Characteristics And Mechanism Of Programmed Cell Death During The Formation Of Pith Cavity In Two Gramineous Plants

Pith cavity is the hollow part among plant internode,which is formed by the gradual disappearance of the central basic tissue cells or pith cells during the growth of the stem in some plants.The pith cavity is common in aquatic plant species and in terrestrial plant species,such as wheat,rice,bamboo and other plants.The formation of the pith cavity is caused by the death of pith cells,it acts as the main channel for gas exchange in aquatic plants,and the consumption of nutrients is reduced during plant growth in terrestrial plants after the degeneration of the pith tissue.In addition,the existence of hollow structures helps the stem become flexible,and ensures better to support for the above-ground parts of the plant.Programmed cell death(PCD)is an important cytological event in plant life activities.During the growth and development of plants,PCD occurs the whole life cycle,and plays an important role in plant morphogenesis,growth and development,sexual reproduction,and environmental stress.To explore the PCD process of the occurrence of pith cavity,young stems at different developmental stages were studied in Oryza sativa L.and Triticum aestivum L.in this study,and the solid stage(Stage 1,S1),cavity occurrence stage(Stage 2,S2),cavity expansion stage(Stage 3,S3)and the maturity stage of pith cavity(Stage 4,S4)were selected,the cytology structure changes of nucleus,vacuole,mitochondria,cell membrane and the cell wall were observed in the process of cell death by light microscope,fluorescence microscope and electron microscope;the death characteristics of the pith cells were analysed.The results showed that the two plants had similar characteristics in pith cell death.At the early stage of stem development,pith cells are small in volume,compact in arrangement,no intercellular space,the nuclei are large and locate in the center of the cell,the cytoplasm is dense.Mitochondria and nucleus structure were complete and the membrane envelope were clear.There are almost no vacuoles in the cell;the cell membrane and cell wall are well organized.With the development of the stem,pith cells increased in size,and many small vacuoles appeared in the cells,and then fuseed with each other to form a large central vacuole.At the same time,most vacuolar membranes of the central vacuoles were invaginated the cytoplasmic matrix and some mitochondria and other organelles into vacuoles,and these bodies wrapped by the vacuolar membrane were degraded under the action of vacuolar hydrolase.At this stage,nuclear pyknosis,chromatin agglutination and nuclear membrane envelope rupture were occurred.The organelles were shifted to the edge of the cell.The vacuole was also begun to degrade,mitochondria were swelled and the envelope membrane was ruptured further.The cell membrane was separated from the cell wall,and the cell membrane wrinkled into the cytoplasm in many places.At the same time,a large number of vesicular structures were observed.The cell wall finally degraded,which was the final event of pith cell death.The results of DAPI staining and TUNEL detection showed that the pith cell nucleus was round and regular in the early stage of stem development.DAPI staining was uniform and TUNEL detection was negative.With the development of the stem,cavities appeared in the pith tissue and further expanded,DAPI staining showed nuclear structure malformation and uneven staining,and TUNEL detection was positive.These results suggested that pith cell death is a typical PCD process during the form of pith cavity.In order to further explore the specific mechanism of PCD in pith cells,transcriptome sequencing was performed on the pith tissues of young stems at different developmental stages to trace the genes related to PCD in Triticum aestivum L.The hierarchical clustering was performed on the relationships between the samples at four developmental stages and the expression of related genes,and the clustering results were presented by using heat map.The results showed that the expression levels of most PCD-related genes were up-regulated during cavity occurrence and expansion,and some genes began to be up-regulated during solid phase.However,during the maturity of pith cavity,almost all of these related genes were down-regulated,indicating that PCD events did not occur during pith cavity maturity.These PCD-related genes were classified by function,and their expression levels were detected and verified by q RT-PCR.The results showed that the results of q RT-PCR were consistent with the results of transcriptome sequencing.Through the transcriptome sequencing,we found the expression level of ACC synthase gene(ACS)was rised significantly in the solid phase,which is related to the fact that ACS is a key enzyme in the upstream pathway of ethylene synthesis,and the expression level of the ACC oxidase gene(ACO),the key rate-limiting enzyme in ethylene synthesis,was relatively backward in time and space,and reached the maximum expression level in cavity emergence stage.Western blotting was used to locate ACO,and it was found that the enzyme had the highest content in the cavity occurrence stage.This result was consistent with the results of transcriptome sequencing and q RT-PCR verification,which further indicated that ethylene was involved in the PCD process as a signal molecule.The relative expression level of calmodulin gene(Ca M)and calreticulin gene(CRT)was high during the solid stage and cavitary emergence stage,indicating that calcium ions as signaling molecules were involved in the PCD process.The The relative expression level of ACS,ACO,Ca M and CRT genes were high at solid stage and cavitary emergence stage,indicating that calcium ions and ethylene as upstream signaling molecules were involved in the process of PCD in pith cells during the development of stem in Triticum aestivum L..Among the ROS-related genes,the relative expression levels of CAT,POD and GST were high during cavity occurrence and expansion stage,indicating that a large amount of ROS was produced in the cells during the PCD process of pith cavity formation.The relative expression level of CELLULASE gene was the highest during the period of cavity expansion,which was consistent with the final degradation event of cell wall in the PCD of pith cells.In addition,Apoptosis-Inducing Factor(AIF),Cytochrome c Oxidase Subunit 6B(COX6B)and Alternative Oxidase 1a(AOX1a),the endonuclease genes BIFUNCTIONAL NUCLEASE 1(BFN1)and RIBONUCLEASE 3(RNS3)were involved in PCD of pith cells.The expression of PBA1 and Apoptotic protease activating factor 1(Apaf-1)during the solid phase may be related to the fate of pith cells which determined to develop PCD in the early development.Nuclear DNA of the pith tissue at different stages of development was observed by DNA gel electrophoresis.It was found that the DNA bands were significantly tailed during the cavity emergence and expansion stages in two plants,which indicated that DNA degradation occurred during these two stages,which indicates that the death of pith cells is a typical PCD process.The results of Caspase 3-like immunohistochemistry indicated that the Caspase 3-like was located in the nucleus at the early stage of development,which may be closely related to chromatin concentration and DNA fragmentation.Western blotting showed that the content of Caspase 3-like was high during the cavity emergence and expansion stages in pith cells,indicating that Caspase 3-like played an important role in the PCD process of pith cavity formation.The mitochondria were stained by rhodamine 123(Rh123)at different developmental stages,and it was found that the mitochondrial fluorescence intensity was decreased in the cavity occurrence stage and expansion stage,indicating that the mitochondrial membrane structure was destroyed in these two stages,which was consistent with the results of the volume swelling and membrane structure destruction of the mitochondria in the cavity occurrence stage.The localization of mitochondrial Cytochrome c(Cyto c)was studied by immunohistochemistry,western blotting,immunofluorescence and immunocolloidal gold electron microscopy.The immunohistochemistry result showed that Cyto c was positive in the cytoplasm during the cavity emergence and enlargement stages.Mitochondria solution and cytoplasmic matrix were studied by western blotting,the result showed that Cyto c content from mitochondria solution was less during the cavity occurrence stage and expansion stage.Howere,cytoplasmic matrix contained a lot of Cyto c in the two periods,which showed Cyto c have been released from mitochondria to cytoplasmic matrix in these two periods.Immune colloidal gold electron microscopy was used to further locate the spatial displacement of Cyto c,and it was found that Cyto c golden particles mainly existed in mitochondria at the early stage of stem development,and were released from mitochondria to cytoplasm with stem development.The results obtained from the above different methods all suggested that Cyto c was released from mitochondria into cytoplasmic matrix with the rupture of mitochondrial membrane envelope during the formation of pith cavity.This study indicated that mitochondria and Cyto c played an important role in the PCD process of pith cells in Triticum aestivum L..