Studies On The Characters And Control Signals Of Programmed Cell Death In Mixed Bud Of Apical-bud-senescence Chestnut(Castanea Mollissima BI.)

The low yield, low utilization rate of land and light energy were the mostest restriction problem of China’s chestnut industry. The apical-bud-senescence chestnut is characterized by the apical mixed bud of fruit bearing shoot, which usually senescence and abscission after flowering and the basal latent bud will develop to new fruit bearing shoot in next year. This has the advantages of facilitating intensive cultivation and increasing production. However the mechanism of mixed bud senescence still remained unknown by now.In this study, the time course of cell death leading to mixed bud wilting and abscission on chestnuts were first observed, then hallmarks of programmed cell death (PCD) were studied in apical mixed bud undergoing senescence including DNA degradation, nuclear morphology, and cell death ratio and cell ultrastructure morphology, and then the contents of endogenous hormones, mineral elements contents in mixed bud undergoing PCD of chestnut were tested by HPLC, GC and FAAS in order to study the mode of action of these contral signal factors in the process of PCD and wether these control singals could induce the PCD in mixed bud of chestnut. The main results were as follows:1. The apical mixed bud in shoots be senescenced was the direct cause of character ’apical-bud-senescence’of chestnut. The stage that mixed buds undergoing senescence was subdivided into five distinguished stages by visible symptom.2. The results showed that DNA degradation occurred in apical mixed bud senescence prior to visible wilting. And nuclear stained with propidium iodide indicated that the fluorescence and diameter of nuclear significantly decreased in mixed bud undergoing senescence of apical-bud-senescence chestnut. Compared to the control chestnut buds, the cell apoptotic ratio, determined by flow cytometry, significantly increased in apical mixed buds undergoing senescence. Further cell ultrastructure detection proved that some characteristic of apoptosis including chromatin condensation, tonoplast invagination and DNA degradation occurred at an early stage of mixed bud senescence. This paper confirmed that PCD did occur in mixed bud of apical-bud-senescence chestnut, and what’s more the type of PCD is vacuolar cell death rather than necrosis.3. When in the progress of mixed buds undergoing PCD in apical-bud-senescence chestnut, contents of ZT and ZR, which was belonged to CTK, declined gradually with the DNA degradation more and more seriously during mixed bud senescence, and it indicated that ZT and ZR have a negative regulatory role in the PCD, the deletion of content of them may be the essential factors of PCD in bud, then spraying KT-30also proved that CTK has an inhibitory role in bud PCD. Content of IAA sharply rose suddenly5d before DNA degradation, and kept high content during the whole DNA degradation, then sharply decreased with the end of DNA degradation in bud. So IAA may be had the negative regulation role in PCD. Changes of ABA in mixed bud of apical-bud-senescence chestnut was similar to Eth, contents of the two hormones sharply rose suddenly5d before DNA degradation in mixed bud, and kept high content, which indicated that ABA and Eth were the induced factors of PCD in mixed bud, then the test of spraying ethylene and ABA indicated that ethylene could induce death of bud quickly, and ABA had the double role in bud cell death. Analysis of Eth/ABA indicated that Eth and ABA have a wave-type dynamic balance, with the sharply increasing of Eth/ABA, the balance was broken by Eth increased and induced physiological changes of PCD in chestnut bud, meantime the start time of PCD was the same to the time of Eth/ABA sharply increased, so Eth/ABA increased could be used as physiololgical markers of the begin of execution in PCD. In summary, in the process of PCD in mixed buds of apical-bud-senescence chestnut, changes of IAA, ABA, and Eth contents were earlier than DNA degradation, and Eth maybe was the main control signals in mixed buds undergoing PCD of apical-bud-senescence chestnut.4. When in the progress of mixed buds undergoing PCD in apical-bud-senescence chestnut, the PCD process accompanied by degradation of N, P contents, and the degradation time of N, P contents was the same to start time of PCD. With the beginning of the PCD in bud, the content of K presented the upward trend, content of Ca2+sharply increased when PCD started, and significantly higher the former content, then come to the maximum value in the time when DNA degraded most seriously. Content of Fe increased before DNA degradation, and then sharply increased with the beginning of PCD, come to the maximum value in the time when DNA degradation accompanied with appearance of DNA ladder was most seriously30d after bloom in apical mixed bud senescence. The study indicated that with the DNA degradation in the PCD, content changes of N、P、K、Ca and Fe were different with CK, which could indicated that they maybe involve in PCD, and deletion of content of N, P may be the essential factor of PCD, increasing of content of Ca maybe increase the activity of related endoenzyme, which promoted PCD process. Content of Fe sharply increased before DNA degradation, and kept a high content level during DNA degradation, which indicated that Fe may be the positive control signals of PCD.The datas obtained here may promote the PCD research on woody plants and lay the foundation for artificial regulation of mixed bud senescence so as to remodel the development pattern of fruit branches of chestnut.