Dynamic Changes Of Cytoskeletons And Localization Of Acpase In The Sieve Elements And Tracheary Elements Of Developing Wheat (Triticum Aestivum L.) Caryopsis

Sieve elements(SEs)in caryopsis of wheat(Triticum aestivum L.)are responsible for the transport of nutrients.Tracheary elements(TEs)are responsible for the transport of water and mineral ion.Previous study have shown that SEs in the developing caryopsis of wheat undergo a special process of PCD.The mature SEs not only keeps the cell activity,but also transporting nutrients and storing in the endosperm of wheat caryopsis.In this study,The multiple biological technology such as transmission electron microscopy,ultracytochemical technology,indirect immunofluorescence labeling were used to observe the dynamical changes of cytoskeleton and ACPase activity during the development of SEs and TEs in the caryopsis of wheat,and understand their relationship between those changes and PCD.The main results were as follows:1.The microstructure and ultrastructure result displayed that sieve elements distributed as semi-circular at the outside of tracheary elements,and the organelles were wrapped by the autophagosome for degradation in the developing sieve elements.The mature sieve element retains some organelle fragment and is alive.While in tracheary elements,once the tonoplast rupture,the cytoplasm were degraded by the phagophore in the cell and the cytoplasm was completely degraded.Finally,fully degraded tracheary elements are obviously dead.At 6 and 7 DAF(DAF,day after flowering),the cell wall dissolved more than one place,and forming the hole in the end wall.2.The SEs and TEs at different stages of development were studied by acridine orange / propidium iodide fluorescence staining.The result shows that the cell wall of SEs and TEs was dyed green,and the nucleus was dyed red.The nuclei of most SEs degraded mainly in 3 to 5 DAF.While during the development of TEs,the degradation of nuclei mainly at 2 DAF,and then the nuclei has been fully degraded at3 DAF.3.Immunofluorescence labeling of microtubule combined with the use of confocal microscope show that the microtubules of SEs distributed in the middle side of two cells or around the cell wall at 2 DAF,while the microtubules of TEs distributed in the cell or close to the cell wall.The microtubules of SEs and TEs increased with the cell wall thickening.The microtubules of SEs around the cell wall decreased at 5 DAF.While in TEs,the transverse microtubules appeared at 4 DAF,and microtubules increased at 6 to 7 DAF.Immunofluorescence labeling of microfilament combined with the use of confocal microscope show that the network-like arrangement of microfilament and short actin aggregates of SEs distributed in the cells or around the cell wall at 2 DAF,while the network-like arrangement of microfilament appeared during the development of TEs.The microfilament of SEs and TEs increased with the cell wall thickening.The microfilament of SEs around the cell wall decreased from 5 DAF and eventually degraded.At 4 DAF transverse arrangement of microfilaments appeared during the development of TEs.The transverse microfilament of TEs decreased at 6 to 7 DAF.4.Localization of ACPase showed that ACPase activity can be found in the mitochondrion vacuolated by endoautophagy,organelles such as endoplasmic reticulum after the tonoplast rupture.In the early stages of the development of sieve element,ACPase activity was detected only in vacuole,where there were a few cytoplasms.With the development of the sieve element,ACPase activity was detected not only in mitochondria,but also in the plasmodesmata of the mature SEs.In conclusion,the development of SEs and TEs in wheat appeared the typical characteristics of PCD,such as the nucleus degradation,inclusions missing and so on.They had different way to degrade cytoplasm,and it is a process of microautophagy in sieve elements.While in TEs,the cytoplasm were degraded by the phagophore in the cell,and the inclusions of TEs degraded quickly than in SEs.From the point of morphological changes,the cell wall dissolved more than one place,and forming the hole in the end wall during the development of TEs.What's more,the mature SEskeep the cell activity,while the TEs were dead cells.The dynamic changes of microtubules and microfilaments were related to programmed cell semi-death of SEs and programmed cell death of TEs in the developing caryopsis of wheat.The distribution of cytoskeleton in SEs and TEs regulated the formation of cell wall.ACPase activity was related to the degradation of cytoplasm.The acid phosphatase acitivity of autophagy provides the further evidence for the degradation of cytoplasm.At the same time,ACPase activity distributed on plasmodesmata may also be involved in the transport of substances and the transmission of information between cells.