present study, there was a significant decrease of Ca²⁺ content in all the tissues with salinisation of soil. 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 interactions, precipitation and increase in ionic strength that reduce the activity of Ca²⁺ (Janzen and Chang, 1987). 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 photosynthase (Marschner and Cakmak, 1989).  Results suggested that in extreme saline habitats N,   K and  s Ca  are limiting factors for the growth of A. officinalis. Khan and Aziz (2001) suggested the growth of this mangrove species would be better if fresh water from Indus river is allowed mixing with seawater near Karachi coast in Pakistan.

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