Aerenchyma Formation And Their Relationship To Programmed Cell Death In Three Plants

Aerenchyma is composed of gas-filled cavities and their surrounding cells. Aerenchyma formation is an adaptive strategy for wetland plants in the long evolution process. The main function of aerenchyma is as a channel to transport oxygen from overground to inner roots. By now, aerenchyma was divided into lysigenous and schizogenous, but the knowledge is mainly reported at histological levels and lack of cytological essence from developmental points. The characteristics of programmed cell death (PCD) and regulatory mechanism during aerenchyma formation are still unknown from cyto-morphological and molecular biology points of view. The aims of this research are to study the aerenchyma formation and PCD process in three plants, the effects of ethylene signal to aerenchyma formation, using light microscope, laser scanning microscope, immunocytochemistry and molecular biological techniques. In the present work, the rule of initiation and development, aerenchyma type, cyto-morphological characters during the developmental process of aerenchyma formation are investigated. The aims are to receal regulatory mechanism of ethylene, ROS and caspase3-like protein during aerenchyma formation from the view of plant anatomy, cytology and molecular biology, to provide new evidence for discussing the signal control route in the development of aerenchyma formation, and also providing theoretical evidences for gene regulating.In this paper, we systematically studied aerenchyma morphologisis and their correlation to the programmed cell death in Trapa pseudoincisa, Nelumbo nucifera and Helianthus annuus, and some important results were listed as follows.The main reason of space enlargement of aerenchyma in T. pseudoincisa petiole was formed by cells separation, and accompany with cell shrink and degradation at the late of formating period. During expanding and mature stage, one reason of the enlargement of aerenchyma cavity was cell volume expanded, another reason was part of diaphragm cell died, which lead to aerenchyma cavities melt to be a large one. Furthermore, DNA fragmentation was obvious during aerenchyma formation detected by TUNEL assay. Tonoplast rupture, vesical appears, organelle degradation and nucleus degradation were investigated from ultrastructure. All these evidences comfirmed cell death happened during aerenchyma formation. Therefore, aerenchyma in T. pseudoincisa petiole is lysigenous.The aerenchyma formation in T. pseudoincisa petiole belongs to developmental regulated PCD. The precursor cells initiated PCD originating from meristematic cell. The nuclei degradation was the first hallmark of PCD. A weak of TUNEL-positive nuclei were first detected in solid phase. Deformed nuclei, condensed chromatin and the appeared vesicles were the detectable events. The rupture of tonoplast was preceded by condensation of chromatin. Then, the number of vesicles increased in the cytoplasm, and some of which contained organelle materials. Next, protoplast began to degrade by the role of hydrolase released from vacuole. Organelles degraded after the degradation of tonoplast, which belong to metaphase event. Plastid, which has intact morphology in the late phase of organelle degradation was completely degraded with interact with membrane vesicles. The degradation of plasma membrane was the last key event during aerenchyma formation process. The cell wall became loosen in the expanding phase, but did not degraded completely, and continue to maintain the structure of aerenchyma in petiole.The main reason of space enlargement of aerenchyma in N. nucifera root was formed by cells separate and the expandeding volume. During expanding and mature stage, one reason of the enlargement of aerenchyma cavity was cell volume expanded and cell morphology changed, another reason was part of diaphragm cell died, which lead to aerenchyma cavities melt to be a large one along the radial direction. Furthermore, DNA fragmentation was obvious during aerenchyma formation detected by TUNEL assay. Tonoplast rupture, vesical appears, organelle degradation and nucleus degradation, cell wall degeneration were investigated from ultrastructure. All these evidences comfirmed cell death happened during aerenchyma formation. Therefore, aerenchyma in N. nucifera petiole is lysigenous. The aerenchyma formation in N. nucifera petiole belongs to developmental regulated PCD. The tonoplast rupture was the first hallmark of PCD. A weak of TUNEL-positive nuclei were first detected in solid phase. Deformed nuclei and condensed chromatin were the early detectable events.the number of vesicles increased in the cytoplasm, and some of which contained organelle materials in the metaphase. Next, Organelles degraded after the degradation of tonoplast. The disturbed membrane system and reformed Organelles were observed in this phase. The degradation of plasma membrane preceded the degradation of cell wall.protoplast rapid to degrade after plasma membrane rupture. Cell wall degradation was the last key event during aerenchyma formation.Aerenchyma was induced by ethylene in the hypocotyl of H. annuus. To form the earliest space, the cells in special area of cortex first happened to plasmolysis and cell deformation. During expanding and mature stage, cell wall degradation was observed by light microscope. Aerenchyma formation was the result of cell lysis. Furthermore, analysis from the results of TUNEL assay (DNA fragmentation) and ultrastructure (Tonoplast rupture, vesical appears, organelle and nucleus degradation, cell wall degeneration). All these evidences comfirmed cell death happened all the time during aerenchyma formation. Therefore, aerenchyma formation in H. annuus hypocotyl is lysigenous.The aerenchyma formation in the hypocotyl of H. annuus belongs to environmentally induced PCD. Deformed cell wall, plasma membrane shrink and condensed chromatin were the early detectable events. Then, the number of vesicles increased in the cytoplasm, and some of which contained organelle materials. These evidences indicated that vesicles played an important role during aerenchyma formation. It resembled self-autophagy in animal PCD process. Organelles degraded after the degradation of tonoplast, which belong to metaphase events of PCD. Mitochondria and plastid remain integrity in the late other organelle degradation. The degradation of plasma membrane preceded the degradation of cell wall. Cell wall degradation, which became thinner and hyalinize was the last key event during aerenchyma formation. Aerenchyma was formed under waterlogging in T. pseudoincisa, N. nucifera and H. annuus. Two of the former species are aquaticc botany, in which aerenchyma formation was regulated in hypoxia environmental and genetic factor. When21%oxygenwas applied in the root of N. nucifera, the area of aerenchyma is smaller than the control from the root tip to root-hair zone. Therefore, aerenchyma formation is one mechanism of improve hypoxia stress, and aerenchyma formation was inhibited in some extent under sufficient oxygen.Ethylene induce aerenchyma formed early in N. nucifera root and H. annuus hypocotyl, and the area of aerenchyma induced by ethylene is larger than the control. The inhibitor of ethylene synthesis (1-MCP) could inhibite aerenchyma formation in some extent. So ethylene, as a signal molecules, direct involved in aerenchyma formation in N. nucifera root and H. annuus hypocotyl.ROS induced aerenchyma formation in1day in H. annuus hypocotyl. Meanwhile, The inhibitor of ROS (DPI) could inhibite aerenchyma formation in some extent. So ROS, as a signal molecules, direct involved in aerenchyma formation in H. annuus hypocotyl.The inhibitor of ROS (DPI) could inhibite waterligging-, ethylene-and ROS-induced aerenchyma formation. The inhibitor of ethylene (1-MCP) could inhibite waterligging-and ethylene-induced aerenchyma formation, but it could not inhibite ROS-induced aerenchyma formation. Therefore, ethylene is the upstream signal moleculars during PCD process of aerenchyma formation in H. annuus. The PCD signal transfer to downstream by ROS, and PCD take place lastly.The express level of caspase3-like was increased induced by ethylene. The activity of caspase3-like was consistent with ethylene and ROS signals or the inhibitor signals. Ethylene as the upstream signal of ROS. Caspase3-like, which activated by ethylene and ROS, regulat the initiation of PCD and aerenchyma formation.