Studies On The Biological Basis Of Cell Death Of Soybean Axes By Artificial Aging

Seed aging is accompanied with cell death. However, there were only very fewpublished papers concerning cell death taking place during seed aging. To get a betterunderstanding of the mechanism of cell death during seed aging, cell death events ofsoybean seeds "zhongdou27" being artificially aged at40°C was used as experimentalmaterials. Tetrazole and Trypan blue staining and NMT techniqes are used to assess theviability of cells from different parts of the axis, and ultimately make it clear that whichpart of the axis die first upon seed aging. Using a fluorescence dying kit, we can tellwhether the cells die by way of programmed cell death (PCD) or necrosis. The mainresults are as follows:1. Tetrazole staining indicated that artificially aging induces the cells die of thesoybean axes. Tetrazole staining and Trypan blue staining showed a decline in theproportion of viable axes with seed aging. The two staining methods confirmed that thecell death of each region of the soybean axes was not synchronized. Cells within theradicle (including the root cap, apical meristem) died first. The firt quarter from the roottip of the axes is regarded as the key zone for cell death during seed aging.2. TUNEL staining indicated that the aging induced cell programmed death (PCD).PCD mainly took place in the seed death key zone, with the radicle meristem as thetrigger piont, and then gradually extended to the germ end. The number of PCD cellsincreased with aging. The results of the TUNEL staining were not exactly the same withthat of viability staining, indicating that in addition to PCD, necrosis might occur in agedseeds too.3. NBT staining showed that in unaged seeds being imbibed for24h, mitochondriahas been activated and superoxide accumulated, the latter might be a normal byproductof seed germination or was required for normal germination. While no accumulation ofsuperoxide was detected in aged axes, especially not in the seed death key zone. DABstaining showed a similar pattern to viability staining, indicating that hydrogen peroxidemight take part in cell death by acting as a 'trigger' to initiate the aging-induced PCD.Using NMT technology, Ca²⁺influx was detected in24h-imbibed soybean axes. Ca²⁺ might be required for normal metabolic activities. The flux of Ca²⁺was significantlydifferent among various regions of the axe of unaged seed, with the maximum influx atthe seed death key zone, which might suggest that the metabolism of these cells wasvery active. There was no significant difference of Ca²⁺influx at the root tips betweenaged and unaged seeds. However, Ca²⁺influx at the seed death key zone of aged axesreduced significantly. Outflux of K⁺from the axes cells was detected using NMTtechnique. There was an obverious increase in K⁺outflux with aging. Therefore, duringseed aging, cells from the seed death key zone died earlier than other cells might berelated to the accumulation of hydrogen peroxide, changes in flux of Ca²⁺and K⁺.