Role of Aluminium and Phosphorus on the Secretion and Behaviour of Acid Phosphatase in Callus Culture of Brassica juncea

 

R. Malathi¹_, G. Melchias², S.Chandrasekar¹ and A.Cholarajan³*

¹Department of Biotechnology, Bharathidasan University College, Perambalur-621 212

²Department of Biotechnology, St. Joseph College, Trichy –620 002

³P.G. Department of Microbiology, Meenakshi Chandrasekaran College of Arts and Science, Pattukkottai – 614 626, Tamilnadu, India.

 

ABSTRACT:

The role of aluminium and phosphorus on the secretion of acid phosphatase in the callus culture of Brassia juncea was studied.  Callus culture derived from leaves of mustard (Brassica juncea) plants.  Proliferated calluses were subjected to P-deficient as well as P-sufficient conditions in the presence and absence of aluminium.  The 10 days old calluses were harvested and so the culture medium in which they were cultured, for analysis of acid phosphatase, protein and amino acid content.

 

INTRODUCTION:

Aluminium (Al) is an important rhizotoxic metal. Aluminium is not present in metallic forming nature, but its compounds are present in almost rocks and soils. In soils Al is present as complex in the form of aluminosilicate. The acid soils release Al in the form of hexa aqua-aluminium (Al (H₂0)₆) which are determine to plants limiting growth and productivity. Lower concentration of Al been reported to bring about beneficial effect in certain crops.  In higher concentration of Al, whether supplied or naturally occurring, limit plant growth and metabolism.  Aluminum toxicity is an important limitation to crop productivity.

 

MATERIAL AND METHODS

Brassica juncea was grown in sterile MS medium for the production of explants (Murashige and Skoog, 1962). After six days of growth, its leaves were used as explants and transferred to MS medium for callus culture in the composition of MS medium with 1 ppm 2,4-D.  The proliferating calluses were subculture on the fifteenth day for the experiment.  The experiment was design that series I represented the control (with phosphorus (P) and without Al). Series II was used to impose P-deficiency stress.  Series III and IV were 250 and 500 µm of Al as (Al₂ (SO₄)₃, 16 H₂O) in the presence of P, and series – V represented Al stress (250 µm) in the absence of phosphorus.  The pH was maintain to 5.8.Callus were harvested on the 10^th day of applying stress (Plate – 1).  Fresh tissue was used for the assay of amino acid (Moore and Stein, 1948) protein (Lowry et al., 1951) and acid phosphatase (Ikawa et al., 1964).

 

(Plate-1)

I. Control II. P-deficiency condition III. 250 µ mol Al stress 1V. 500µmol Al stress V. Al stress with absence of P

 

 

RESULTS AND DISCUSSION:

Callus subjected to aluminium stress showed reduced protein content in the presence as well as absence of phosphorus.  The amount of protein secreted by the callus into the medium.  However increased by aluminium and phosphorus stress.  But when both stresses were applied together, secretion of protein into the medium was reduced by 32 percent (Table-1).

 

In the callus tissue phosphorus deficiency reduced the protein content by about 13 percent, compared to control.  The reduction was one percent higher by 250 µ mol aluminium, but at 500 µ mol aluminium the reduction in protein content was 32 percent.  When both the stresses were applied simultaneously, the reduction in callus protein content was by a lower magnitude 25.37 percent.

 

Individual stresses of aluminium and phosphorus deficiency increase the protein content.  The increase was 27 percent by phosphorus deficiency, 64 percent by 250 µ mol aluminium and 33 percent 500 µ mol as aluminium in on the other hand, when both the stresses were applied simultaneously, the reduction in protein content.  In the medium was lowered by 32.54 percent compared to non-stressed control.

 

For, P-deficiency reduced the amino acid content by 55.75 percent while 250 µ mol aluminium reduced it by 66.60 percent.  The reduction however was only 38.96 percent by 500 µmol.  But when stresses were given together amino acid levels dropped by about 19.72 percent (Table- 2).

 

 

Table -1 Effect of P-deficiency and / or Al stress on the protein content of Brassica juncea leaf callus

 

 

 

Table- 2 Total free amino acid content of Brassica juncea leaf callus subjected to P-deficiency and / or Al stress.

 

Table -3 Changes in enzyme activity (µ moles of P – nitro phenol released      min^-1) of acid phosphatase as affected by P-deficiency and / or Al stress.

 

 

Acid phosphatase activity

The enzyme acid phosphatase activity was analyzed in the callus and also in the medium (Table -3).  Phosphorus deficiency increased the activity of the enzyme by 1.76 percent per gram callus tissue.  Al stress in the absence and presence of phosphorus reduced the enzyme activity.  The activity dropped by about 35.69 percent and 52.42 percent respectively by 250 and 500 µmol Al.  When Al was supplemented with P stress the fall in enzyme activity was over 52.85 percent.  A similar trend was observed when the activity was calculated for mg protein.

 

On the other hand, the response of the enzyme secreted into the medium was differential.  P stress reduced the activity by about 9 percent and 250 µmol Al reduced it about 7 percent.  But the activity was enhanced by over 15% in    500 µ mol Al and 2.96% by both stresses together.

 

The enzyme activity in the medium was calculated as the function of the protein content of the callus tissues.  This showed as 68 percent activity in the control, 62 percent by P stress, 69.5 percent and 90.5 percent for 250 and 500 µmol Al stress, while the activity was 67.7 percent under the combined stress condition. Brassica juncea callus under P-deficiency and Al stress showed reduced protein content.

 

CONCLUSION:

Reduction in protein content of the callus may be due to reduced protein synthesis and /or secretion of the protein into the medium. Reduction in amino acid content must be due to two reasons: reduced amino acid synthesis, and exhaustion of amino acid for protein synthesis. Since the change in level of amino acid of the callus was in a pattern similar to protein level of the medium but not of the callus. It could assume that reduction in the callus protein levels was due to the secretion of the proteins from the callus into the medium. Secretion of organic acids and acid phosphatase as an innate quality of plants that grow under P-deficient conditions has been reported by many authors (Hirata et al., 1982; Tarafdar and Jungk, 1987.) This is also believed to be a wide spread adaptive mechanism of plants in order to grow better in P-deficient soils (Tadano et al., 1993). Hence, it could be assumed that the callus exhibited the same ability under similar conditions of P-deficiency. Tadano et al.,(1993) reported that when P is withdrawn from the medium and is made inaccessible for plants, a particular isoenzyme form ( acid phosphatase) of a larger molecular size is secreted in appreciate quantity to hydrolyze organic P compounds (phosphate ester) in the rhizospere and release inorganic phosphorus. This view was supported by Haussling and Marschner (1989).

 

REFERENCES:

1.       Hirata, H., Hisaka, H., and Hirata, A.1982. Soil science plant Nutrition. 28: 543-552.                 

2.       Haussling, M., and Marschner, H. 1989. Biolo.Fertile.soil.8 :128-133

3.       Ikawa, T.K., Nisizawa, Z., and Miwa,T. 1964.  Specifications of several acid phosphatase from plant source. Nature. 203:939.   

4.       Lowry, D.H., Rosebrough, Far, A.L., and Randel, R.J.1951. Protein measurement with Folin-Phenol reagent. J.Biol.Chem.193:265-275.

5.       Moore, S. and Stein, W.H.1948. Photometric ninhydrin methods for use in the chromatography of amino acids. Biol Chem. 179: 367.

6.       Murashige, T. and Skoog, F.1962.A revised medium for rapid growth and bioassays with tobacco tissue culture. Physio .Plant.15:473-497.               

7.       Tarafdar, J.C. and Jungk, A.1987.Biol.Fertil Soils, 3:199-204.

8.       Tadano, T., Ozawa, K., Sakai, H., Osaki, M and Matsui, H.1993. Secretion of acid phosphatase by the roots of crop plants under phosphorus-deficient conditions and some properties of the enzyme secreted by lupin roots. Plant and Soil. 155/156; 95-98.

 

 

 

Received on 28.06.2012                             Accepted on 29.07.2012        

©A&V Publications all right reserved