Preliminary Study On The Growth And Development Of Arabidopsis Protoplasts Under Heat Stress

It was important for agricultural production to understand correctly the mechanism that plant responded and adapted to change in environment. The heat stress was one of the serious climatic for plant growth and development. Hence, it was necessary to study the plant response and the way of signal transduction under heat stress. Numerous studies had demonstrated that heat stress could lead to fluctuations in the calcium signaling and cytoskeleton depolymerization in plant cell. What exactly is the influence of the change on plant cell growth and development? The study mainly concerned on Arabidopsis protoplasts growth response under heat stress at the cell level, included Arabidopsis protoplasts morphology change, cell viability, cell wall building and cell division after 7 day's culture. According to the results, enrich the evaluation of heat tolerance in Plant protoplasts.At the same time, the study showed what role calcium signaling and cytoskeleton played in cell changes under heat stress, and the mechanism of Arabidopsis protoplasts response and the way of signal transduction was discussed . The research content and the result were as follows:â‘ The optimum treatment of Arabidopsis Protoplasts isolation and culture: leaves of 27 day-old plants were taken for protoplast isolation, which was subjected to low temperature pretreatment at 4℃for 24h, then incubated at 28℃for 14 hours in enzyme solution, finally centrifuged at 600r/min for 10min three times. Freshly isolated mesophyll protoplasts were suspended in 0.5 M mannitol at a density of 4-6 x 105/mL, and embedded in B5 medium containing B5 salts and vitamins, 1% sucrose, 1% agar, 1.0mg/mL 2,4-D, 0.5mg/mL BA. The initial protoplasts'density was of 2×10⁵ protoplasts/mL. B The isolation procedure described resulted in 2.91×10⁶ protoplasts/g and of the protoplasts 84.87% were viable (Fig, 2.1-2.3) and after 7 days'culture of the protoplasts 51.48% were viable and 11.48% divided. (Fig,2.5,2.6)â‘¡Exogenous Ca²⁺ pretreatment played an active role in Arabidopsis protoplasts growth and development. Plasmamembrame-channel blocker La3+, intracellular channel blocker ruthenium red (RR), verapamil and Ca²⁺ chelator EGTA all inhibited Arabidopsis Protoplasts growth and development. The treatment and results were as follows: Arabidopsis protoplasts were heated at 52℃for 10min, exogenous Ca²⁺ pretreatment resulted in of the protoplasts 65.67% were viable and all calcium channel blockers pretreatment less than 15.00%(Fig,3.1,3.2). Heated at 37℃for 10min, embedded and cultured for 7 days, exogenous Ca²⁺ pretreatment resulted in of the protoplasts 68.92% were viable, 18.23% divided and of the cell wall 79.69% regenerated (fig,3.4-3.7). All calcium channel blockers pretreatment less than 10.00%, doing not divided and less 30%.â‘¢Arabidopsis protoplasts were incubated with oryzalin and Cytochalasin D or only oryzalin, which promote the growth of them after heat stress. The treatment and results were as follows: Arabidopsis protoplasts were heated at 52℃for 10min, the cytoskeleton disrupting drug, oryzalin and cytochalasin D or only oryzalin, lead to increase 5 times of the protoplasts vitality (Fig,4.1,4.2). Heated at 37℃for 10min, embedded and cultured for 7 days, increase 50% of the number of protoplasts which were viable, divided and of the cell wall regenerated respectively(Fig,4.3-4.5). The cytoskeleton disrupting drug, cytochalasin D, had no significant effects on morphology, viability, growth and development.â‘£Arabidopsis protoplasts were incubated with cytoskeleton disrupting drug and calcium channel blockers, which reduced the active role of cytoskeleton disrupting drug for protoplasts growth and development. That caused destruction of cell morphology, reduction of viability, inhibition of the cell wall regeneration and cell division. The treatment and results were as follows: Arabidopsis protoplasts were heated at 52℃for 10min , and La+Oy+CD led to of the protoplasts 16.67% were viable, and Oy+CD 68.27%(Fig,4.6). Heated at 37℃for 10min, embedded and cultured for 7 days,La+Oy+CD lead to of the protoplasts 18.32% were viable, 36.78% of cell wall regenerated and inhibited cell division. Oy+CD led to of the protoplasts 72.92% were viable ,78.97% of cell wall regenerated and 19.68% cell divided(fig,4.7,4.8)Based on the experimental results, fluctuations in the calcium signaling and cytoskeleton depolymerization in plant cell had a positive impact on Arabidopsis Protoplasts growth and development under heat stress. They might interact with each other and affect plant cell response to heat stress.