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Feb 11, 2016 - 313 J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328. Could Sodium Benzoate Enhance Broad Bean Salinity.

JCBPS; Section B; February 2016-April 2016; Vol.-6; No.-2; 313-328.

E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online at www.jcbsc.org Section B: Biological Sciences CODEN (USA): JCBPAT

Research Article

Could Sodium Benzoate Enhance Broad Bean Salinity Tolerance? I. Seedling Vigor, Membrane Features, Antioxidant Enzymes and Osmolytes Bardees Mohammad Mickky Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt, P.O. Box: 35516, Fax: +20 50 2246254, Tel: +20 1009828025 Received: 01 February 2016; Revised: 11 February 2016; Accepted: 16 February 2016

Abstract: Seed presoaking is a simple strategy exploited to improve seedling performance and alleviate the ill impact of salinity which poses a serious obstacle to agricultural production. A germination trial was conducted to evaluate sodium benzoate (SB) potentiality to adverse seawater (SW) strain on Vicia faba seedlings. Salt stress reduced germination capacity, root length, number of adventitious roots and seedling vigor index (SVI) while SB could enhance these parameters. Furthermore, SW prolonged the serial time required for germination but it was shortened by SB. Salinity also declined membrane stability index (MSI) and phospholipid content accompanied by promoted lipid peroxidation and membrane leakage (ML). Application of SB could attenuate lipid peroxidation and ML with corresponding upgrade in MSI and phospholipids. Besides, salinization activated catalase, peroxidase, ascorbic peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, glutathione reductase and superoxide dismutase with further increment in the activity of the first five enzymes by SB. Moreover, SW increased osmotic pressure (OP) caused by more organic osmotica (soluble sugars, proline, keto acids and citric acid) as well as inorganic ions (chlorides, sodium, calcium and phosphorus). SB could induce additional augmentation in OP on account of the assemblage of organic and inorganic osmoprotectants. More interestingly and via a novel statistical parameter "Contribution Index", enzymatic antioxidant system followed by 313

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

Could….

Bardees Mohammad Mickky

membrane stabilization and finally osmoregulation were ranked in order of their contribution to overall trend of seedling performance inferred by SVI. Thence, the results herein suggest seed presoaking in SB as an efficient approach to promote bean salt tolerance. Keywords: broad bean; sodium benzoate; seawater; seedling vigor; contribution index INTRODUCTION Broad bean (Vicia faba L.) is an annual protein-rich legume grown for human and animal consumption with a diverse distribution under variable agro-climatic circumstances that may render the productivity of other crops1. Nevertheless, it is crucial to investigate the response of such a strategic crop to salinity and how to ameliorate stress impact on its growth especially with most cultivated lands all over the world affected by salinity. Coinciding with this problem, the shortage in the available fresh water imposes studying how to make use of the predominant seawater (SW) in irrigation. Accordingly, establishing crop varieties that can withstand in saline habitats is an effectual attitude in enhancing sustainable agriculture2. It is well demonstrated that salinity could hamper plant performance due to both osmotic and ionic stress. Moreover, oxidative stress associated with salinity originates due to the overproduction of reactive oxygen species (ROS). Excess ROS can impede routine cellular metabolism via lipid peroxidation in membranes, denaturation of proteins and breakdown of nucleic acids 3. All of these events would be reflected on the plant growth pattern more notably during germination and seedling growth; the highly critical phase in the plant lifetime most amenable to the hostile influence of salt stress4. The behavior of seedlings prone to salinity is often intricate with different degrees of susceptibility or tolerance to such a deleterious factor. Salt-tolerant plant cultivars tend to activate the expression of genes responsible for membrane stabilization, antioxidant defence and osmotic adjustment 5. Therefore, several strategies have been employed to induce such adaptive responses and hence improve germination under salt stress. Among those, seed presoaking is a simple cheap technique that does not entail any complicated equipment, so it could be counseled to fulfill maximal germination under in vivo conditions6.As one of the chemical agents uncommonly utilized in seed presoaking, sodium benzoate (SB) was recorded as a component of a proprietary technology to enhance seed germination and seedling development under salinity7. Likewise in earlier studies, spraying water-stressed corn leaves with SB reduced the oxidative damage8. Also, seed presoaking in SB incited wheat germination with increased root length, shoot length and root/ shoot ratio under five salt concentrations 9. Similarly, SB application inhibited ABA-induced senescence, lipid peroxidation and H2O2 content in rice plants 10. Therefore, the present study pursues to appraise the role of seed supplementation with SB, as a presoaking agent, in improving broad bean germination and seedling metabolic behavior under salinity stress caused by graduated levels of seawater. Furthermore, the present study involves the establishment of a novel potent statistical parameter that estimates the individual contribution of some crucial plant physiological criteria; namely membrane stabilization, enzymatic antioxidation and osmoprotection, to the overall seedling performance under the studied conditions. MATERIAL AND METHODS Plant Material and Growth Conditions: Seeds of broad bean (Vicia faba L., var. Giza 3, Egyptian origin with G.1*NA29 pedigree) were surface sterilized using 0.01 M HgCl 2 solution followed by 314

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

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Bardees Mohammad Mickky

thorough washing with distilled water. The seeds were then divided into three groups. Seeds of the 1 st group were soaked in distilled water to serve as control, while those of the 2 nd and 3rd ones were soaked in 0.25 and 0.50 mM SB; respectively each for 8 hours. The seeds were then allowed to germinate at 25±2OC in dark-painted plastic boxes. Throughout 7 days, each group of the three was sub-divided into three sets, one for the unstressed seedlings being supplemented with distilled water when required while the 2nd and 3rd sets were treated with 10% and 25% SW so as to obtain nine treatments. Estimation of Seedling Vigor: Germination capacity was estimated as the percentage of seeds germinated by the end of the experiment, herein after 7 days 11. In addition, root length together with the number of adventitious roots was measured. The three parameters were combined to express seedling vigor index (SVI) following Abdul-Baki and Anderson12 where; SVI = germination capacity (%) × root length (cm) + number of adventitious roots Also, the time required for germination of the first seed (T0) as well as one quarter (T25), half (T50), three quarters (T75) and all (T100) the seeds was followed. Estimation of Membrane Features: Membrane characteristics addressed in the present investigation include membrane stability index (MSI), membrane leakage (ML), the amount of membrane phospholipids and the degree of membrane lipid peroxidation. Determination of MSI: According to Sairam et al.13, samples were cut into pieces, washed three times with distilled water and placed in distilled water in two sets. One set was kept at 40C for 30 minutes and the second in a boiling water bath for 15 minutes and electric conductivity (EC) for each was measured. MSI was expressed in percentage as: MSI = (EC40 / EC100) × 100 Determination of ML: Samples were cut, washed three times with distilled water and placed in distilled water followed by centrifugation for 80 minutes at 300 rpm. Electric conductivity divided by fresh weight as a percentage represents the total ion leakage14. Determination of phospholipids: The plant material was extracted twice with trichloroacetic acid (TCA), twice with ethanol and then thrice with ethanol/ ether mixture. The extract was incinerated with H2SO4 to quantify inorganic phosphorus as described by Humphries 15 using H2SO4, ammonium molybdate and stannous chloride with measuring the optical density at 710 nm. Determination of lipid peroxidation: Lipid peroxidation was determined by estimating malondialdehyde (MDA) content16. The plant tissue was extracted with TCA followed by addition of TCA containing thiobarbituric acid, heating then cooling to read absorbance at 532 and 600 nm and using an extinction coefficient of 156 × 10-3 µM-1 cm-1. Estimation of Antioxidant Enzymes Activity: Antioxidant enzymes were assayed in the plant extracts prepared as described by Agrawal and Shaheen17 in phosphate buffer at pH 6.8 for glutathione reductase (GR), catalase (CAT), peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL); and pH 7.8 for ascorbic peroxidase (APX) and superoxide dismutase (SOD). Assay of GR (EC 1.8.1.7.): According to Goldberg and Spooner18, approximately 0.05 ml enzyme extract was mixed with 1 ml phosphate buffer combined with EDTA, 0.1 ml glutathione and 0.1 ml NADPH. The change in absorbance at 340 nm was then recorded. 315

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

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Assay of CAT (EC 1.11.1.6.): As described by Devi19, 1 ml enzyme extract was mixed with 2 ml H2O2 and 3 ml phosphate buffer. After incubation at 27°C for 5 minutes, 1 ml H2SO4 was added and the residual H2O2 was then titrated against KMnO4 until the pink color persisted. Assay of POX (EC 1.11.1.7.): POX reaction mixture contained 3 ml pyrogallol, 0.1 ml enzyme extract and 0.5 ml H2O2 with recording the increase in absorbance at 420 nm20. Assay of APX (EC 1.11.1.11.): APX reaction mixture contained 0.83 ml ascorbic acid, 0.13 ml H2O2 and 0.04 ml enzyme extract with measuring the decrease in absorbance at 290 nm21. Assay of PPO (EC 1.14.18.1.): As described by Devi20, PPO reaction mixture contained 2 ml phosphate buffer, 1 ml pyrogallol and 1 ml extract. The reaction mixture was incubated for a minute at 25°C then stopped by adding 1 ml H2SO4, and the absorbance was read at 420 nm. Assay of SOD (1.15.1.1.): According to Nishikimi et al.22, 1ml of working reagent (phosphate buffer, nitroblue tetrazolium and NADH) was combined with 0.1 ml enzyme extract and 0.1 ml phenazine methosulphate then the increase in absorbance at 560 nm was recorded. Assay of PAL (EC 4.3.1.24.): PAL reaction mixture contained 2 ml phenylalanine, 0.9 ml distilled water and 0.1 ml enzyme extract. The increase in absorbance at 270 nm was recorded 23. Estimation of Osmolytes Content: Osmolytes were quantified in plant-water extract prepared by heating the dry tissue powder with distilled water at 90ºC for an hour with shaking followed by centrifugation. The pellet was re-extracted and the combined supernatants were raised up. Determination of osmotic pressure of the extract: The electric conductivity of the plant-water extract was measured using conductivity meter to directly express the osmotic pressure. Determination of total soluble sugars: The extract was treated with freshly-prepared anthrone reagent in a boiling water bath for 10 minutes with reading the cooled samples at 625 nm24. Determination of proline: The method adopted was that of Bates et al.25. To the plant extract, glacial acetic acid and ninhydrin reagent were added followed by heating at 100ºC for 60 minutes then glacial acetic acid was added followed by cooling and measuring optical density at 510 nm. Determination of keto acids: The plant extract was vigorously mixed with 2, 4- dinitrophenylhydrazine then ethyl acetate was added to be shaken well. To ethyl acetate extract, Na 2CO3 was added with shaking then NaOH was added to the aqueous extract, shaken and estimated at 510 nm26. Determination of citric acid: Following the method of Snell and Snell27, a deproteinizing solution of HgCl2 and ZnSO4 was added to an aliquot of the plant extract followed by filtration then 10N HCl and 6.2% FeCl3 solution were added to measure the color developed at 445 nm. Determination of ionic contents: According to Hansen and Munns28, chlorides were quantified by titration against AgNO 3. As described by Chapman and Pratt 29, sodium was determined with flame photometer while calcium was measured by titration against EDTA using murexide indicator. As mentioned before, phosphorus was estimated following Humphries 15. Statistical Analysis: Ten replicas were taken for germination parameters assay while three were chosen for other investigations; and only the mean values were represented. A test at P ≤ 0.05 was performed using CoHort/ CoStat software where the replica of treatments were applied to descriptive analysis determining standard deviation with completely randomized analysis of variance so that small letters were 316

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

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denoted, where different letters refer to significant variation with higher degree of variation as the letters are far from each other .A novel statistical parameter "Contribution Index; CI" was introduced to assess which of the estimated criteria contributed more to the overall trend of the seedling performance. For saltstressed treatments, correlation coefficient (r) and the coefficient of determination (r 2) relating SVI with each parameter of the estimated criteria were estimated. For each criterion, contribution coefficient was obtained in % as the average of 100 r2 for the included individual parameters then contribution index was calculated as the percentage of each contribution coefficient in relation to 100%. RESULTS AND DISCUSSION Changes in Seedling Vigor: Perusal of the results cleared that seawater significantly decreased germination capacity, root length, the number of adventitious roots and subsequently seedling vigor index of bean seedlings with delaying germination onset as signified by T0, T25, T50, T75 and T100 (Figures 1 and 2). The inhibitory action of salinity at higher dose (25% SW) was generally more pronounced than that at lower dose (10% SW). Application of SB could paramountly enhance germination and seedling growth attributes with more inductive effect of SB at 0.50 mm than at 0.25 mm Comparable findings about the inhibitory effect of salinity on germinated broad bean were reported elsewhere 30-31. Also, SB was similarly recorded to potentially mitigate the reverse impact of salinization in other investigations7-32. Germination capacity (%)

Seedling vigor index a

ab

1250

b

b

b

1000

bc c

750

d

d

500 250

ab

bc

c

a

bc

d

bc

c

d

0

Treatment Root length (cm)

Number of adventitious roots a

ab

20

ab bc

15

10

ab

ab

ab

ab

a d

c d

ab

bc

cd

bc

ab

d

5

0

Treatment

Figure 1: Effect of seed presoaking in sodium benzoate on germination parameters of seawater-stressed broad bean seedlings. Vertical bars represent standard deviation with different letters referring to significant variation at P ≤ 0.05. 317

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

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T 0 (hour)

T 25 (hour) a

a

28

b bc 21

bcd

cd

cd

cd

d

a abc

ab

14

bc

bcd

bcde

cde de

e

7

0

Treatment T 50 (hour)

T 75 (hour)

T 100 (hour)

65

a a

52

de

c

a

d

ef

f

39

b

b

c

b

d

d

b c

d 26

de

a

b c

e

13

0

Treatment

Figure 2: Effect of seed presoaking in sodium benzoate on time required for germination of first seed (T0) as well as 25% (T25), 50% (T50), 75% (T75) and 100% (T100) of the seeds of seawater-stressed broad bean. Vertical bars represent standard deviation with different letters referring to significant variation at P ≤ 0.05. Seed germination and the consequent seedling growth is the most salt-sensitive juncture that depends mainly on water availability in the growing medium to mediate softening of the seed coat and activation of hydrolytic enzymes that catalyze cleavage of stored food33. However and as noticed in the present study, saline habitats could hamper seed germination and seedling development either directly due to ionic stress or indirectly by osmotic stress. Furthermore oxidative stress associated with salinity causes overproduction of reactive oxygen species (ROS) that can damage vital cellular components leading to disruption of seedling metabolism that would be reflected as retarded growth pattern of the stressed seedlings34. Though not extensively, the efficacy of SB to positively interfere with plant metabolism 318

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

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under stress conditions was recorded since SB is a precursor of salicylate which has a potent ability to protect plants growing in saline conditions as well as other abiotic stresses 7. Moreover, earlier results established by Gaballah and Gomaa 35, working on two broad bean varieties, proved that SB application to salinized plants could stimulate water conservation and/or decrease water loss. In addition, the ROSscavenging activity of SB especially toward the hydroxyl radicals could contribute to its promotive action on stressed plants32. Changes in Membrane Features: Cellular membrane is the prime receptive of stress; and so membrane features have long been considered as an indicator of stress perception. It is obvious from the data that increasing seawater doze lowered MSI and phospholipid content of broad bean seedlings with marked increase in ML and lipid peroxidation. Treatment with SB corrected the stress-induced damage to the cell membrane as was evident from the significant increment in MSI and phospholipid content along with the marked reduction in ML and lipid peroxidation. Moreover, the effect of SB was directly proportional to the level of its concentration except in case of lipid peroxidation in 10% SW-stressed seedlings and phospholipids of their unstressed relatives (Figure 3). Matching this trend, salt stress has been found to substantially modify membrane structure and permeability36-37. Also and in accordance with our results, SB could improve the growth of salt-stressed broad bean by reducing lipid peroxidation35. Membrane stability index (%) 100

a

ab

b

Membrane leakage (%) ab

b

c

a

b d

80 60 40

d

20

f

c

g

d

a

f

b

e

0

Treatment Lipid peroxidation (umol MDA g-1 f wt) 0.25

a b

bc

c

a

b

0.15

de

e

c d

cd de

a

a

0.20

0.10

Phospholipids (mg g-1 f wt)

a

de

bc de

0.05 0.00

Treatment

Figure 3: Effect of seed presoaking in sodium benzoate on membrane features of seawater-stressed broad bean seedlings. Vertical bars represent standard deviation with different letters referring to significant variation at P ≤ 0.05. 319

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Lipids and proteins are the major constituents of biomembranes but it is well documented that it is the phospholipid component that determines the critical biological characteristics of the cell membrane 38. Under salinity, modifications in membrane integrity, as usually indicated by MSI and ML, could be attributed to qualitative and quantitative alterations in membrane lipids. The reduction recorded in membrane phospholipids in parallelism with higher rate of lipid peroxidation in salt-suffering plants may be on one hand ascribed to the deleterious effect of ROS excessively generated under stress conditions. On the other hand, such depletion in membrane phospholipids may promote maintenance of membrane integrity as well as cellular homeostasis in salt-tolerant plants unlike their sensitive synonyms lacking such an adaptive response39. Changes in Antioxidant Enzymes: In response to salinity, some plants can develop various arrays of enzymatic defence system to scavenge ROS, thereby protecting cells from oxidative damage. In the present study, seawater induced marked activation of all the assayed antioxidant enzymes, except for 10% SW that had no significant influence on the activity of CAT and SOD as well as 25% SW on APX. Generally, high salt concentration caused more pronounced effect than the lower concentration. Excluding GR and SOD, SB enhanced the activity of all the considered enzymes in salt-stressed seedlings; and this effect intensified with raising SB concentration (Figures 4 and 5). Catalase (Unit g-1)

Polyphenol oxidase (A) a

0.6

b e 0.4

a b

b f

b

c

d g

cd

a

c e

cd

c

d 0.2

0.0

Treatment Peroxidase (delta A)

Ascorbic peroxidase (delta A)

0.3

a

b

c

d e

f 0.2

g

h f

a

b i g

f

e

c

d

f

0.1

0.0

Treatment

Figure 4: Effect of seed presoaking in sodium benzoate on catalase, polyphenol oxidase, peroxidase and ascorbic oxidase activity of seawater-stressed broad bean seedlings. Vertical bars represent standard deviation with different letters referring to significant variation at P ≤ 0.05. 320

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

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Phenylalanine ammonia lyase(Unit ml-1)

Glutathione reductase (Unit ml-1)

0.15

a 0.12

b

b c

0.09

cd

de

e

e

f 0.06

0.03

de

de

b

c

d

e

a

b

c

0.00

Treatment Superoxide dismutase (Unit ml-1) Superoxide dismutase (Unit ml-1)

350

a b

280

c 210

b c e

de

cd e

140

70

0

Treatment

Figure 5: Effect of seed presoaking in sodium benzoate on phenylalanine ammonia lyase, glutathione reductase and superoxide dismutase of seawater-stressed broad bean seedlings. Vertical bars represent standard deviation with different letters referring to significant variation at P ≤ 0.05. Similar studies showed that salt-tolerant cultivars usually have enhanced antioxidant enzyme activity under salt stress compared with those of salt-sensitive ones40. This behavior has been demonstrated in numerous plants41-42. Nonetheless, Gaballah and Gomaa 35 noticed that SB brought about slight increase in the activity of SOD in broad bean. When ROS accumulates because of salt stress, chain reactions commence where dismutation of superoxide radical is catalyzed by SOD into O 2 and H2O2. The latter is then scavenged by CAT that decomposes H 2O2 into H2O and O2 or by POX that cleaves H2O2 by oxidation of co-substrate such as phenolics. Instead, H 2O2 can be detoxified through ascorbate/glutathione cycle which involves oxidation/reduction of ascorbate and glutathione through APX and GR; respectively43. In addition, PAL catalyzes phenylalanine conversion to trans-cinnamic acid, the first step in the biosynthesis of anthocyannins which show potent activity in ameliorating the deleterious effects of

321

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ROS. Furthermore, PPO utilizes O2 to oxidize phenolic compounds to their respective quinones, so higher PPO activity may indicate more degradation of toxic substance accumulated during stress 42. Changes in Osmolytes: Among the multifarious plant responses to salt stress, osmolytes accumulation could be considered as an index to evaluate salt tolerance of different plant species, genera and even cultivars. In the present study, SW significantly increased due to the marked increase recorded in the amount of organic osmotica (total soluble sugars, proline, keto acids and citric acid) as well as the inorganic ions (chlorides, sodium, calcium and phosphorus). Also, SW at 25% concentration had more powerful effect than that at 10%. Seed presoaking in SB induced additional increase in OP as well as the amount of both organic and inorganic osmolytes but the application of SB was much more effective with 10% SW-stressed seedlings than with those treated with 25% SW (Figures 6 and 7). Similar reports about osmoregulation in plants under salt stress were previously documented 44-45. OP (EC in mS)

Proline (mg g-1 d wt)

Keto acids (mg g-1 d wt)

1.5

a

c

0.9

f

g

f

a b

f

g

c b

d c

c e

g

e

f

h g

a

b

d

0.6

0.3

b

c

d

e

1.2

0.0

Treatment Total soluble sugars (mg g-1 d wt)

Citric acid (mg g-1 d wt)

60

a

b c

45

h 30

d

e

f

g

i

15

f

e

d

b

d

a

c

c

b

0

Treatment

Figure 6: Effect of seed presoaking in sodium benzoate on osmotic pressure and organic osmolytes of seawater-stressed broad bean seedlings. Vertical bars represent standard deviation with different letters referring to significant variation at P ≤ 0.05.

322

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Chlorides (mmol g-1 d wt)

Sodium (mmol g-1 d wt) a

3.2

2.4

b 1.6

f

f

g

h

0.8

ef

a

bc de

c

d

e

ab

cd

cde

f

g 0.0

Treatment Calcium (mmol g-1 d wt)

Phosphorus ([mmol g-1 d wt]*10) a

0.28

b c

c

c

0.21

d e

0.14

ef

f 0.07

a d f

e

e

d

c

c

b

0.00

Treatment

Figure 7: Effect of seed presoaking in sodium benzoate on ionic content of seawater-stressed broad bean seedlings. Vertical bars represent standard deviation with different letters referring to significant variation at P ≤ 0.05. Compatible solutes have been recorded as multi-functional molecules often accumulate in stressed cells to serve as potent osmotica that balance the reduced water potential in the plant cell vacuoles with that of the extracellular environment. These solutes can also mitigate the negative effects of stress on enzymatic activity without disturbing proteins and nucleic acids structure and function; thus serving as effectual protectants of essential macromolecules. Moreover, they can act as non-enzymatic antioxidants by scavenging free radicals, particularly the highly-damaging hydroxyl radicals. In addition, they can stabilize biomembranes and also provide available source of nitrogen, carbon and reduction equivalents during stress recovery30-46. Contribution Index: After assessing the behavior of broad bean seedlings when irrigated with diluted seawater particularly after seed presoaking in SB, it was such an important issue to conceive which of the estimated physiological indices (i.e., membrane stabilization, enzymatic antioxidant defence system or osmoregulation) could contribute more to the overall seedling performance inferred by SVI. This could 323

J. Chem. Bio. Phy. Sci. Sec. B, February 2016-April 2016; Vol.-6; No.-2; 313-328.

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Bardees Mohammad Mickky

by statistically achieved using contribution coefficient and contribution index. It could be noticed that contribution coefficient statistically calculated for the estimated membrane characteristics (71.87%) was lower than that for antioxidant enzymes (74.50%) but higher than that for osmolytes (69.69%). Accordingly, contribution index was output as 33% for membrane features, 35% for antioxidant enzymes and the residual 32% was recorded for the studied osmolytes (Table 1).

Coefficient of Determination (r2)

Average Coefficient of Determination ( Aver r2)

Contribution Coefficient ( CC = 100 * Aver r2)

Contribution Index (CI = relative % of CC)

1

100

100 %

71.87

(71.87 × 100)/ (71.78+74.50+69.69) = 33.26 %

74.50

(74.50 × 100)/ (71.78+74.50+69.69) = 34.48 %

69.69

(69.69 × 100)/ (71.78+74.50+69.69) = 32.25 %

Physiological Criterion

Physiological Parameter

Correlation Coefficient (r)

Seedling Vigor

SVI

1

1

MSI

0.9161

0.8393

Phospholipids

0.8151

0.6644

Lipid Peroxidation

- 0.8530

0.7275

ML

- 0.8022

0.6436

CAT PPO

0.9286 0.8994

0.8623 0.8088

POX

0.8740

0.7638

APX

0.8697

0.7563

PAL

0.6711

0.4504

GR

- 0.8725

0.7613

SOD

- 0.9012

0.8121

OP Total Soluble Sugars Proline

0.9475

0.8977

0.8168

0.6671

0.8105

0.6569

Keto Acids

0.8871

0.7870

Citric Acid

0.8988

0.8078

Chlorides

0.8623

0.7435

Sodium

0.7811

0.6101

Calcium

0.8477

0.7186

Phosphorus

0.6191

0.3833

Membrane Features

Antioxidant Enzymes

Osmolytes

0.7187

0.7450

0.6969

Table 1: Contribution coefficient and contribution index of some physiological criteria related to seedling vigor index of seawater-stressed broad bean seedlings under the effect of sodium benzoate presoaking. 324

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Could….

Bardees Mohammad Mickky

CONCLUSION As an illation, SB could be recommended as a powerful presoaking agent to enhance seed germination and seedling development of broad bean irrigated with dilute seawater. Such promotive effect lies in its potentiality to orderly induce antioxidant enzymatic machinery, membrane stabilization and osmoprotection. REFERENCES 1. A.K. Singh, B.P. Bhatt, Faba bean (Vicia faba L.): A potential leguminous crop of Indi, 2012. 2. X. Li, C. Guo, J. Gu, W. Duan, M. Zhao, C. Ma, X. Du, W. Lu, K. Xiao, Overexpression of VP, a vacuolar H+-pyrophosphatase gene in wheat (Triticum aestivum L.), improves tobacco plant growth under Pi and N deprivation, high salinity, and drought. J Exp Bot, 2014, 65, 683-696. 3. D. Prakash, G. Upadyay, P. Pushpangadan, Antioxidant potential of some under-utilized fruits. Indo Global J Pharm Sci, 2011, 1, 25-32. 4. R. Seth, S. Kendurkar, In vitro screening: An effective method for evaluation of commercial cultivars of tomato towards salinity stress. Int J Curr Microbiol Appl Sci, 2015, 4, 725-730. 5. R.D. Satbhai, R.M. Naik, Osmolytes accumulation, cell membrane integrity, and antioxidant enzymes in sugarcane varieties differing in salinity tolerance. Sugar Tech, 2014, 16, 30-35. 6. M.B. Fredj, K. Zhani, C. Hannachi, T. Mehwachi, Effect of NaCl priming on seed germination of four coriander cultivars (Coriandrum sativum). Eurasia J Biosci, 2013, 7, 21-29. 7. J.J. Wargent, D.A. Pickup, N.D. Paul, M.R. Roberts, Reduction of photosynthetic sensitivity in response to abiotic stress in tomato is mediated by a new generation plant activator. Plant Biology, 2013, 13, 1-1. 8. B. Yan, Q. Dai, X. Liu, S. Huang, Z. Wang, Flooding-induced membrane damage, lipid oxidation and activated oxygen generation in corn leaves. Plant Soil, 1996, 179, 261-268. 9. N.K. Roy, A.K. Srivastava, Effect of presoaking seed treatment on germination and amylase activity of wheat (Triticum aestivum) under salt stress conditions. Rachis, 1999, 18, 46-51. 10. K.T. Hung, C.H. Kao, Nitric oxide acts as an antioxidant and delays methyl jasmonate– induced senescence of rice leaves. J Plant Physiol, 2004, 161, 43-52. 11. Y.A. El-Kassaby, Improving lodgepole pine select seed utilization through understanding germination behavior. For Gen Consulting Ltd; 2006. 12. A.A. Abdul-Baki, J.D. Anderson, Relationship between decarboxylation of glutamic acid and vigour in soybean seed. Crop Sci, 1973, 13, 222-226.

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45. G.S. Ranganayakulu, G. Veeranagamallaiah, C. Sudhakar, Effect of salt stress on osmolyte accumulation in two groundnut cultivars (Arachis hypogaea L.) with contrasting salt tolerance. Afr J Plant Sci, 2013, 7, 586-592. 46. B.M. Mickky, Biotechnological aspects of economic maximization and characterization of medically-active phytochemicals from Medicago sativa plants. Ph D Thesis, Faculty of Science, Mansoura University, Mansoura, Egypt, 2012.

Corresponding author: Bardees Mohammad Mickky Botany Department, Faculty of Science, Mansoura University, Mansoura, Egypt,

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