Study On Programmed Cell Death Of Sieve Elements And Pericarp Cells In Developing Caryopsis Of Triticum Aestivum L.

Phloem sieve elements(SEs) in abdominal of wheat caryopsis have been considered the mayor conductive tissues for the transport of nutrients.During the development of SEs,cellular organelles selectively disappeared,such as nuclei,ribosomes,Golgi apparatus and vacuoles;while other organelles selectively survived,so the SEs retains a intact plasmalemma,mitochondria,and stacked endoplasmic reticulum,all of which occupy a parietal position within the living cell.It is probable that the development of SEs is attributable to the phenomenon of programmed cell death(PCD) according to previous studies,this postulate,however requires further evidence.It is undisputed that the vacuole disappears during maturation,but the precise time and function that vacuole rupture occurred is unclear.Also,it is unclear how cell organelle degradation ceased and whether nuclei containing fragmentary DNA.Pericarp cells in cereal crops are generally have a large vacuole with little cytoplasm in cell.Pericarp cells development accompanied with starch accumulation,and cell inclusions disaggregated gradually,it become empty cells at last.Not only for ovule and seed protection,pericarp cells may also act as a place for transit and temporary accumulate nutrients.No detailed report for PCD in pericarp cells were found,and the association between starch accumulation and PCD is also unclear.In this study,the classic PCD detection methods were used in the developing process of SEs and pericarp cells to solve the questions above.The main results are as follows.1.SEs at different stages of development were studied by TUNEL staining and acridine orange/ propidium iodide staining.The earliest stage at which any TUNEL-positive signals could be detected was at 3 DAF,with the number of such signals reached a maximum at 4 DAF,and the nuclei had an obscure appearance,then TUNEL-positive signals disappearred and SEs became mature cells at 6 DAF.The results above showed that the development of SEs may be PCD process.In this process,nuclear degradation and DNA fragmentation were observed.2.SEs at different stages of development were studied by transmission electron microscopy.Results showed that the development of SEs were typical PCD process,but mature SEs survived PCD and formed functioning cells for nutrient transport.It exhibited as:Nuclei degradation,chromatin condensation,the remaining condensed chromatin was localized to the periphery of the nucleus,and the nucleus was observed to maintain its original shape even after the nucleic acids had degraded.Cell vacuolation was observed,and after the nuclei disappearred,SEs were highly vacuolated.Vacuoles contained abundant electron-dense materials and could selectively removing certain organelles.Immediately prior to or at the onset of vacuole rupture,few electron-dense materials were detected,and the rupture of the tonoplast did not affect the cell contents that survived,indicating vacuole may empty the included hydrolytic enzymes prior to rupture or that the enzymes loose their activity.Mitochondria were found to be enveloped by a single cisternae of the ER and degraded in this way.The mature SE retains a intact plasmalemma,mitochondria, plastids,all of which occupy a parietal position within the cell.A large number of the plasma membranes formed cavities containing electron dense material through invagination of the plasmalemma toward the SE cavum were observed in mature SEs,some of them lost their integrity and released their electron dense materials,indicating nutrients transporting may be performed by apoplast pathway.3.Ultracytochemical localization of Ca²⁺-ATPase showed that,in the development of SEs,no reaction products of Ca²⁺-ATPase were seen in cell wall and tonoplast. Significant changes of reaction products of Ca²⁺-ATPase were detected in cell membrane, and the least reaction products were observed at 3 DAF,at this time,TUNEL-positive were first observed,indicating Ca²⁺-ATPase may be associated with DNA fragmentation.5.Pericarp cells at different stages of development were studied by TUNEL staining and acridine orange/ propidium iodide staining.TUNEL-positive nuclei were detected at 5 days before flowering;the numbers of such signals decreased gradually at 2-7 DAF. The nucleus gradually reduced at 4 DAF and disappearred at 15 DAF.Amyloplast significantly increased at 4-11 DAF,then decreased rapidly.The results above showed that nuclear degradation and DNA fragmentation were observed in developing pericarp. and it may be PCD process.6.Pericarp cells at different stages of development were studied by transmission electron microscopy.After 2 DAF,nuclei had significant features of degradation,such as chromatin condensation,nuclei condensation,nuclei deformation.Then chromatin disappeared gradually,following with complete degradation of nuclei at 15 DAF;in addition,mitochondria degradation,vacuolation and tonoplast disruption were detected, indicating that the development of pericarp cells were typical programmed cell death process,moreover,PCD started early and lasted for a long time,there is a delay in process of PCD.In the PCD process,starch granules synthesized and deposited temporarily in amyloplasts(before flowering) or chloroplasts(after flowering);the degradation of starch granules could provide sufficient energy for pericarp cells to survive,and,when the starch granules disappeared,pericarp cells died rapidly due to lack of energy,indicating that energy supply determined the fate of pericarp cells.Besides,normal mitochondria are necessary for pericarp cells to survive.Apart from the protective function of pericarp,it can function as starch synthesis, storage and degradation as well as nutrient transportation.