IMMUNODETECTION AND COMPARISION OF CELL CYCLE PROTEINS AT DIFFERENT GROWTH STAGES OF CALLUS IN BRASSICA
Department of Molecular Biology and Genetic Engineering
College of Basic Sciences and Humanities , GBPUAT
Pantnagar-263145( INDIA )
Alternaria blight accounts for lower productivity in Brassica sp. Host selective toxin(s) produced by Alternaria brassicae mediates the disease. Based on antagonism observed between the pathotoxin and phytohormone, it was postulated that pathotoxin could be acting through cellular signal transduction pathway, which ultimately affects the regulation of cell cycle. Hence, in present study, Dot blotting was done for detection of the cell cycle proteins using crude protein from 15, 30 and 45 days old calli as antigen and antibodies raised in rabbit against human p53,cyclin, cdc2 p34 and Cdc2(PSTAIRE) as antibody. The analysis of the results of the different dot blots indicated that the expression of the four cell cycle proteins with respect to time interval has a gradation. The expression of p53 increases from 15 days(proliferating stage) to 30 days(stationary growth stage) after that it remains constant up to decaying stage. Concentration of cyclin B1 increased during active growth and was maximum as the callus reaches stationary phase. It goes down during decaying stage. Expression of cdc2 p34 is almost nil at proliferating stage and increases gradually. It is maximum at decaying stage and same is the case with cdc2(PSTAIRE). The results showed that cell cycle proteins are highly conserved and their detection in Brassica sp. , is possible through heterologous antibodies i.e. against human protein in this case. Western blot analysis will lead to deeper insight into cell cycle regulation in Brassica sp.
KEY WORDS Brassica campestris, Alternaria blight, Pathotoxin, Cdc proteins; Cyclin
Rapeseed and mustard belonging to cruciferae are one of the important commercial crops of India ( Hindu Survey of Agriculture, 1997 ). The oil seed Brassica is susceptible to number of pathogenic diseases among which the most important and devastating disease is alternaria blight, caused by Alternaria brassicae . The disease accounts for 50% loss in yield. Plant breeders have been trying albeit without success so far to develop disease resistant line for many years through conventional plant breeding methods. Modern biotechnological methods which allow molecular manipulation at will can successfully be utilized to develop de novo resistance to disease provided that the mechanism of host parasite interaction is understood at molecular level.
The pathogenecity factor for Alternaria blight disease of Brassica is host selective toxin(s) produced by A.brassicae . Toxins include two major components which have been indentified and purified by Sephadex G-10 , LH-20 chromatography , HPLC and G8 column. These two components are chlorotic toxin which is cyclodepsipeptide , destruxin B and necrotic toxin which is perhaps polyketide in nature ( Tyagi, 1991 ). Besides host specific toxins, phytohormones were also suspected to be significant in the establishmrnt of the disease. Green island in chlorotic lesion is formed due to cytokinin like activity elaborated by the pathogen itself. Intrenstingly a fraction of extract from infected leaves that could induce roots suppresses the action of chlorotic toxin on foliar bioassa ( Agarwal et. al. 1994). The observed antagonistic effect of two structurally different entities viz. Pathotoxin and phytohormone on mitotic index of calli from Brassica juncea (Pandey,1996) strongly suggests the possible involvement of signal transduction cascade which converge at single site.
Antagonistic effects of pathotoxinm and phytohormone further suggests the role of hormonal signaling in cell proliferation which involves cell cycle mechanism. Therefore, it is quite necessary to study cell cycle machinery to assess its phtopathogenecity. Understanding the normal regulation of cell proliferation and cell death in plant system will be a breakthrough in the field of Plant Biotechnology. Hence, in the present study attempts have been made to compare the expression of cell cycle proteins at different stages of growth of Brassica calli.
Callus induction and Maintenance
Hypocotyl segments were cut in to small pieces (3-4 mm ). These pieces were teased with the help of scalpel prior to transfer into MS medium supplemented with BAP and NAA. Induction of callus was observed after 10 days and its growth was monitored at 5 days interval up to 45 days.
Measurement of growth
The callus volume and growth doubling time were estimated as described by Arora (1983).
Callus protein extraction
The protein was extracted from callus of Brasssica as described by Feiler and Jacobs (1990) with slight modifications. The protein was estimated by dye binding method (Bradford, 1976).
Dot blot ELISA
Dot blot ELISA, an efficient technique for rapid detection of proteins, was used for detection of cell cycle components during different stages of growth of Brassica calli. The human anti- cdc p34(C-19), anti cdc (PSTAIRE), anti cyclin B1( H 433) and anti p53 antibodies were used in present study as heterologous immunoprobes for detection of cell cycle ccomponentsin Brassica.
The cell cycle as well as apoptotic proteins involved in cell proliferation and cell death pathways have been highly conserved from yeast to mammals. Therefore, to detect the presence or absence of the cell cycle proteins in different growth stages of callus of Brassica, immunoblot techniques (Gershoni,1988) can be used by taking the callus proteins of different stages as antigen and antibodies raised in animal system against those specific proteis of human system . Dot blot technique is the most suitable for this detection because it is rapid process and semi-quantitative technique (Samuel et al. ,1988 ). In cell cycle machinery, the proteins playing the major role in regulation of cell cycle are the cyclins, Cdc’s and p53. The cyclins have been reported in plants (Dahl et. al. ,1995 ).Their molecular weight is not yet known .The Cdcs have not yet been fully characterized in plants. An earlier report on p53 in Brassica is from our Laboratory (Khandelwal, 1997). Therefore, it was decided to use immunoprobe to detect the presence of cell cycle protein in the calli of B. juncea .
The analysis of the results of the different dot blots indicated that the expression of the four cell cycle proteins with respect to time interval has a gradation as given in following Table1.
Table 1. Immunodetection of cell cycle proteins during different stages of growth of Brassica calli
Expression levels with time
Proteins 15 days 30 days 45 days
p53 ++ +++ +++
Cyclin B1 + +++ ++
Cdc2p34 - ++ ++++++
Cdc 2 ( PSTAIRE) - + +++
(- Negligible expression, + Very low expression, ++ Low expression , +++ High expression, +++++ Very high expression )
The results showed that expression of p53 increases from 15 days (proliferating stage ) to 30 days (stationary growth stage ) but then afterwards remains constant up to decaying stage. Concentration of cyclin B1 increased during active growth and was maximum as the callus reaches stationary phase. It goes down during decaying stage. Expression of Cdc p34 is almost nil at proliferating stage and increases gradually. It is maximum at decaying stage and same is the case with cdc2 (PSTAIRE).
It is obvious from the above results that cell cycle proteins are highly conserved and their detection in Brassica spp. Is possible through heterologous antibodies i.e. against human protein in this case. The PSTAIRE region seems to be also conserved. These studies have laid foundation for getting deeper insight in cell cycle regulation in Brassica spp. With the use of western blots. The initial result of western blot in our laboratory further suggested the differential expression of cdc-2 proteins in response of different cytokinins viz. BAP and Kinetin. Understanding of regulation of cell cycle proteins and their multiplicity in different hormonal signaling will not only be helpful for development of new agronomic traits in terms of productivity but also for development of de novo resistance.
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