Indian Journal of Experimental Biology Vol. 39, May 2001, pp. 469-475

Amelioration of NaCl stress in Pisum sativum Linn.

Ali Abdel-Aziz Abdallah EI-Mashad & Ehab Abdei-Razik Kamel

Department of Biological Sciences & Geology, Faculty of Education, Ain Shams University, Cairo, Egypt

Received 27 May 1999; revised 22 December 2000

Activity of endogenous auxins and growth inhibitors, gibberellins and cytokinins was observed in the extracts of seedlings of Pisum sativum under NaCI stress. After 6 days of germination, when Pisum sativum seedlings were subjected to low concentration of NaCl (50 mM) or boron (10 ppm) increased the endogenous growth regulating substances. Higher concentration of NaCl ( 150 mM) decreased endogenous level of growth regulators, length of the root and shoot, and fresh and dry weights of seedlings, whereas boron increased the parameters except endogenous growth regulators. Mitotic index and some abnormalities were observed in the treated plants. SDS-PAGE banding pattern of Pisum sativum seedlings extracted in tris-glycine and tris-HCl showed that lower concentration of NaCl increased the number of protein bands, while the higher concentration decreased these protein bands. Combination of boron and NaCl (150 mM) caused an increase in total number of protein bands compared with the total number of bands recorded by using NaCl ( 150 mM) alone.

Effect of NaCl stress is harmful at early stages of germination 1, which is concentration dependent and species specific. Salinity causes reduction in length of root and hypocotyl, germination and fresh weight of seedling, and at higher levels of salinity delayed initiation of germination and lipid breakdown2

•

Adverse effects of salinity or any other similar stress conditions were attributed by some investigators to lower endogenous levels of cytokinins3

·5 and I or to

imbalance hormonal contents6• Garate et ae, studied

the effect of boron on pea cv. argona. He has reported a quick increase in plant weight with the addition of boron. Increase in different growth parameters in Vigna sinensis has also been reported8

• Levitt9 has reported that salinity adversely affects the protein metabolism due to decrease in protein synthesis, accelerated proteolysis, decrease in availability of amino acids and denaturation of enzymes involved in protein synthesis. Salt stress induces the accumulation of free amino acids with concomitant decrease in protein content in Raphanus sativus10

•

In the present paper, the effect of NaCJ and boron on phytohormone activities, protein pattern and mitotic division of seedlings of Pisum sativum has been presented.

Materials and Methods Pisum sativum cv. sugary, Hordeum vulgare cv. g

118, Andropogon sorghum cv. g 142 and Xanthium brassilicum Hell. were used in the present study. Seeds of these plants were obtained from the Agriculture Research Center, Ministry of Agriculture,

Giza. Sodium chloride and boric acid used were of analytical grade.

Seeds of uniform size of Pisum sativum were selected, sterilized for 2 min in sodium hypochlorite solution (1 %) and washed with distilled water. These seeds were divided into six groups (G 1-G6), each group comprises 100 seeds. These seeds were soaked for 12 hr at 25°±1°C either, in distilled water (control; G1); boron (10 ppm; G2); NaCl (50 rnM; G3); NaCl (150 rnM; G4); boron (10 ppm) with NaCl at a concentration of 50 rnM (G5) or 150 rnM (G6). Seeds were then germinated in petri dishes (15 em diam) lined with moistened filter paper at 25°±1 °C. Samples of the growing seedlings (20 seedlings) were collected after 6 days of germination. Extraction of the growth substances was done as described earlier11

, and activity of auxins 12

, growth inhibitors 12, giberellins 13

,

and gibberellin-like substances 13, and cytokinins 14

was estimated.

Cytological studies were conducted using root tips of germinating seeds. Five root tips were taken of each treatment in addition to control, fixed in Camoy·s fixative (1 :3 acetic : alcohol) for 24 hr and stored in alcohol (70%) at 4°C. The fixed roots were hydrolyzed for 6-8 min in 1 N HCl at 60 °C, stained with Feulgen stain and squashed in a drop of acetic acid (45%) 15

•16

• Mitotic index was calculated by counting number of dividing cells from 5 different root tips. For each treatment about 3000 cells were counted. Total percentage of abnormalities was calculated and types of abnormalities were

470 INDIAN J EXP BIOL, MAY 200 1

photographed usmg Carl-Zeiss photomicroscope III (x X 2000).

Seedling proteins of untreated and treated Pisum sativum seedli ngs were analyzed using SDS-PAGE (Ref. 17). For electrophoretic analysis the seedling proteins were extracted in two different buffers (tris­glycine, pH 8.2; and tris-HCI, pH 8.0) to obtain a good separation of polypeptide bands, and electrophoresis in 12 and 12.6% of polyacrylamide gel was undertaken respectively. Gel Pro-Analyzer software version 2.0 was used to determine the molecular weight of each protein band.

Results and Discussion High concentration of NaCI (150 mM) decreased

the root and shoot length of the treated Pisum sativum seedli ngs, however, an increase was observed in NaCI (50 mM) or boron {I 0 ppm) treated seeds {Table 1). Combination of NaCI and boron caused variable changes in these characters. This indicated that the

120 115

110

95

Control

IAA IAN ++ ++

10ppm B

IAA IAN ++ ++

0/J 1/J 0.0 0.5 1.0

"' .. ...

.. 110 ..1 ~ 105 0

50 mMNIICI

i 115t

~ 100 L-----------u '(; 95 ....__ __ --''-------' .c ii4l c:

SOmMN.CI +10ppm B

! 150mMNaCI 1~miiiN.CI

~:~t~[~ 0.5 1.0 0.0 o.s 1.0

Fig. I -Changes in auxins and growth inhibitor contents of NaC I and boron treated and untreated Pisum sativum seedlings after 6 days germination.

low concentration of NaCI or boron treatment caused not only cell enlargement but also cell divi sion, while higher concentration of NaCI (1 50 mM) reduced both cell division and cell enlargement of root and shoot of Pisum sativum seedlings. An increase in auxins and gibberellins concomitantly with high contents of cytokinins was observed in the seedlings treated with NaCI (50 mM), boron (10 ppm) or combination of NaCI and boron . Decrease contents of endogenous hormones in response to highelf dose of NaCI may be attributed to presence of endogenous growth inhibitors that retarded the cell div ision and cell enlargement of the root and shoot of Pisum sativum seedlings (Fig. 1). Also, increase in fresh and dry weight of Pisum sativum seedlings in response to NaCI (50 mM), boron ( 10 ppm) or combination of them may be due to greater water uptake. While the significant reductions in fresh and dry weight of seedlings with the high concentration of NaCI {150 mM) may be due to decreased water uptake or can be attributed to the combined action of low level of

Control 10 pPm B

GA:J ++

0,5 1.0

!50mM NaCI +10ppm 8

1SOmAf NaCI +10 ppm 8

f~ 0.5 1.0

Fig. 2--Changes in gibberellin contents of NaCI and boron treated and untreated Pisum sativum seedlings after 6 days germination.

MASHAD & KAMEL: AMELIORATION OF NaCI STRESS IN PISUM SATIVUM LINN. 471

auxins, gibberellins and cytokinins and the high content of the growth inhibitors.

That salinity causes reduction in the length of root and hypocotyl of Pisum sativum seedlings has been reported earlier2

. On the other hand, El-Mashad8 has found that in Vigna sinensis, low concentration (10 ppm) of boron, significantly increases some growth parameters. It has been noted that dry weight of root and shoot of barley decreases with increasing NaCI (Ref. 18). Also, Kord and Khalil 19 have reported decrease in seed germination and seed water uptake with increasing salinity in wheat and pea.

Cytological observations and the data obtained from the analysis of root tips treated with NaCI and boron are summarized in Table 2 and 3. Seeds treated with NaCI (50 mM), boron (10 ppm) and NaCI (50 mM) +boron (10 ppm) had increased mitotic indices,

while high concentration of NaCI (150 mM) decreased the mitotic index . Percentage of chromosomal abnormalities increased by the treatment of seeds of Pisum sativum by low concentration of NaCI, boron or both together (Table 2).

Maximum per cent of abnormal cells (29.8) was scored in seeds treated with NaCI (150 mM), whereas the treated seeds with boron (10 ppm) and combination of NaCI (50 mM) and boron were found to have less per cent of abnormal cells (Table 2). Abnormalities like disturbance, stickiness, C­metaphase and micronuclei were observed which were maximum in the seeds treated with NaCI (150 mM ; Table 3, Plate 1 ).

Increase in extractable indole auxins of Pisum sativum seedlings in response to NaCI (50 mM), boron (10 ppm) or combination (Fig. 1) might result

Table !-Effect of NaCI and boron on growth parameters of Pisum sativum seedlings [Values are mean of 10 samples]

Concentrations Root Shoot Fresh weight Dry weight length length of seedling of seedling (em) (em) (g) (g)

A 9.5 * 3.3 * 0.88 * 0.162 * B 10.1 * 3.5 * 0.92 * 0.1 72 * c 2.9 1.5 0.56 0.120 D 5.7 2.5 0.76 0.134 E 10.5 * 4.4 * 1.02 * 0.196 *

Control 6.7 3.0 0.82 0.152

5 % 0.48 0.14 O.Q25 0.004 L.S.D. at

1 % 0.69 0.2 1 0.036 0.006

* Significant at I%. Treatment given: A- NaCI (50 mM); 8- NaCI (50 mM) +boron (10 ppm); C- NaCI (150 mM); D- NaCI (150 mM) +boron (10 ppm); and E- boron (10 ppm].

Table 2-Mitotic index and percentage of the different mitotic phases in Pisum sativum seedlings treated with NaCI and boron

Total No. of No. of %of Prophase Metaphase Anaphase M.I.% Concentrations cells divid. abn. abn. +Telophase

exam. cells cells cells No. % No. % No. %

A 3355 297 28 9.4 121 40.7 66 22.2 110 37.0 8.86 * B 3490 315 29 9.2 110 34.9 100 31.8 105 33.3 9.03 * c 3568 188 56 29.8 44 23.4 36 19.2 108 57.5 5.27 D 3632 236 32 13.6 60 25.4 88 37.3 88 37.3 6.50 E 3240 295 25 8.5 125 42.4 65 22.0 105 35.6 9.10 *

Control 3375 252 5 2.0 108 42.9 41 16.3 103 40.9 7.47

5 % 0.25 L.S.D. at

1 % 0.36

* Significant at l %. A, B, C, D, E- as indicated in Table I.

Table 3-Different types of abnormali ties at interphase and mitotic phases of Pi sum sarivum seedlings treated with NaCI and boron

Prophase Metaphase Anaphase + Telophase Interphase Concentrations Di st. Stick Dist. Diag C- meta Lag. Stick Dist Lag. Brid Micro Stick Micro Mult Divid.

nucl. nucl. nucl nuc!.

A 3 2 5 3 6 3 2

B 3 3 4 7 2 4 2

c 7 5 8 9 2 3 3 3 2 2 3 6 2

D 4 4 5 4 2 2 2 2 2 2

E 5 4 2 8 3 2

Control

A, B, C, D, E - as indicated in Table I .

Table 4-Protein electrophoretic pattern of Piswn sarivum seedlings treated with boron and NaCI extracted by Tris-glycine and Tri s-HCI buffers

No. of bands

I 2 3 4 5 6 7 8 9 10 II 12 13 14 iS 16 17 18

M.W. (KD) 71.10 65.30 62.00 56.40 53.00 41.00 37.30 34.00 32.10 30.20 28.00 25.00 22.50 2 1.00 19.30 !7.00 16.00 14.00

Total no. of bands

A

+

+ + + + +

+ + + + + + + + + 15

Tri s-glycine Buffer. B C

+ + + +

+ + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + 15 18

D

+ + + + + + + + + + +

+

13

E

+ + + + + + + + + + +

+ + + 14

F

+ + + + + + + + + + +

+ + + 14

Tris-HCI Buffer No. of M.W. bands (KD)

I 114.1 2 0 3 99.20 4 92.00 5 85 .20 6 71.00 7 62 .00 8 53.00 9 47.50 10 43.30 ll 41.00 12 37.30 13 34.00 14 30.00 15 29.00 16 28.00 17 22.20

2 1.00 Total no. of bands

A

+

+ + +

+ + + + + + +

+ + +

13

B

+ + + +

+ + + + + +

+

+ + +

14

c

+ + + + + + + + + + + + + + + + +

17

D

+

+

+

+ + + + + + + +

II

E

+ + + +

+

+ + +

+

+ + +

12

control; B- boron ( l 0 ppm); C- NaCJ(50 rnM); D- l"/aCI i 150 mM); E- NaCI (50 mM)+boron ( 10 ppm) and NaC1 ( 150 rnM)+boron ( I 0 ppm)

F

+ +

+

+ +

+ + +

+

+ +

+

12

Total no.

28

29

56

32

25

5

+>. -...)

N

z 0 > z tTl X '"C w 0 _r 3::: )>

-< N

8

MASHAD & KAMEL: AMELIORATION OF NaCI STRESS IN PISUM SATIVUM LI NN. 473

....;

.. '< " ' '

' ... • . "~ ~ L . .

~ ~ f :._:.:: ~

. ~ . ---•

(\.

• I . · ...

D • . .:'"" IJ , . f) t • ~

.r·'*

·-. '

(! .. ~ft .•

a .....

' ~ ' l , .:.... .e•A

1 ..

~ ~ ·~

~ ""' ... ,. <

' . ~ '

.• [I] ,.,..>;·.

I

i ;

m Plates !-Types of abnormal phases observed in root tips of Pisum sativtml treated with NaCI & boron. [(I) Disturbed prophase with lagg ing chromosome; (2) Disturbed prophase; (3) Slight sticky prophase; (4) Disturbed metaphase; (5) Diagonal metaphase; (6) C­metaphase: (7) Lagging chromosome at metaphase; (8) Sticky metaphase; (9) Disturbed anaphase; ( I 0) Chromosome bridge; ( II ) Diagonal anaphase with lagging chromosome; ( 12) Micronucleus at te lophase; (13) Micronucleus; (14) Multinucleated cell; and ( 15) Divided nucleus].

474 INDIAN J EXP BIOL, MAY 2001

Control

"'[ ~::.._ " ... 0

~ 0

"' "' "' =

o.o a.s 1.0

~ :::[--50 mM NaCI

c 105

"' c ~ 100 .. >. 0 " ... 0 ..c 0. 150 mM NaCI

~ 110[ ~ 105 I ._

::2 100 lllpil~~~-r,.......~4-. L.. u

95

0.0 o.s 1.0

~ 0.0

10ppm B

0.5

150 mM NaCI + 10 ppmB

Kp

1.0

[?7 ~~~~·¥

0.0 1.0

Fig. 3-Changes in cytokinin of NaC I and boron treated and untreated Pisum sativum seedlings after 6 days germination.

from either decreasing the destruction of auxins by reducing the activity of IAA-oxidase or by increasi ng the biosynthesis of auxins. The first view has been supported by EI-Mashad8 who has reported that significant decrease in the activity of IAA-oxidase when the seeds of Vigna sinensis were treated with boron (10 ppm) . The second view was supported by Gauch and Duggcr20 and Devlin and Withman21 who have suggested that boron pl ays a role in DNA sy nthesis in meri stems, which has been implicated in cellular differentiation and development, The common features of boron deficiency in plants are the death of stem and root tips, abscission of flowers, and increase in endogenous hormone and metabolic activities of plants . In this respect one can sugges t thar boron may interfere with hormone metabolism particularly auxins.

Higher concentration of NaCl (!50 mM) decreased the biosynthesis of g ibberellins, while NaCI (50 mM), boron (I 0 ppm) and combination between them increased the g ibberellin biosynthesis (Fig. 2) . Cresswell and Nelson22 have found that boron and GA3 innuence the a-amylase activity in germinated

0 2

f>7 -

30 -

20-1-

14-4- . .. ~· -~ _ _::_ __ __

Tris -giY<:in..: l l uffn

MAB CDE F

Tris-IICI ButTer

Fig. 4-Eiectrophoretic banding pr fi les of seedling proteins ex tracted in Tri s-g lycine and Tris-HC I buffers of Pi sum sativ11111 treated with NaC I and boron. [M-Ma.-ker; A-control; 8 -boron ( I 0 ppm); C-NaCI (50 mM); D-NaCI (I SO mM); E-NaC I (50 mM) + boron ( I 0 ppm); and F-NaC I ( 150 mM) + boron (I 0 ppm)].

Phaseolus vulgaris seeds. They have sugges ted that boron apparently has a regulatory role in the synthesis of GA3.

NaCI (150 mM) reduced t e cytokinin content of seedlings of Pisum sativum as compared wi th the controls. Treatment with NaCI (50 mM), boron (1 0 ppm) and combination of NaCI + boron increased the cytokini ns (Fig . 3). Adverse effects of salinity or any other s imil ar stress condi tions ascribed by some

MAS HAD & KAMEL: AMELIOR ATION OF NaCI STRESS IN PISUM SA TIVUM LI NN. 475

investigato rs as being mainly due to lower endogenous level o f cytokinins4

·5

·13

. However, increase in activity of cytokinins in shoots and roots has been reported in Vigna sinensis8

.

Variation in SDS-PAGE banding pattern of proteins (Fi g. 4) ex tracted from Piswn sativum seedlings treated by NaCl and boron and extracted in tri s-glyc ine (pH 8 .2) and tri s-HCJ (pH 8.0) are expressed by band numbers g iven in Table 4 . Low concentration of NaCI (50 mM) increased the total number of protein bands ( 18 and 17) when buffers compared with the cont ro l ( I 5 and 13), While, high concentration of NaCI ( I 50 mM) decreased the nu mber of protein bands ( 13 and II ). Additi on of boron to NaCl recovered the stress of salinity as seen the similar number o f protein bands compared with contro l (Table 4).

The stress ameliorati on conferred by boron may be medi ated by increase in endogenous growth regul ators, protein synthes is and os moregulation.

References I Levi ll J, in Germination and seedling growth under salinity

and moistu re stress, Response of plants to environmental stress. Vol. II (Academic Press. New York) 1980, 132.

2 Kayani S A, Naqv i H H & Ting I P. Sa linity effec t on germinati on and mobi li za ti on of reserves in jojoba seed, Crop Sci, 30 ( 1990) 704.

3 Ben-Zioni A, Itai C & Vaadia Y, Water and sa lt stresses, kinetin and protein synthesis in tobacco leaves. Plant Physiol, 42 ( 1967) 36 1.

4 ltai C & Vaadia Y, Kinetin-like ac ti vity in root ex udate of water-stressed sunll ower pl ants. Physiol Plcnll, 18 ( 1965) 94 1.

5 lta i C & Vaad ia Y. Cytok inins ac tt vtty in water stressed shoots, Phn11 Physiol. 47 (!97 1) 87.

6 Pri sco J T & O'Leary J W, Enhancement of intac t bean leaf senescence by NaCI salini ty, Physiol Pl(// lf , 27 ( 1972) 95.

7 Garate A. Esteban E, Lucena J J. Carpena R 0, Bonill a I & Barrow N J, Effect of boron on nodul ated and non-nodul ated pea pl :lllts, Plant So il Sci, 54 ( 1993) 4 17.

8 El-Mashad A A A, Effect of certain micronutrient elements 011 plan/ growlh and 111etabolism. Ph.D. Thes is, subm itted to A in Shams Uni v., Cairo, Egy pt ( 1998).

9 Lev itt J, in Protein, nucleotide and ribonucleic acid metabolism during germination unuer water stress, Response of plants lo enviro111ne111al stress, Vol. II Monograph (Academic Press New York). 1972. 365.

10 Muthukumarasamy M & Panneerselvarn R, Triazo le induced protein metaboli sm in the salt s;ressed Raphanus smil'us seedl ings. J Indian Bot Soc, 76 (!997) 39.

II Gazit S & Blumenfe ld A, Cytokin in an inhi bitor ac tivities in the avocado frui t mesocarp, Plant Phvsiol, 46 ( 1970) 334.

12 Foda H A & Radwan S S A. Straight growth test for hormones and inhibitors using coleoptiles of some Egyptian plants. Ain Shams Sci Bull, 8 (1962) 38 1.

13 Bently J A & Mowatt . Act ivity of gibberellins GA 1 to GA9 in the Avena firs t bioassay and location afte r chromatography. Ann Bot, 30 ( 1966) 165.

14 Esashi Y & Leopold A C. Cotyledon expansion as a bioassay fo r cytok inin, Plant Physiol, 44 ( 1969) 61 8.

15 Darlington CD & La-Cour L F. Stai ning and mount ing. The handling of chromosomes. 6' 11 ed it ion (G org Allen & Unwin Ltd, London) 1976.

16 Fukui K & Nakayama S, Plant chromosomes at metabolic phase, Plant chromosomes: Labora101y methods (CRC Press Inc. Boca Raton, New York, London. Tokyo) 1996.9.

17 Weber K & Osbon M, The reli abi lity of molec ular weight determinations by dodecy l sulphate-I)olyacry lamide gel electrophoresis. J Bioi Chem, 244 ( 1969) 4406.

18 Hu rkman W J & Tanaka C K, The effects of sa lts on the pattern of protein synthesis in barley mots. Plant Physiol. 83( 1987) 5 17.

19 Kord M A & Kh alil M S, Salini ty stress and enzymatic activ it ies du ri ng seeds germination. Egypl J Physiol Sci . 19 ( 1995) 225.

20 Gauch H G & Dugger W M, The phys iologica l action of boron in higher plants; a review and interpretat ion. Mm)'land Agric Exp Sta Tech Bull (A), ( 1954) 80.

2 1 Dev lin R M & Wi th man F H, Root elo nga ti on and nuc leic ac id metabolism, Plant physiology (CBS Publishers and Distributors, New De lhi, India) 1986, 15 1.

22 Cresswe ll C F & Nelson H. The inlluence of boron on the RN A level, a-amylase activity and level of sugars in germin ati ng Themeda triwu/ra Forsk. eed, Ann Bot. 37 (! 973) 427.