Financial support: Ministry of Science, Research and Technology of Iran.

Keywords: Programmed Cell Death (PCD), rice, senescence.

Present address: #Research Centre for Applied Sciences, Jahade Daneshgahi, Shahid Beheshti University, Evin, Tehran, Iran. Tel: 0098-21-2402097 (ext. 213). Fax: 0098-21-9403884. §European Commission, DG12, Office SDME 9/28, 200 Ruela Lai, 1049, Brussels, Belgium.

In this paper some molecular aspects of a process called "senescence" is dealt with. Senescence comes under a broader expression "programmed cell death" (PCD). This is the final stage of an organism's life and many genes and gene products (enzymes) are involved in it. Apoptosis is another type of PCD, used mostly to describe a specific type of cell death in animal cells; and the same as senescence, is highly organized and programmed. Many scientists have been trying to discover if a similar or identical type of programmed cell death, like apoptosis exists in plant kingdom and so far, there have been some positive indications.

To discover the possible causes of senescence, different systems have been employed. In this study, we used rice cell suspension culture as a model system. Cell suspension cultures are clumps of cells maintained in a liquid nutrient medium on a shaker. The cells have to be transferred into fresh media in particular intervals (called subculture), in our case once a week. To start the investigation, the cells were not subcultured and maintained in the same media for up to 4 weeks. Every week, the cells were collected through filtering a sieve and their fresh weight, protein content, viability (viable cells)  and nuclease activity were measured. Nucleases are a group of enzymes involved in the catabolism (breakdown) of nucleic acids, DNA and RNA and found to be specifically activated during both senescence and apoptosis. Fresh weight was measured by the difference between the initial and final weights. Protein content was obtained by comparing a determined volume of protein extract against a standard curve. Viability was assayed by a test called TTC. A substrate in a buffer was added to 5 mg of cells and kept overnight at 25ºC. Active and viable cells would convert the colorless substrate into a red substance, which the absorbance of the colour was measured at 495 nm and viability was expressed as the absorbance/g of cells. Nuclease activity was determined by measuring the absorbance of free nucleotides (released during 20 min periods of incubation of single stranded DNA substrate by the activity of present nucleases) at 260 nm, and expressed as dA (absorbace) at 260/mg of protein/ min.

Cell growth was rapid between weeks 1 and 2, decreased slightly between weeks 2 and 3 and showed a sharp decline between weeks 3 and 4. Protein content and cell viability also declined, but nuclease activity increased from week 1 to week 2, was almost constant up to week 3 and the second increase occurred between weeks 3 and 4. Different ions and chelators (EDTA and EGTA) were tried on the nuclease activity found in the cells. Amongst all ions used, zinc had a stimulating effect and EDTA had an inhibitory effect. To visualize the nucleases in the cell extracts, a gel assay was used. Two nucleases with the molecular weights of 26 and 53.5 KDa were detected. The activity of the 53.5 KDa increased, but the 26 KDa nuclease remained relatively the same with the age of the cells.

Cell cultures have found great importance in regard to understanding mechanisms underlying different types of PCD. In this regard, they have served as model systems in getting basic knowledge and extending the information onto whole organisms. They have been used in studying xylogenesis, hypersensitive response, starvation, osmotic stresses and senescence. In this study, one of the landmarks of apoptosis observed in animal cells, i.e an increase in the activity of specific nucleases was detected in senescing rice suspension cultures.