Study On Abscission Bark Formation Process And Mechanism Of Eucalyptus Grandis×E. Urophylla

The traditional abscission bark forming theory holds that:“due to the formation of newperiderm in the bark, the rhytidome that located outside of new periderm die because waterand nutrient supplying was cut off, finally, the rhytidome peeled off from the trunk with theinfluence of wind and rain”. From the theory, we could see that the new periderm wasimportant for the rhytidome to abscission. The forming of abscission zone and abscissionlayer was the core in studying plant tissue abscission. As for the forming of abscission bark,the periderm was just as abscission zone, and the few cork layers that located outermost ofnew periderm was just as abscission layer. Therefore, studying the forming process andmechanism of abscission bark was to study the abscission zone or periderm forming process.For enriche and complete the theoretical on abscission bark forming, reveal the formingprocess and mechanism of cork and cork cambium in periderm, improve performance forincrease utilization of abscission bark, the forming process and mechanism of abscission barkshould be studied.This study based on the basic theory of bark abscission and periderm formation in woodscience, guided by microstructure and ultrastructure changes of abscission zone. Each stage ofabscission bark forming was studied as follows: The microstructure and ultrastructure wasstudied for grasp the changing process of abscission zone cells; The PCD was studied forconfirm the nature of abscission zone cells death; Distribution and activity change of cell wallhydrolases was studied for understand cell wall loosen and separation mechanism ofabscission zone cells; The dynamic change of ROS level and antioxidant enzyme activity wasstudied for finding the change mechanism of abscission zone cells. The results showed that:（1） The living bark was consist of vascular cambium, secondary phloem and periderm，the sieve tube, companion cells, crystal-containing cells, phloem fibers, phloem rays, andphloem parenchymas arrayed in second phloem with some rule; The extractive content ofhot-water,1%NaOH and alcohol benzene in living bark was11.89%、18.89%、1.85%respectively, and the content of cellulose, hemicelluloses, lignin in living bark was45.90%,27.00%,11.49%respectively; It could be judged from FTIR spectra that the crystal incrystal-containing cells was calcium oxalate monohydrate.（2） The abscission zone forming process was dynamic. The forming process ofabscission bark was divided into six stages based on abscission zone cell structure andsequence, including before cell dedifferentiation stage, cell dedifferentiation stage, corkcambium forming stage, cork forming stage, radial expanding layer forming stage andabscission layer cells separating stage. The main cell features at each stage as follows:○1The parenchymal cells of abscission zone were not activity grounds for their big vacuole, little andmarginalized cytoplasm and small nucleus before cell dedifferentiating stage.○2Theparenchymal cells were dedifferentiating for regain fission ability at cell dedifferentiatingstage, with the feature of cytoplasm increase rapidly, small vacuole, big and centered nucleuswith obviously nucleoli and dense nuclear pore.○3The parenchymal cells were dividing inamitosis to forming cork cambium at cork cambium forming stage, with the feature ofabundant cytoplasm, big and obviously nucleoli. The forming process of new cell wall wasinvested at the same time.○4The cork cambium dividing cork cells outward and phelloderminward in motosis at cork forming stage. The PCD feature appeared in cork cells when theymature, at the same time, the corner cells were disappearing with obviously PCD feature atcork forming stage.○5There were several features at radial expanding layer forming stage,such as, the cork cambium expanding in radial direction and becoming radial expanded layer,its radial cell wall broken and recombined during expanding; The cork cells appearedobviously PCD features; The U shape thicken in cork cells that opening toward rhytidomewas forming.○6There were several features at abscission layer cells separating stage, suchas, the abscission layer cells began to separate from periderm; The cells of radial expandedlayer were filled by amorphous substance and cell wall was thickened by suberinlamella; Thesecond radial expanded layer was formed with suberificated cell wall; The U shape thickenwas strengthened with obviously laminated structure.（3） After the abscission zone cells feature was studied, the follows are discovered.○1The cork cambium in the abscission zone came from dedifferentiated parenchyma cells;○2There was no starch grains accumulation at cell dedifferentiating stage, which different fromtissue culture;○3The primary cell wall forming process was investigated at cork cambiumforming stage;○4There was a phenomenon of radial cell wall broken and restructure atradial expanding layer forming stage, which provided visual evidence for chemical bondfracture theory when cell wall loosening;○5There was no direct relationship among cellsuberization, cell death and bark abscission;○6There were obviously difference betweenbark abscission and other organ （flower, leaf, fruit） abscission in direction, structure andabscission layer location of abscission zone.（4） The ultrastructure features detected by TEM showed the death of abscission zonecells was active and programmed, but not passive. The results of TUNEL and DNA ladder testshowed that abscission zone cells had obviously PCD feature at cork forming stage, andcontinuously deepen with development of abscission zone cells. The results of TUNEL andDNA ladder test are accorded to PCD features that observed through ultrastructure. theseprovided enough evidence for abscission zone cells PCD. The ultrastructural feature showed that the abscission zone cells dying was a PCDprocess, this conclusion was further proved by DNA ladder and TUNEL test. The suberizationprocess only induced the PCD, however, the real reason of abscission zone cells abscissionwas cell wall degradation by pectinase.（5） Cellulase mainly located in primary wall. It was important for hydrolyzing celluloseof abscission zone cell wall at cell dedifferentiation stage and radial expanding layer formingstage, but unimportant for abscission layer cells separation. Pectinase mainly located inmiddle lamella and corners among the cells. It was important for hydrolyze middle lamella atcell dedifferentiation stage, radial expanding layer forming stage and abscission layer cellsseparating stage. Middle lamella was hydrolyzed by pectinase which was the primary cause ofabscission layer cells separation.（6） Through analysis the microstructure, ultrastructure, reactive oxygen species（ROS）level dynamic, and combined with modern theory, the results showed that the role of ROS inabscission bark formingwas important because of the following reasons:○1·OH couldincrease cells’ volume by loosen cell wall,, and accelerate the deep and speed of cork cellsdeath.○2O₂－could induce abscission zone cells death directly and convert O₂－to H₂O₂,for regulate abscission zone cells division and differentiation.○3H₂O₂promoted cellsdedifferentiation during abscission bark forming process, participates cell wall formation andinitiate PCD at cork forming stage and radial extending layer forming stage, which promotedethylene produce for accelerating abscission zone cells abscission at abscission layerseparating stage.（7） Through analysis the microstructure, ultrastructure, reactive oxygen species level andantioxidase activity dynamic, combined with modern theory, the results showed that the roleof antioxidant enzymes in abscission bark forming was important because of the followingreasons:○1POD could promote abscission zone cells dedifferentiate at cell dedifferentiationstage, and participate cell wall formation at cork forming stage and radial expanding layerforming stage, and promote respiration for accelerating abscission zone cells senescence atabscission layer separating stage.○2PAL participated cell wall formation of abscission zoneand polyphenol accumulation.○3SOD could regulate abscission cells division anddifferentiation by convert O₂－to H₂O₂.○4CAT could regulate ROS ratio by convert H₂O₂to O₂and H₂O, for participating abscission cells division and differentiation.（8） Based on the results in this thesis, the abscission bark forming theory of Eucalyptusgrandis×E. urophylla was porposed as followed. A new abscission zone （periderm） wasformed in Eucalyptus grandis×E. urophylla bark every year, after abscission zone growned,the PCD happened in cork cells, at the same time, the outer layer of cork cells was degraded by pectinase, lead to the cork cells separated from periderm, therefore, rhytidome lost touchwith stem, under the role of wind, rain and sun, rhytidome ruptured and fall off frm stem,become abscission bark. Cell wall hydrolases, ROS and antioxidant enzyme all play animportant role in abscission zone forming and abscission layer separating.（9） The abscission bark was consist of periderm and part of secondary phloem，there weresieve tube, companion cells, phloem fibers, phloem rays, and phloem parenchymas in the partof second phloem; The outside face smoother and the inside face appeared grain with age, thethickness and arrear also increased with age; The content of cellulose, hemicelluloses, ligninincreased with age, and the extractive content of hot-water,1%NaOH and alcohol benzenedecreased with age; The main characteristic absorption peaks were about cellulose，hemicelluloses, lignin and calcium oxalate monohydrate in FTIR spectra of abscission bark.