Research On The Aluminum Stress And Regulation Effect About Onion Based On Patch Clamp Technique

Nowaays,aluminum toxicity in plants brought by acid soil has been a main issue all over the world.Most plants in acid soil were aluminum toxicosis.And It indirectly effect the absorption of water and nutrition.So it has been thought that aluminum toxicity is a main factor that limits the agricultural productivity of the world's acid soil area. K⁺ is the most abundant and necessary univalent cation in the plant cells. Al toxicity inhibit root K⁺ absorption and reduce the root K⁺ quantity. Al³⁺,cation channel blocker,can block K⁺ and Ca²⁺ channel of wheat root cells.If blocking is too much time,it will appear the symptom of K⁺ absence.The effects of Al³⁺ on inward K⁺ channels at plasma membrane in epidermal cell protoplasts within the Allium cepa L. were studied by using the whole-cell patch-clamp recording mode.According to the difference of stimulation rate, we established Al -Ca Concentration model . It provides a hardware for the proper concentration of calcium including its spray.The results show as below:(1) Objective to optimize the isolation condition of epidermal cell protoplasts within the Allium cepa L. The protoplasts were released by digesting cell walls with cellulase and pectolase, and were purificated from the crude extracts by centrifugation and suspending in sucrose solution. The isolation efficiency of the protoplasts (in different condition) was decided by counting their number with globulimete. The results showed that enzyme,osmotic pressure stabilizer(D-Mannitol),pH,enzymolysis time significantly affected isolation of protoplasts from Epidermal Cell. According to orthogonal experiment design of enzyme,osmotic pressure stabilizer,pH,enzymolysis time ,we got the optimal isolation condition by the number of protoplasts.Results The optimal isolation condition is showed in results: enzymolysis time 2 h,pH5.5, D-Mannitol0.6 mol/L, 1.5% cellulose and 0.1% pectolyase and 0.1%BSA,temperature 28℃.(2) Inward currents in epidermal cell protoplasts within the Allium cepa L. were investigated using the whole-cell patch-clamp technique. The properties of these currents were similar to those seen in inward rectifying K⁺ channels in other plant tissues. And we found that low concentrations of AlC1₃ increased inward K⁺ currents. High AlC1₃ concentrations decreased inward K⁺ currents relative to the low concentration when voltages were most negative. (3)We found that inward K⁺ channels currents were both depressed by extracellular and intracellular AlCl₃ in a concentration-dependent manner.The results show that AlCl3 inhibited the inward K⁺ channels of epidermal cell protoplasts within the Allium cepa L. The dose response was fitted by the Michalis-Menten relationship characterized by an inhibitory constant Ki of 59.65μmol/L(outside membrane) and 1.85×10^-8mol/L (inside membrane) respectively, suggesting that intra-cellular Al³⁺ had a stronger inhibitory effect on outward K⁺ currents than extra-cellular Al³⁺ did. We conclude that Al³⁺ inhibits K⁺ uptake by internally blocking inward K⁺ channels.(4) Ion currents are record and measured with all kinds of Al³⁺ and Ca²⁺ concentration combination at certain voltage.Also the stimulation rate that Ca²⁺ impacts the Al³⁺currents is calculated, and as a bridge (stimulation rate is above 300 percent), Al-Ca Concentration model is established:a good method that guides us to find the proper Ca²⁺ concentration which can effectively relieve the ion stress is given ,including a curve relation between the Al³⁺concentration and relative [Ca²⁺]_max,[Ca²⁺]_min and [Ca²⁺]optimal,also with their curves fitting equations.The establishment of Al -Ca concentration model strongly provides us many theoretical basis and data reference for the fact that imposing certain concentration calcium improver can effectively relieve the Al stress Al,also it is of great theoretical value for study the anti- Al stress mechanism of crops at the level of ion channels.