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Journal of Stress Physiology & Biochemistry, Vol. 8 No. 4 2012, pp. 236-252 ISSN 1997-0838 Original Text Copyright © 2012 by Kaouther, Ben Fredj, Mani, Hannachi
ORIGINAL ARTICLE
Impact of salt stress (NaCl) on growth, chlorophyll content and fluorescence of Tunisian cultivars of chili pepper (Capsicum frutescens L.)
Kaouther Zhani*, Ben Fredj Mariem, Mani Fardaous and Hannachi Cherif
University of Sousse, Department of Horticulture and Landscape, Higher Institute of Agronomy. 4042 Chott Mariem, Tunisia
*E-Mail: [email protected]
Received September 3, 2012
Salinity is considered as the most important abiotic stress limiting crop production and plants are known to be able continuing survive under this stress by involving many mechanisms. In this content, the present study was carried out to evaluate the impact of NaCl on some physiological and biochemical parameters in five Tunisian chili pepper (Capsicum frutescens L.) cultivars: Tebourba (Tb), Somaa (Sm), Korba (Kb), Awald Haffouzz (AW) and Souk jedid (Sj). Thus, an experiment of five months was carried out under greenhouse at Higher Institute of Agronomy, Chott Meriem, Tunisia and stress is induced by NaCl at 7 concentrations (0, 2, 4, 6, 8, 10 and 12g/l). Results showed that increasing salinity stress, for all cultivars, had a negative impact on roots (length, fresh and dry weights) and leaves (number and area). Also, chlorophyll (a and b) amount in addition to quantium yield (Fv/Fm) decreased significantly. However, biosynthesis of proline in leaves is activated. Awlad Haffouzz and Korba cultivars succefully tolerated highest salinity level by accumulating more proline in leaves and maintaining usually higher values in all parameters in opposition to Souk jedid cultivar. Taken together, our data partly explain the mechanism used to ovoid salt stress by pepper plants when excessive in the culture medium.
Key words: chlorophyll content, leaves, NaCl, pepper, proline, quantium yield, roots
ORIGINAL ARTICLE
Impact of salt stress (NaCl) on growth, chlorophyll content and fluorescence of Tunisian cultivars of chili pepper (Capsicum frutescens L.)
Kaouther Zhani*, Ben Fredj Mariem, Mani Fardaous and Hannachi Cherif
University of Sousse, Department of Horticulture and Landscape, Higher Institute of Agronomy. 4042 Chott Mariem, Tunisia
*E-Mail: [email protected]
Received September 3, 2012
Salinity is considered as the most important abiotic stress limiting crop production and plants are known to be able continuing survive under this stress by involving many mechanisms. In this content, the present study was carried out to evaluate the impact of NaCl on some physiological and biochemical parameters in five Tunisian chili pepper ( Capsicum frutescens L.) cultivars: Tebourba (Tb), Somaa (Sm), Korba (Kb), Awald Haffouzz (AW) and Souk jedid (Sj). Thus, an experiment of five months was carried out under greenhouse at Higher Institute of Agronomy, Chott Meriem, Tunisia and stress is induced by NaCl at 7 concentrations (0, 2, 4, 6, 8, 10 and 12g/l). Results showed that increasing salinity stress, for all cultivars, had a negative impact on roots (length, fresh and dry weights) and leaves (number and area). Also, chlorophyll (a and b) amount in addition to quantium yield (Fv/Fm) decreased significantly. However, biosynthesis of proline in leaves is activated. Awlad Haffouzz and Korba cultivars succefully tolerated highest salinity level by accumulating more proline in leaves and maintaining usually higher values in all parameters in opposition to Souk jedid cultivar. Taken together, our data partly explain the mechanism used to ovoid salt stress by pepper plants when excessive in the culture medium.
Key words: chlorophyll content, leaves, NaCl, pepper, proline, quantium yield, roots
Salinity is a major environmental factor determining plant productivity and plant distribution. It affects more than 10 percent of arable land and salinization is rapidly increasing on a global scale, declining average yield for most major crop plants by more than 50 percent (Bray et al. 2000). Salt stress occurs in areas where soils are naturally high in salt and precipitation is low
(Neumann, 1995) and/or where irrigation, hydraulic lifting of salty underground water, or invasion of sea water in coastal areas brings salt to the surface soil that inhabit plants. Globally 20% of irrigated land and 2.1% of dry land agriculture suffers from the salt problem and NaCl is the predominant salt causing salinization (Munns and Tester 2008). Salinity adversely affects germination, growth,
physiology and productivity by reducing the ability of plants to take up water causing imbalance in osmotic potential; ionic equilibrium and nutrient uptake (Niu et al. 1995). Further, it facilitates severe ion toxicity by depositing high concentration of Na+ which causes membrane disorganization, inhibition of cell division and expansion. In addition, it impairs a wide range of cellular metabolism including photosynthesis, protein synthesis and lipid metabolism (Alia-Mohanty and Saradhi, 1992; Ashraf, 1994). Lichtenthaler et al. (2005) found that salt stress was responsible for decreased biosynthesis of chlorophyll and inefficiency of photosynthesis (Munns, 2002) all of which ultimately leading to lowered economic productivity. The decline in photosynthesis due to salinity stress could be due to lower stomata conductance, depression in carbon uptake and metabolism, inhibition of photochemical capacity or a combination of all these factors (Mundree et al. 2009). Chlorophyll fluorescence has proved particularly useful in salinity-tolerance screening programs (Jimenez et al. 1997) because the effects of salt damage can be detected prior to visible signs of deterioration (West, 1986).
To perceive the incoming stresses and rapidly regulate their physiology and metabolism, plants evolved mechanisms that allow them to cope with them (Zhang et al. 2006) by synthesis and accumulation of a number of compatible solutes called "osmolytes". These osmolytes include proteins, carbohydrates, amino acids and quaternary ammonium compounds (Ashraf, 2004) which are accumulated in plants at high concentrations to alleviate enzyme inactivity or loss of membrane integrity due to water deficiency (Schwab and Gaff, 1990). Proline is a key osmolyte which helps plants to maintain cell turgor (Hsu et
al. 2003, Seki et al. 2007). A large number of plant species accumulate proline in response to salinity stress and this accumulation may play a role in defense against salinity stress that is why in some cases higher proline content could be correlated with abiotic stress tolerance (Premachandra et al. 1992). The role of proline in cell osmotic adjustment, membrane stabilization and detoxification of injurious ions in plants exposed to salt stress is widely reported (Hare et al. 1999; Kavi Kishor et al. 2005; Ashraf and Foolad, 2007).
Pepper is one of the most widely grown vegetable in the world. World production of pepper is estimated at 23.2 million tones and the largest producer is China with 11.5 million tons, or nearly 50% (FAO, 2003). In Tunisia, pepper is widely grown in all regions both in the field and under greenhouse and occupies the 4th largest area planted by gardening. In 2009, the area for growing peppers in Tunisia is approximate 18900 ha and production reached 296000 tones corresponding to a yield of 16 t / ha. This production allows the country to rank second place in the African export after Morocco and the fourth in the world after Spain, Hungary and Germany. However in the harvest season, peppers are exposed to many biotic (virus, champignon) and abiotic conditions especially salinity which had negative effect on pepper growth (Ibn Maaouia-Houimli et al. 2008), yield and fruit quality (Ibn Maaouia-Houimli et al. 2011) since pepper is a sensitive salt-tolerant crop (2 g/l).
Hence the present study was initiated to evaluate the effect of NaCl treatment in five accessions of chili pepper on growth, chlorophyll content, fluorescence and proline synthesis in order to better understand their differences on salt stress tolerance and select tolerant accession.
MATERIALS AND METHODS
In Higher Institute of Agronomy, Chott Meriem, Tunisia, the study was carried out under greenhouse characterized by an area of 170 m2 (20 m*8.5 m) and 25°C/18°C day/night temperature. This greenhouse is covered with plastic film (low density polyethylene) and cemented by its side. Seeds of five accessions: Tebourba (Tb), Somaa (Sm), Korba (Kb), Awald Haffouzz (Aw) and Souk jedid (Sj) were sterilized for 20 mn in sodium hypochloride solution (5%) and then rinsed 3 times with distilled water. Ten seeds for each cultivar were sown on February 15th 2012, at depth of 2 cm, in plastic pot (20 cm diameter and 25 cm height) filled with peat, sand and topsoil (1/3:1/3:1/3). Pots were left in greenhouse on bricks. Once the seeds have germinated, we kept a single plant on which the trial continues. For two months (April and May), plants were watered with saline water at seven levels of NaCl concentrations (0, 2, 4, 6, 8, 10 and 12g/l). During culture, plants were not fertilized but were processed by Talastar (80cc/hl) preventively and curatively against aphids using a Knapsack sprayer. Salinity stress effect was studied by measuring root length, fresh and dry weights of roots, number of leaves, leaf area, chlorophyll content (a and b), quantum yield (Fv/Fm) and proline amount in leaves. Dry weight was determined after drying into oven at 80°C for 48 h. Leaf area was measured by planimeter (Area Meter 3100). Chlorophyll (a and b) content was determined by Arnon method (1949) at 663 nm and 645 nm according to the following equations:
Chl a (mg/gFW) = 12,7 (OD 663) - 2,63 (OD 645) Chl b (mg/gFW) = 22,9 (OD 645) - 4,86 (OD 663)
Proline content is estimated by Bates et al. (1973) method at 520 nm by UV
spectrophotometer. Ratio (Fv/Fm) was measured using portable fluometer (FI 1500) when leaves were dark-adapted for 30mn, then dark fluorescence (F0), maximal fluorescence (Fm) and photochemical yield (Fv/Fm) were recorded (Fv=Fm-F0).
Pots were disturbed in completely randomized design with three replications and data analysis was done using "SPSS software 13.00". Duncan's Multiple Range test was used to compare between means and determine significance between variables (P < 0,05).
RESULTS
Root length
In control, root length varied from 25 cm (Tb cv) to 30.3 cm (Sj cv). The addition of sodium chloride in water reduced root length in all cultivars (Table 1). This reduction becomes more pronounced at highest salt concentration. Thus, at 12 g/l, the root is shorted by 72, 78, 85, 88 and 92 % respectively for Kb, Aw, Sm, Tb and Sj cv.
Roots fresh and dry weights
According to Figure 1, in control plant, pepper root system weights 19.6 g (Aw cv) to 25.9 g (Sj cv). The presence of NaCl is accompanied with a significant reduction in root fresh weight in all cultivars as well as NaCl concentration is increasing. At the highest concentration (12 g/l), the fresh weight decreased till 60% in Aw cv to 82 % in Sj cv. The same decrease is observed for dry weight of roots (Figure 2) where the decrease compared to control was 40, 46, 67, 74 and 85 % respectively for Aw, Kb, Sm, Tb and Sj cv. This impact on roots is more illustrated in Figure 3.
Number of leaves
Number of leaves is significantly affected by salt stress (P<0.05); it declines in all cultivars by
increasing NaCl concentration (Table 2). At the highest level, pepper plants don't produce more than 3.5 (Tb cv) to 9.15 (Kb cv) leaves; it is a respective decrease of 86 and 69% compared to control.
Leaf area
Salt stress affects negatively leaf surface in plants of pepper of all cultivars in study. Indeed, in control, leaf area ranged from 10.92 cm2 for Sj cv to 21.5 cm2 for Tb cv. By addition salt (2 g/l), leaf area decreased to 9.55 cm2 and 19.14 cm2 respectively in Sj and Tb cv (Table 3). This drop increases gradually as one increases the concentrations of NaCl in water. At the highest concentration, leaf area did not exceed 1.01 cm2 for Sj cv corresponding to a significant decrease of 91%.
Chlorophyll content
Leaves of control plants contain about 1.44 to 3.65 mg/gFW for chlorophyll a (Table 4) and about 0.89 to 1.84 mg/gFW for chlorophyll b (Ttable 5) respectively for Aw and Sj cultivars. Among the five cultivars of chili pepper in study, increase of NaCl concentration is accompanied by a decline of chlorophyll synthesis. Thus, at 12g/leaves contains only 0.11 mg/gFW of chlorophyll a (Sj cv) and 0.07 mg/gFW of chlorophyll b (Aw cv) corresponding to a
respective decrease of 97 and 85%. This very low content results in chlorosis (Figure 4).
Chlorophyll fluorescence (photochemical efficiency)
Salt application affected negatively maximal efficiency of PSII photochemistry (Fv/Fm) measured in the dark adapted leaves of the five cultivars studied. Thus, in absence of NaCl, (Fv/Fm) ratio was in the range of 0.760 to 0.807 respectively for Sj cultivar and Aw cv (Table 6). NaCl addition (2 g/l) declines of about 15% (Sj cv) occurred for plants irrigated with distilled water. This decline was increased by 26% (Tb cv) at the highest NaCl concentration when results showed that Kb cv exhibit the highest quantum yield (0.635) differing with those of Sj cv (0.603).
Proline content
According obtained results in Table 6, leaf proline in all cultivars increase significantly (p<0.001) with the increase of salt concentration in irrigation water. This increase was more at 12 g/l NaCl treatment when the five cultivars displayed very high values: 0.094 (Tb cv), 0.096 (Sj cv), 0.206 (Aw cv), 0.214 (Sm cv) and 0.415 mg/gFW (Kb cv) corresponding respectively to 3.6, 2.1, 6, 4 and 7.15 times to the level found in controlled plants.
Table 1. Root length (cm) of five cultivars of chili pepper watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/l)
NaCl (g/l)
0 2 4 6 8 10 12
Tebourba 25.003 21.70ab 19.3 0bc 13.40c 7.00d 5.00de 3.10f
Somaa 28.10a 24.0011 20.10bc 17.50° 10.25d 6.00e 4.20f
Korba 27.3 0a 25.4011 22.80c 19.30cd 15.20e 10.50lg 7.70g
Awlad haffouzz 26.203 23.10b 13.00c 18.70d 12.20e 9.30* 5.80g
Souk jedid 30.30a 28.10ab 21.20c I5.30d 10.80e 7.001 2.50g
Means followed by the same letter are not significantly different at 5% level according to Duncan test.
Table 2. Leaves number of five cultivars of cliili pepper watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g 1)
NaCl (g/1)
I) 2 4 6 8 10 12
Tebourba 24.66a 20.3 3b 18.66c 15.55ccl 12.87de 8.78e 3.50f
Somaa 33.16a 30.26b 23.16c 17.1 ld 14.13d 9.16e 5.30f
Korb a 29.66a 21.66b 18.16c 16.33ccl 14.83d 11.01* 9.161
Awlad haffouzz 31.66a 25. 01b 20.13c 14.82de 11.66el 9.131 4.1 lg
Souk jedid 40.10a 34.33b 25.50c 17.33d 13.16" 7.25f 5.13§
Means followed by the same letter are not significantly different at 5% level according to Duncan test.
Table 3. Leaf area (cm2) of five cultivars of chili pepper watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/1)
NaCl (g/1)
0 2 4 6 8 10 12
Tebourba 21.50a 19. 14b 14.24c 12.87c 9.78d 7.78e 2.50t
Somaa y* bo as 13,02at> 11.89b 9.39b 7.85c 4.49d 2.50e
Korba 18.30a 15.60b 12. 67c P Lh oo p- 9.50d 5.96e 5.011
Awlad haffouzz 20.26a 17.60b 15.20bc 8.53c 7.93cd 6.34d 5.39e
Souk jedid 10.92a 9.5 5ab & OO 5.79c 4.46d 3.1 lde 1.01e
Means followed by the same letter are not significantly different at 5% level according to Duncan test.
Table 4. Chlorophyll a content (mg/gFW) of five cultivars of chili pepper watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/1)
NaCl (g/1)
0 2 4 6 8 10 12
Tebourba 3.34a 2.68b 2.18bc 1.83c 1.59c 0.25d 0.14e
Somaa 3.3 T 3.14b 2.65c 1.26d 0.79e 0.451 0.23g
Korba 2.86a 1.31b 1.05c 0.87cl 0.61el 0.521 0.421
Awlad haffouzz 1.44a 1.3 5b 1.23bc 1.18c 0.52d 0.33e 0.221
Souk jedid 3.65a 3.50bc 3.12c 2.74d 1.64e 0.581 0.1 lg
Means followed by the same letter are not significantly different at 5% level according to Duncan test.
Table 5. Chlorophyll b content (mg/gFW) of five cultivars of chili pepper watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/1)
NaCl (g/1)
0 2 4 6 8 10 12
Tebourba 1.76a 1.64ab 1.40b 0.98c 0.72d 0.41e 0.211
Somaa 1.03a 0.86b 0.61c 0.54c 0.35d 0.21e 0.171
Korba 1.77a 1.57b 1.25c 0.80d 0.53e 0.321 0.2 lg
Awlad haffouzz 0.89a 0.8 lab 0.72b 0.43c 0.22d 0.13e P o
Souk jedid 1.84a 1.61b 1.3 lc 0.85d 0.61e 0.311 0.13s
Means followed by the same letter are not significantly different at 5% level according to Duncan test.
Table 6. Effect of NaCl (0, 2, 4, 6, 8, 10 and 12 g/1) on PS-II efficiency (Fv/Fm) of five cultivars of chili pepper watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/1)
NaCl (g/1)
0 2 4 6 8 10 12
Tebourba 0.775 0.712 0.689 0.671 0.656 0.641 0.573
Soman 0.785 0.687 0.674 0.669 0.653 0.631 0.591
Korba 0.792 0.751 0.723 0.711 0.671 0.657 0.635
Awlad haffouzz 0.807 0.747 0.714 0.698 0.681 0.652 0.621
Souk jedid 0.760 0.746 0.708 0.684 0.665 0.621 0.603
Table 7. Effect of NaCl (0, 2, 4, 6, 8, 10 and 12 g/1) on leaf pro line content (mg/gFW) in five cultivars of chili pepper watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/1)
NaCl (g/1)
(1 2 4 6 8 10 12
Tebourba O o NJ G\ 0.031e 0.051d 0.067cd 0.073bc 0.082° 0.094a
Somaa 0.053E O o 0.127e 0.169de 0.193c 0.207b 0.214a
Korba 0. 058g 0.1211 0.178e 0.205d 0.35 lcd 0.385b 0.415a
Awlad haffouzz 0.034g o © a 0.081e 0.115d 0.134c 0.187b 0.206a
Souk jedid 0.046e 0.053d 0.068c 0.073c -fi CO o © 0.09 lb 0.096a
Means followed by the same letter are not significantly different at 5% level according to Duncan test.
30
0 2 4 6 8 10 12
NaCl (g/1}
Figure 1. Roots fresh weight (g) of five Tunisian chili pepper cultivars watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/1)
0 2 4 6 S 10 12
NaCl (g/1)
Figure 2. Roots dry weight (g) of five Tunisian chili pepper cultivars watered during 60 days with NaCl (0, 2, 4, 6, 8, 10 and 12 g/1)
Figure 3. Effect of NaCl on roots of Tunisian chili pepper, Kb cv: reduction in length and biomass
Figure 4. Effect of NaCl on leaves of Tunisian chili pepper, Aw cv: reduction in leaf area and appearance of chlorosis
DISCUSSION
Salinity treatment during two months using NaCl (0, 2, 4, 6, 8, 10 and 12 g/l) in five Tunisian cultivars of chili pepper (Tb, Kb, Sm, Aw and Sj) had significant effect on the vegetative growth where the highest concentration of NaCl resulted in severe reduce on the corresponding parameters in study. Similar results were reported in potato (Kerkeni,
2002) for root length, in canola (Byubordi, 2010) for leaf area and in groundnut (Mensah et al. 2006) for number of leaves. Al Thabet et al. (2004) working on sprout, Singh et al. (2007) on peanut, Farhoudi and Tafti (2011) in soybean and Keshavarzi (2011) on savory found also that under salinity stress plant growth is inhibited due to the low water potential, ion toxicity and imbalance excreted by salinity (Greenway and Munns, 1980).
As consequence, a decrease in photosynthetic pigment content was observed with increasing salt concentration in all cultivars. At 12 g/l, we noted a reduce of 93% and 97% respectively in chlorophyll b and chlorophyll a content compared to control (Sj
cv). This result is in agreement with those of Tewari and Singh (1991) in lentil, Beinsan et al. (2003) in bean, Iqbal et al. (2006) in wheat, Chen and Yu (2007) in Glycine max seedlings, Moussa Helal
(2006) in maize, Molazem et al. (2010) in corn, Malik et al. (2010) in cucumber, El Iklik et al. (2011) in tomato and Rahdari (2012) in Purslane,
Parida and Das (2005) suggested that decrease in chlorophyll content in response to salt stress is a general phenomenon which led to disordering synthesizing chlorophyll and appearing chlorosis in plant. According to Rao and Rao (1981), NaCl stress decreased total chlorophyll content of the plant by increasing the activity of the chlorophyll degrading enzyme: cholorophyllase, inducing the destruction of the chloroplast structure (BlumenthalGoldschmidt and Poljakoff-Mayber, 1968) and the instability of pigment protein complexes (Dubey, 1997). In another study, Ali et al. (2004) attributed this reduction in chlorophyll concentration by NaCl to the inhibitory effect of the accumulated ions of various salts on the biosynthesis of the different
chlorophyll fractions.
Working on potato and gladiolus leaves, Mehouachi (1993) and Bettaieb et al. (2008) respectively affirmed that the decrease of chlorophyll content is due to a decrease of photosynthetic activity. Based on this last hypothesis, we choose to study photosynthetic activity through the measurement of chlorophyll fluorescence estimated by the ratio (Fv/Fm) which is considered as an indicator of the efficiency of Photosystem II (Maxwell and Jonhson, 2000). Baker (1991) confirmed that photosystem II (PSII) plays a key role in the response of leaf photosynthesis to environmental perturbation.
The maximum photochemical efficiency (Fv/Fm) indicates the capacity of absorption of excitation energy by leaves and it is usually decreasing thereafter as a consequence of leaf senescence and decrease of photosynthetic assimilation (Barbagallo et al. 2003). In our study, the ratio (Fv/Fm) showed parallel trend with chlorophyll a and chlorophyll b content. Increasing salinity level is accompanied by a significant reduce in Fv/Fm ratio below 0.8.: a normal value reported for most the plants and showing the health and vigor of the plant (Bjorkman and Demmig-Adams, 1995) while value below 0.8 indicates that plants are experiencing stress conditions (Schreiber et al. 1995). DeEll et al. (1999) confirmed this and reported that the ratio for a normally functioning leaf varies between 0.75 and 0.85 and a decline in this ratio is indicative of photoinhibitory damage. Our results can be related to some earlier findings in which it has been observed that salt stress has significant effect on PSII photochemical activity, e.g., in strawberry (Rahimi and Biglarifard, 2011) and maize (Suriyan and Chalermpol, 2009). However, there are some reports that suggest that salt stress may not causes
changes in Fv/Fm ratio in wheat (Akram et al. 2006) and pepper (Ibn Maaouia Houimli et al. 2008). The reduced quantum yield as obtained in our study may result from a structural impact on PS II (Everard et al.1994; El-Shintinawy, 2000;) induced by salinity which affects reaction centers of PSII either directly (Masojidek and Hall, 1992) or via an accelerated senescence (Hasson and Paljokoff-Mayber, 1981; Kaya et al. 2002;).
In fact, chlorophyll fluorescence is related to only Chlorophyll a, but in our research, the decrease in Fv/Fm ratio throughout the experiment in all cultivars in study coincided also with a decrease in Chlorophyll b content under salt-stress conditions. The result agrees with those of Lutts et al. (1996) in rice and Rahimi and Ali (2011) in strawberry. Kocheva et al. (2004) suggested that decrease in Chlrophyll b content in leaves might lead to structural/conformational changes in the PS-II antennae as chlorophyll b is mainly associated with PS-II antenna. Hall and Rao (1999) reported that analysis of fluorescence characteristics such as quantum yield reflects the properties of the chlorophyll molecules and their interaction with the external environment and also with associated physiological processes. It has been used since long by crop physiologists to evaluate response of various crop species to determine the influences of abiotic stresses at various stages of plant growth to have a quantitative assessment to be used in ranking plant species for their tolerance and/or sensitivity towards environmental stresses (Maxwell and Johnson, 2000). Thus, the varieties studied can be ranked in order of decreasing tolerance as follows: Kb, Aw, Sm, Tb and Sj.
As a response to salt stress, leaves of pepper plants accumulate proline, a common phenomena observed in all organisms ranging from bacteria to
higher plants to survive both water deficit and high salinity (Ahmad and Jhon, 2005). Thus, proline content increased significantly in the leaves of all the genotypes of the chili pepper in study as the salt concentration increased. Our result is supporting findings in Triglochin bulbosa (Naidoo and Naidoo, 2001), barley (Sadeghi, 2009), mulberry (Harinasut et al. 2000 ; Kumar et al., 2003), mangrove (Parida et al. 2002), maize (Cicek and Cakirlar, 2002), sorghum (De Lacerda et al.
2003), Phaseolus aureus (Misra and Gupta, 2005), Morus alba (Ahmad et al. 2007), Sesamum indicum (Koca et al. 2007), cotton (Desingh et al. 2007), wheat (Khan et al. 2009; Shafi et al. 2011), Paulownia imperialis (Astorga and Melendez, 2010), Atriplex (Ouiza et al. 2010), rapessed (Farhoudi, 2011) and Chookhampaeng (2011) in pepper, where salt stress resulted in extensive proline accumulation.
Proline is an organic solute known to be involved in osmoregulation which reduces the cell osmotic potential to a level to provide high turgor potential for maintaining growth (De Lacerda et al. 2005; Ashraf and Harris, 2004; Chaum et al. 2004). Also, apart protection of macromolecules from denaturation and carbon and nitrogen reserve for stress relief, proline has several other functions during stress: e.g. osmoprotection (Kavi Kishor et al. 2005), free radical scavenger and antioxidant activity (Sharma and Dietz, 2006). It is also compatible with other cytoplasmic components and can be easily converted to glutamate that takes part in the synthesis of other essential amino acids (Rains, 1981). Recently, Matysik et al. (2002) reported that proline accumulation protects plants against free radical induced damage by quenching of singlet oxygen. In many species like Alfalafa (Fougere et al. 1991; Petrusa and Winicov, 1997),
rice (Lutts et al. 1996) and mulberry (Ramanjula and Sudhakar, 2001), a positive correlation between magnitude of free proline accumulation and salt tolerance has been suggested as an index for determining salt tolerance potentials between cultivars. In our study, a similar behavior in the seedling of chili pepper was also observed: proline accumulation in the salt tolerant pepper cv. Korba was significantly higher than that in the salt sensitive one (cv. Souk jedid).
CONCLUSION
In conclusion, our study showed that salt stress at higher concentration, especially 12g/l NaCl is harmful to vegetative growth of the five cultivars of pepper since the root length, fresh and dry weight of roots, number of leaves and leaf area decreased significantly. In addition to this morphological features, photosynthetic parameters (quantum yield and chlorophyll content) were adversely affected while proline amount in leaves was activated and increased by increasing salinity level. Awlad haffouzz and Korba cultivars of chili pepper are classified as salt tolerant whereas cv Souk Jedid as susceptible based on various parameters studied. It's thus apparent from the present investigation that salinity stress tolerance in pepper is attributed to the biosynthesis of proline which makes plants able to continue growth even under higher salinity level. However, literature affirmed that there is no single parameter could be suggested as sole factor responsible for salinity stress tolerance; it is the combination of many characters. Thus, in future, a mineral analysis (Na+, Cl", K+ and Ca2+) and measurement of soluble protein content and percent soluble sugar are worthy to be studied.
REFERENCES
Ahmad, P., Jhon, R. (2005) Effect of salt stress on growth and biochemical parameters of Pisum sativum L. Arch. Agron Soil Sci., 51, 665-672.
Ahmad, P., Sharma, S. and Srivastava, P.S. (2007) In vitro seletion of NaHCO3 tolerant cultivars of Morus alba (Local and Sujanpuri) in response to morphological and biochemical parameters. Hort. Sci., 34, 115-123.
Akram, M., Farooq, S., Afzaal, M., Naz, F. and Arshad, R. (2006) Chlorophyll Fluorescence in different wheat genotypes grown under salt stress. Pak. J. Bot, 38(5), 1739-1743.
Al Thabet, S.S., Leilah, A.A., and Al-Hawass, I. (2004) Effect of NaCl and incubation temperature on seed germination of three canola (Brassica napus L.) cultivars. Scientific of King Faisal University (Basic and Applied Sciences), 5(1):81-92.
Alia-Mohanty, P. and Saradhi, P.P. (1992) Effect of sodium chloride on primary photochemical activities in cotyledonary leaves of Brassica juncia. Biochem Physiol., 188, 1-12.
Ali, Y., Aslam, Z., Ashraf, M.Y. and Tahir, G.R. (2004) Effect of salinity on chlorophyll concentration, leaf area, yield and yield components of rice genotypes grown under saline environment. International Journal of Environmental Science & Technology., 1 (3), 221-225.
Arnon, D.I. (1949) Copper enzyme in isolated chloroplasts, polyphenoloxidase in Beta vulgaris. Plant Physiol., 124, 1-15.
Ashraf, M. (1994). Organic substances responsible for salt tolerance in Eruca sativa. Biol. Plant, 36, 255-259.
Ashraf, M. (2004) Some important physiological selection criteria for salt tolerance. Flora, 199,
361-376.
Ashraf, M. and Foolad, M.R. (2007) Roles of glycinebetaine and proline in improving plant abiotic stress tolerance. Environ. Expt. Bot., 59, 206-216.
Ashraf, M. and Harris, P.J.C. (2004) Potential Biochemical Indicators of salinity tolerance in plants. Plant Sci., 166, 3-16.
Astorga, G.I., and Melendez, L.A. (2010) Salinity effects on protein content, lipid peroxidation, pigments,and proline in Poulownia imperialis and Paulownia fortune grown in vitro. Electronic Journal of Bitechnology., 13(5), 115.
Baker, N.R. (1991) Possible role of photosystem II in environmental perturbations of
photosynthesis. Physiol. Plant., 81, 563-570.
Barbagallo, R., Oxborough, K., Pallett, K. and Baker N.R (2003) Rapid, non invasive screening for perturbations of metabolism and plant growth using chlorophyll fluorescence imaging. Plant Physiol., 132, 485-493
Bates, L.S., Waldren, R.P. and Teare I.D. (1973) Rapid determination of free proline for water-stress studies. Plant Soil., 39, 205-207.
Beinsan, C., Camen, D., Sumalan, R. and Babau M.
(2003) Study concerning salt stress effect on leaf area dynamics and chlorophyll content in four bean local landraces from Banat area. Faculty of Horticulture, 119, 416-419
Bettaieb T., Denden M. and Mhamdi M. (2008) Régénération in vitro et caractérisation physiologique de variants somaclonaux de glaïeul (Gladiolus grandiflorus Hort.) tolérants aux basses températures Tropicultura, 26 (1), 10-16.
Bjorkman, O. and B. Demmig-Adams (1995)
Regulation of photosynthesis light energy capture,conversion, and dissipation in leaves of higher plants. In E.D. Schulze and M.M. Caldwell (eds.), Ecophysiology of Photosynthesis. Berlin, Heidelberg, New York, Springer-Verlag, pp.17-47.
Blumenthal-Goldschmidt, S. and Poljakoff-Mayber, A. (1968) Effect of substrate salinity on growth and submicroscopie structure on leaf cells of Atriplex halimus L. Australian Journal of Botany, 16(3), 469-478.
Bray, E.A., Bailey-Serres and Weretilnyk E. (2000) Responses to abiotic stress. In: Buchanan B , Gruissem W and Jones R (eds.), Biochemistry and Molecular Biology of Plants. American Society of Plant Physiology , Rockville, pp. 1158-1203.
Bybordi, A. (2010) The Influence of Salt Stress on Seed Germination, Growth and Yield of Canola Cultivars. Not. Bot. Hort. Agrobot. Cluj, 38 (1), 128-133
Chaum, S., Kirdmanee, C. and Supaibulwatana K.
(2004) Biochemical and physiological responses of thai jasmine rice (Oryza sativa L. ssp. indica cv. KDML105) to salt stress. Sci. Asia., 30, 247-253.
Chookhampaeng, S. (2011) The Effect of Salt Stress on Growth, Chlorophyll Content Proline Content and Antioxidative Enzymes of Pepper (Capsicum Annuum L.) Seedling. European Journal of Scientific Research, 49 (1), 103-109.
Chen, X.Q. and Yu, B.J. (2007) Ionic effects of Na+ and Cl on photosynthesis in Glycine max seedlings under iso osmotic salt stress. J. Plant Physiol. Mol. Biol., 33(4), 294-300
Cicek, N. and Cakirlar, H. (2002) The effect of salinity on some physiological parameters in
two maize cultivars. Bulg. J. Plant Physiol., 28(1-2), 66-74.
De Lacerda, C.F., Cambraia, J., Oliva, M.A., Ruiz, H.A. and Tarquino Prisco, J. (2003) Solute accumulation and distribution during shoot leaf development in two sorghum genotypes under salt stress. Environ and Expt. Bot., 49, 107-120.
De Lacerda, C.F., Cambraia, J., Oliva, M.A. and Ruiz, H.A. (2005) Changes in growth and in solute concentrations in sorghum leaves and roots during salt stress recovery. Environ. Exp. Bot., 54, 69-76.
DeEII; J.R., Van Kooten, O., Prange, R.K. and Murr, D.P. (1999) Applications of chlorophyll fluorescence techniques in postharvest physiology. Hort. Re, 23, 69-107.
Desingh, R. and Kanagaraj, G. (2007) Influence of salinity stress on photosynthesis and antioxidative systems in two cotton varieties. Gen. Appl. Plant Physiol., 33, 221-234.
Dubey, R.S.(1997) Photosynthesis in plants under stressful conditions. In Pessarakli, M. (Ed.), Handbook of photosynthesis. New York, Marcel Dekker, pp. 859-875.
El-Iklil, Y., Karrou, M., Mrabet, R. and Benichou, M. (2002) Effet du stress salin sur la variation de certains metabolites chez Lycopersicon esculentum et Lycopersicon sheesmanii. Canadian Journal of Plant Science., 82(1), 177183.
El-Shintinawy, F. (2000) Photosynthesis in two wheat cultivars differing in salt susceptibility. Photosynthetica., 38, 615-620.
Everard, J.D., Gucci, R., Kann, S.C., Flore, J.A. and Loeschner, W.H. (1994) Gas exchange and carbon partitioning in the leaves of celery
(Apium graveolens L.) at various levels of root zone salinity. Plant Physiol., 106, 281-292.
Farhoud, R. (2011) Effect of Salt Stress on Physiological and Morphological Parameters of Rapeseed Cultivars. Adv. Environ. Biol., 5(8), 2501-2508.
Farhoudi, R. and Tafti, M.M. (2011) Effect of Salt Stress on Seedlings Growth and Ions Homeostasis of Soybean (Glysin Max) Cultivars. Adv. Environ. Biol., 5(8), 2522-2526.
Fougere, F., Le Rudulier, D. and Streeter, J.G. (1991) Effects of salt stress on amino acids, organic acids, and carbohydrate composition of roots, bacteroids, and cytosol of alfalfa (Medicago sativa L.). Plant Physiol., 96, 1228-1236.
Greenway, H. and Munns, R. (1980) Mechanisms of Salt Tolerance in Nonhalophytes. Annu. Rev. Plant Physiol., 31, 149-190.
Hall, D.O. and Rao, K.K. (1999) Photosynthesis. Cambridge University Press, Cambridge, UK, pp.174-180.
Hare, P.D., Cress, W.A. and Staden, J. Van. (1999) Proline biosynthesis and degradation: a model system for elucidating stress-related signal transduction. J. Exp. Bot., 50,413-434.
Harinasut, P., Srisunaka, S., Pitukchaisopola, S. and Charoensatapornb, R. (2000) Mechanisms of Adaptation to Increasing Salinity of Mulberry: Proline Content and Ascorbate Peroxidase Activity in Leaves of Multiple Shoots. ScienceAsia., 26, 207-211.
Hasson, E. and Poljakoff-Mayber A. (1981) Does salinity induce early aging of pea tissue?. Oecologia., 50, 94-97.
Hsu, S.Y., Hsu Y.T. and Kao, C.H. (2003) The effect of polyethylene glycol on proline accumulation in rice leaves. Biol Plant., 46, 73-78.
Ibn Maaouia-Houimli, S., Denden, M., Dridi-Mouhandes, B. and Ben Mansour-gueddes, S. (2011) Caractéristiques de la croissance et de la production en fruits chez trois variétés de piment (Capsicum annuum L.) sous stress salin. Tropicultura, 2011, 29, 2, 75-81.
Ibn Maaouia Houimli, S., Denden, M. and Ben El Hadj S. (2008) Induction of salt tolerance in pepper (Capsicum annuum) by 24-epibrassinolide. EurAsia J BioSci., 2, 83-90.
Iqbal, N., Ashraf, M.Y., Javed, F., Vicente, M. and Kafeel, A. (2006). Nitrate reduction and nutrient accumulation in wheat (Triticum aestivum L.) grown in soil salinization with four different salts. J. Pl. Nutr., 29, 409-421.
Jimenez, M.S., Gonzalez-Rodriguez, A.M., Morales,
D., Cid, M.C., Socorro A.R. and Caballero, M. (1997) Evaluation of chlorophyll fluorescence as a tool for salt stress detection in roses. Photosynthetica., 33, 291-301.
Kaya C., Kirnak, H. and Saltalin K. (2002) Supplementary calcium enhances plant growth and fruit yield in strawberry cultivars grown at high (NaCl) salinity. Sci. Hort., 93, 65-72.
Kavi Kishor, P.B., Sangam, S., Amrutha, R.N., Laxmi, P.S., Naidu ,K.R., Rao, K.R.S.S., Rao, S., Reddy, K.J., Theriappan, P. and Sreenivasulu, N. (2005) Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: Its implications in plant growth and abiotic stress tolerance. Curr. Sci., 88, 424-438.
Kerkeni, A. (2002) Microbouturage et Callogenèse de pomme de terre (Solanum tuberosum L.) sous stress salin (NaCI). Mémoire de Diplôme d'Etudes Approfondies en Agriculture Durable, Ecole Supérieur d'Horticulture et d'élevage Chott Meriem, Sousse, Tunisie.
Keshavarzi, M.H.B. (2011) Effect of Salt Stress on Germination and Early Seedling Growth of Savory (Satureja hortensis). Aust. J. Basic & Appl. Sci.,5(2), 3274-3279.
Khan, M.A., Shirazi, M.U., Khan, M.A., Mujtaba, S.M., Islam, E., Mumtaz, S., Shereen, A., Ansari, R.U. and Yasin Ashraf, M..(2009) Role of proline, K/Na ratio and chlorophyll content in salt tolerance of wheat (Triticum Aestivum L.). Pak. J. Bot., 41(2), 633-638.
Koca M., Bor, M., Ozdemir, F. and Turkan, I. (2007) The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environ. Exp. Bot., 60, 344351.
Kocheva, K., Lambrev, P., Georgiev, G., Goltsev, V. and Karabaliev, M. (2004) Evaluation of chlorophyll fluorescence and membrane injury in the leaves of barley cultivars under osmotic stress. Bioelectrochemistry., 63, 121-124.
Kumar, S.G., Reddy, A.M. and Sudhakar, C. (2003) NaCl effects on proline metabolism in two high yielding genotypes of mulberry (Morus alba L.) with contrasting salt tolerance. Plant Sci., 165, 1245-1251.
Lichtenthaler, H.K., Langsdorf, G., Lenk, S. and Bushmann, C. (2005) Chlorophyll fluorescence imaging of photosynthetic activity with the flesh lamp fluorescence imaging system. Phtosynthetica., 43, 355-369.
Lutts, S., Kinet, J. M. and Bouharmont, J. (1996) NaCl-induced senes-cence in leaves of rice (Oryza sativa, L.) cultivars differing in salinity resistance. Ann. Bot., 78, 389-398.
Malik, A.A., Li, W., Lou L., Weng, J. and Chen, Jin-F. (2010) Biochemical / physiological characterization and evaluation of in vitro salt
tolerance in cucumber. Afr. J. Biotechnol., 9(22), 3284-3292
Masojidek, J. and Hall, V. (1992) Salinity and drought stress are amplified by high irradiance in sorghum. Photosynthetica.,
27, 159-171.
Maxwell, K. and Johnson, G.N. (2000) Chlorophyll fluorescence- a practical guide. J. Exp. Bot., 51, 659-668.
Mehouachi, T. (1993) Evaluation de la croissance et de l'activité écophysiologique de la pomme de terre en relation avec le stress nutritif. Thèse de Doctorat. Faculté des Sciences Agronomiques de Gand, Belgique.
Mensah , J.K., Akomeah, P.A., Ikhajiagbe, B. and Ekpekurede, E.O. (2006) Effects of salinity on germination, growth and yield of five groundnut genotypes. Afr. J. Biotechnol., 5(20), 1973-1979.
Misra, N. and Gupta, A.K. (2005) Effect of salt stress on proline metabolism in two high yielding genotypes of green gram. Plant Sci., 169, 331339.
Molazem, D., Qurbanov, E.M. and Dunyamaliyev, S.A.. (2010) Role of Proline, Na and Chlorophyll Content in Salt Tolerance of Corn (Zea mays L.). American-Eurasian J. Agric. & Environ. Sci., 9(3), 319-324.
Moussa H.R.. (2006) Influence of Exogenous Application of Silicon on physiological Response of Salt-stressed Maize (Zea mays L.). Int. J. Agri. Biol.. 8(2), 293-297.
Mundree, S.G., Baker, B., Mowla ,S., Peters, S., Marais, S., Vander Willigen, C., Govender, K., Maredza, A., Muyanga, S., Farran, J.M. and Thomson, J.A. (2002) Physiological and molecular insights into drought tolerance. Afr.
J. Biotechnol., 1, 28-38.
Munns, R. (2002). Comparative physiology of salt and water stress. Plant Cell. Environ., 25, 239250.
Munns, R. and Tester, M. (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol., 59, 651-681.
Neumann, P.M. (1995) Inhabitation of root growth by salinity stress: Toxicity or an adaptive biophysical response. In Baluska, F., Ciamporova, M., Gasparikova O. and Barlow,P.W.(Eds.), Structure and Function of Roots. The Netherlands: Kluwer Academic Publishers, pp. 299-304.
Niu, X., Bressan, R.A., Hasegwa, P.M. and Pardo, J.M. (1995). Ion homeostasis in NaCl stress environments. Plant Physiol., 109, 735-742.
Naidoo, G. and Naidoo, Y. (2001) Effects of salinity and nitrogen on growth, ion relations and proline accumulation in Triglochin bulbosa. Wetlands Ecology and Management, 9(6), 491497.
Ouiza, D., Belkhodja, M., Bissati, S. and Hadjadj S. (2010) Effet du Stress Salin sur l'accumulation de Proline Chez Deux Espèces d'Atriplex Halimus L. et Atriplex Canescens (Pursh) Nutt. European Journal of Scientific Research., 41(2), 249-260.
Parida, A.K. and Das, A.B. (2005) Salt tolerance and salinity effects on plants. Ecotoxicol. Environ. Saf., 60(3), 324-349.
Parida, A., Das, A.B. and Das, P. (2002) NaCl stress causes changes in photosynthetic pigments, proteins and other metabolic components in the leaves of a true mangrove, Bruguiera parviflora, in hydroponic cultures. J. Plant Biol., 45, 28-36.
Petrusa, L.M. and I. Winicov. (1997) Proline status in salt tolerant and salt sensitive alfalfa cell lines and plants in response to NaCl. Plant Physiol. Biochem., 35, 303-310.
Premachandra, G.S., Saneoka, H., Fujita, K. and Ogata, S. (1992) Leaf water relations, osmotic adjustment, cell membrane stability, epi-cuticular wax load and growth as affected by increasing water deficits in Sorghum. J Exp Bot, 43, 1569- 1576.
Rahdari P, Tavakoli S, Hosseini SM (2012). Studying of salinity stress effect on germination, proline, sugar, protein, lipid and chlorophyll content in Purslane (Portulaca oleraceae L.) leaves. Journal of Stress Physiology & Biochemistry., 8(1), 182-193.
Rahimi, A., Biglarifard, A. (2011) Impacts of NaCl stress on proline, soluble sugars, photosynthetic pigments and chlorophyll florescence of strawberry. Advances in Environemental Biology, 5(4), 617-623.
Rains, D.W. (1981) Salt tolerance- New developments. In Manassan J.T. and Briskey,
E.J. (Eds.). Advances in Food Producing Systems for Arid and Semiarid Lands. Academic Press, New York, pp. 14-456.
Ramanjula, S. and Sudhakar, C. (2001) Alleviation of NaCl salinity stress by calcium is partly related to the increased proline accumulation in mulberry (Morus alba L.) callus. J. Plant Biol.,
28, 203-206.
Rao, G.G. and Rao, G.R. (1981) Pigment composition chlorophyllase activity in pigeon pea (Cajanus indicus Spreng) and Gingelley (Sesamum indicum L.) under NaCl salinity. Indian J. Experimental Biol., 19, 768-770.
Sadeghi, H. (2009) Effects of Different Levels of
Sodium Chloride on Yield and Chemical Composition in Two Barley Cultivars. Am.-Eurasian J. Sustain. Agric, 3(3), 314-320.
Shafi, M., , Bakht, J., Javed Khan, M., Aman Khan, M. and R, (2011) Role of abscisic acid And proline in salinity tolerance of wheat genotypes. Pak. J. Bot., 43(2), 1111-1118.
Sharma, S.S. and Dietz, K.J. (2006) The significance of amino acids and amino acid-derived molecules in plant responses and adaptation to heavy metal stress. J. Exp. Bot., 57, 711-726.
Schreiber, U., Bilger, W. and Neubauer,C. (1995) Chlorophyll fluorescence as a non-intrusive indicator for rapid assessment of in vivo photosynthesis. In Schulze E.D. and M.M. Caldwell (eds.), Ecophysiology of Photosynthesis. Berlin, Heidelberg, New York, Springer-Verlag, pp. 49-69.
Schwab, K.B. and Gaff, D.F. (1990) Influence of compatible solutes on soluble enzymes from desiccation-tolerant Sporobolus stapfianus and desiccation-sensitive Sporobolus pyramidalys. J Plant Physiol, 137, 208-215.
Seki, M., Umezawa, T., Urano, K. and Shinozaki, K;
(2007) Regulatory metabolic networks in drought stress responses. Curr Opin Plant Biol, 10, 296-302.
Singh, R. , Issar, D., Zala, P.V. and Nautiyal PC (2007) Variation in sensitivity to salinity in groundnut cultivars during seed germination and early seedling growth . Journal of SAT Agricultural Research, 5(1), 1-7.
Suriyan, C. and Chalermpol, K. (2009). Effect of salt stress on proline accumulation, photosynthetic ability and growth characters in two maize cultivars. Pak. J. Bot., 41, 87-98.
Tewari, T. N. and Singh, B.B (1991) Stress studies in lentil (Lensesculenta M.) II. Sodicity induced changes in chlorophyll, nitrate and nitrate reductase, nucleic acid, proline, yield and yield components in lentil. Plant Soil, 136, 225-230.
West, D.W. (1986) Stress physiology in trees-Salinity. Acta Hortic., 175, 322-329.
Zhang, J., Jia, W., Yang, J. and Ismail, A.M. (2006) Role of ABA in integrating plant responses to drought and salt stresses. Field Crop Research, 97, 111-119.
