Study On The Programmed Cell Death (PCD) Mechanism Of Endosperm Cells And The Effects Of Waterlogging On PCD In Endosperm Cells Of Triticum Aestivum L.

Previous studies suggested that the endosperm cell development is a special programmed cell death (PCD). Environment stress had an important impact on the PCD of endosperm. Waterlogging is an abiotic stress, which seriously affects the yield and quality of wheat. Little is known about the mechanism of PCD of wheat endosperm, and the development of amyloplast and programmed cell death in endosperm cells under waterlogging treatment. We studied these questions with the material of huamai 8 (chinese Triticum aestivum L). Four water treatments were established from anthesis to maturity, i.e.,3 days waterlogging treatment (3 DWT),7 DWT,12 DWT and moderate water supply (the control). Electron-transmission microscopy (TEM), DNA agarose gel electrophresis, TUNEL assay and RT-PCR detection were used to study the mechanism of endosperm PCD. Scanning electron microscopy (SEM) and LUGOL staining were used to observe the development of amyloplasts. TEM, Evans-Blue staining, Terminal deoxynucleotidyl transferase-mediate fluorescein deoxyuridine triphosphate nick-end labeling (TUNEL) assays and DNA agarose gel electrophresis were used to study the progression of PCD under waterlogging. The main results are follows:1. TEM studies showed that the nuclear envelope became degrade at 12 days post anthesis (DPA), and then chromatin condensed without marginalization. Nucleus could be split into several nuclear residues by the cleavage of nuclear envelope and it was hard to find the nucleus at 25 DPA. Mitochondrial cristae began to degrade and endoplasmic reticulum cyclizes at 16 DPA. These phenomena show that nucleus DNA has degraded at 25 DPA.2. TUNEL-positive staining was detected first in the endosperm cells at 12 DPA, and then the number of the positive nuclei increased further and reached to the maximum at 16 DPA.3. DNA agarose gel electrophresis showed that DNA began to degrade after 16 DPA, showing a typical laddering on gel at 30 DPA.4. RT-PCR showed that the mRNA expression level of AGPase changed clearly. It did not express at 3 DPA, and then began to increase at 9 DPA. It got to the maximum at 12 DPA, and then following decreased. It only had a low level at 21 DPA.These results showed that endosperm cells undergo PCD; the genes which are related to the synthesis of starch have been transcribed, so it can supply enough nutrition to the synthesis of starch at late stage of development.5. Waterlogging decreases the level of chlorolhyll and the grains volume, the long waterlogging, the variation is much clearer.6. With the development of endosperm cells, the number of starch granules increased largely, showing an S-type change. Starch granules number are about 20 in the early stage of development (8-10 DPA), but there was a distinct difference in late stage (20 DPA). The numbers of starch granules were 82.5% and 68% of the control respectively under 7 DWT,12 DWT.7. LUGOL staining and SEM showed that the number of starch granules were not reduced with a short-term waterlogging treatment (3 DWT), but a long period of waterlogging (7 DWT,12 DWT) obviously resulted in grains deformation, the decrease of the number of B-type starch granules, a low degree of enrichment.8. Evans-Blue staining analysis showed that waterlogging accelerates the progression of endosperm PCD; it has blue staining on the backside of endosperm. TUNEL-positive staining results suggested that waterlogging accelerated nuclei DNA fragmentation in endosperm cells. The PCD in endosperm cells was accompanied by the internucleosomal DNA fragmentation and it was earlier under the waterloging than the control. The appearance of DNA laddering was detected at 30 DPA with the control and a high level of DNA fragmentation was detected at 20 DPA under 12 DWT. TEM analysis showed the nuclear envelope degraded at 12 DPA in no-waterlogging treatment,10 DPA under 3 DWT,8 DPA under 7 DWT. Mitochondrial cristae swelled and degraded, endoplasmic reticulum degraded happened at 12 DPA in no-waterlogging treatment,10 DPA under 7 DWT otherwise. So, waterlogging accelerated the degradation of nucleus, mitochondrial and endoplasmic reticulum.These results suggested that the grain had a low degree of enrichment and the number of starch granules decreased under waterlogging. It showed the characters of PCD in the endosperm cells, such as the chromatin condensed, the nuclear envelope degraded, mitochondrial cristae swelled and degraded, endoplasmic reticulum degraded, and these phenomena were more extensive under the waterlogging treatment.