| Programmed cell death (PCD) is the vast majority of cells in vivo in a certain developmental stage of gene regulation, independent, orderly process of dying, it is for organizational evolution, organ development and maintenance of the body’s own stability plays an important role. Laser tweezers Raman spectroscopy (LTRS) is a biological photon technology that combines laser tweezers with Raman spectroscopy technology, which applies to research of the suspension cells, organelles, micromolecular particles without destructive or labeling. Yeast is a kind of low unicellular eukaryotes and a simple biological model species. In yeast fermentation process, the byproducts, such as acetic acid, ethanol, formate, glycerin, are toxic to the physiological activity of yeast and prohibitive to the yeast fermentationlf the metabolites of the yeasts further accumulate, massive yeast cells would be death. But so far, it is still not entirely clear how the yeast metabolites induce the cell death process. Preliminary research suggested that the release of mitochondrion inclusions induced by metabolites could start cell irreversible apoptosis.Alstress is a major factor limiting crop growth and yield of plants in acid soils. Gradually, mitochondria and Cyt c play important roles in Caspase activation and cell death after biological cells stressed by aluminum. But intriguing questions, such as if cell apoptosis can be induced by Al, how Aluminum induces the cell apoptosis, how the signal transduction in vivo after cells apoptosis, are not very clear.In this paper, the LTRS was used to real-time monitor the dynamic process of yeast apoptosis and the extracellular mitochondria of yeast and soybean physiological and biochemical changes under Al stress, so as to deepen the understanding of mitochondrial and Cyt c in its regulatory role in apoptosis. The main results are as follows:(1) We used the laser Raman spectroscopy (LTRS) to monitor the process of a single yeast cell’s apoptosis while induced by Al in real-time, and analysed the spectral information of biological macromolecules combined with repeated measurement variance method. The results showed that under the treatment of 200 mmol/L Al, the intensity of raman peak(721,1083,1301 cm’1), (721,872,1445,1604,1657 cm-1), (1301,1445,1746 cm-1) that attribute to nucleic acid, protein and lipid respectively had a decreasing tendency significantly with the extension of treatment time, indicated that the content of nucleic acid, protein and lipid were reduced during the apoptosis process of yeast cells. According to the changed of the intensity of the raman peak of 1604 cm-1, we could acquire the information that the energy metabolism of cells was blocked due to the damage of mitochondrial function. In the process of apoptosis of yeast cells induced by Al. LTRS could record the process of the changing of the physiological based at a single cell level in real time, and we could acquire the real-time information of the changing of intracellular biological macromolecular in the apoptosis process of a cell, in which all information can reflect the physiological and biochemical process of the apoptosis process truly. LTRS can find a sensitive information of the spectral change of the biological macromolecule in a single cell but the information always is concealed while monitoring group cells and LTRS is a effective tool to monitor the process of apoptosis at the level of a single cell in real-time.(2)The mitochondria were isolated after the yeast cells were stressed by Al, and LTRS was used to real-time monitor the mitochondria and recorded the Raman spectroscopy. Combined with the conventional method to measure mitochondrial physiological and biochemical changes, the change of mitochondria biological macromolecules was tested and valided. The results showed that Raman peaks (1064.1130.1301.1445.1657 and 1746 cm-1) associated with mitochondrial lipid were significantly decreased as a function of the increase in the concentration of Al and treatment time, accordingly, yeast mitochondrial malonylaldehyde (MDA) content measured by a conventional method also was observed to increase with the increase in Al concentration and treatment time, indicating that the unsaturated fatty acids was continuous to be degraded in oxidbillity, and leading to a decrease in mitochondrial lipid content. The intensity of characteristic peak (750 and 1130 cm-1) related with Cyt c in the mitochondria significantly decreased with the increase of Al concentration and treatment time, and the reduced content of Cyt c was similar with the the content of mitochondrial Cyt c/a and the Cyt c measured with conventional method, suggesting that the yeast mitochondrial membrane swelled by the stress of Al, leading to the membrane rupture and Cyt c release into the mitochondrial outer.1604 cm-1 is the characterized peak of mitochondrial respiration, with the increase of the Al concentration and treatment time increases, the respiratory peak was observed to decrease gradually, indicating that the mitochondrial activity become weaker, energy metabolism had been hampered. The mitochondrial membrane potential (Δψm) is consistent with the result measured by conventional method. The intensity of the nucleic acid peaks(1081and 1301 cm-1) and the protein peaks (750. 872,1004.1031.1445.1604 and 1657 cm-1) were significantly reduced with the increase of the acetic acid concentration and treatment time. These results illustrated that nucleic acid, protein and lipid content in mitochondria were a downward trend in the process of apoptosis. In cell level, the changes of cell nucleus and mitochondria under aluminum stress in yeast were observed by means of transmission electron microscopy and DAPI staining. The results showed that the nucleus in the cell edge aggregation, nuclear chromatin condensation showed the crescent-shaped distribution inside the nucleus and a dense stain. Mitochondrial shape was deformed and structure was destructed. In molecular level, Cyt c expression profiles with the treatment of aluminum concentration and the time were analyzed by designing C. humicolus Cyt c primers. RT-PCR analysis showed that the expression of Cyt c gene was up-regulated by Al stress, from the genetic level to to reflect the expression of yeast cell under aluminum stress in Cyt c. After treatment by the aluminum while its degradation yeast genome, elevated ROS and caspase-3-like activity of such phenomena further illustrate some typical characteristics change when aluminum induced apoptosis in yeast.(3) With the conventional method to measure mitochondrial physiological and biochemical changes after root tip cells of Tamba black soybean were stressed by aluminum. The results showed that the concentration of mitochondrial malondialdehyde (MDA) increased with the increase of the Al concentration and treatment time, and mitochondrial Cyt c/a ratio, the content of mitochondrial Cyt c and the mitochondrial membrane potential (Δψm) were on the decline after the Tamba black soybean root tip was induced by Al. Mitochondrial permeability transition pore (MPTP) of soybean root tip cells opened unceasingly, mitochondrial membrane potential (Δψm) decreased, mitochondrial membrane integrity is destroyed under Al stress promoting Cyt c off from the inner mitochondrial membrane. A reduced content of mitochondrial Cyt c/a and Cyt c under Al stress suggesting Cyt c may be released from mitochondria to the cytoplasm through the mitochondrial membrane. In cell level, the changes of cell nucleus in soybean root tip under aluminum stress were observed by DAPI staining. The results showed that the nucleus in the cell edge aggregation, nuclear chromatin condensation showed the crescent-shaped distribution inside the nucleus and a dense stain. In molecular level, Cyt c expression profiles with the treatment of aluminum concentration and the time were analyzed by designing Cyt c primers in soybean. RT-PCR analysis showed that the expression of Cyt c gene was up-regulated by Al stress, from the genetic level to to reflect the expression of soybean cell under Al stress in Cyt c. After treatment by the aluminum while its degradation yeast genome, elevated ROS and caspase-3-like activity of such phenomena further illustrate some typical characteristics change when aluminum induced apoptosis in soybean. |
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