Physiological And Biochemical Studies On The Relationship Between Desiccation Injury And Mitochondrion Induced Programmed Cell Death During The Imbibition Of Mungbean Axes

Desiccation tolerance and desiccation sensitivity are tightly related to germination,dormancy,priming and storage age of seeds,thus,they are the important traits in seeds developments and germination.All the past researches on them are focus on the massive base and molecular mechanism on desiccation tolerance,and recognized that desiccation sensitivity is the back side of desiccation tolerance.Programmed cell death(PCD)is verified as one of the most important mechanism in responding to abiotic stress.In the response of hypoxia and drought stress,PCD is proved to play an essential role.According to these research,a hypothetic explanatory model on desiccation sensitivity with mitochondrial pathway PCD were raised.Moreover,demonstration from morphologcial changes,physiological variation,and comparitive transcriptome analysis were executed for test the hypothesis.And all results are list as follows:1.On the establishment of experimental model,water content and growth of mungbean seeds in different imbibition and dehydration were detected.Results showed that preimbibing for Oh,3h,6h,18h,24h can be used as the representative of different desiccation tolerance/sensitive status for their panel position in imbibing moisture variation.And 25h can served as the dehydation time point for its balance water content in all deiccation times.2.On physiological analysis of reactive oxygen species(ROS)and antioxdative system,the production of ROS,activities of antioxdative enzymes(such as,SOD,CAT,APX),and contents of antioxdative substrates(such as,ascorbrat and glutathione)were determined.Results demonstrated that desiccation sensitivity is obviously related to the increasing of ROS production and the decreasing of activities of antioxidative enzymes.And that H2O2 are tightly related to membrane lipids peroxidation,membrane leakage,thus,it should be act as the vital role in ROS attacking or signaling.3.Results of nonenzymatic antioxidative system detection has further defined that the content of basic reductive substrate,such as glutathione is on the wane state in the late preimbibtive desiccation.Based on this,it can be concluded that the role ROS under dehydration should not be oxidative attacking,but the signal tranductor of PCD.4.Results of vetrificative substances detemination indicated that desiccation tolerance is contributed to the variation of nonreductive sugar and proline contents.It also supported the besis of seed priming for their inductive chacteristics.5.Viability staining proved that the seed viability loss should be located in the cambium cells of embryo axes.And morphological researches suggested that cell death in cambium under late imbibitive dehydration can be recognized as PCD for their nuclear and cellular structure variation.And the biochemical analysis of DNA integrity,activities of caspase and endonuclease also proved the proecess of cell death in cambium of mungbean axes under desiccation is a classic PCD procedure.6.Mitochondrial membrane potential test with JC-1 by using FISH and flow cytometer certified that the organelle play an key role in PCD induction during the process of dehydration.Also,it is clear that mitochodial pathway PCD is the main form of cell death in response to dehydation.Otherwise,the results of cytoplasmic calcium timecourse prompted that Ca2+ play an important role in PCD induction.And,the timecourse also suggested that upstream of Ca2+ signaling should be water potential varication cause by desiccation and the downstream should be ROS and potential turnover(PT)channel on mitochondrion membrane.7.Mitochodial pathway PCD is the main form of cell death in response to desiccation and its main procedure should include 4 stage:(1)desiccation induction,(2)Ca2+ signaling and mitochondrial electron transportation breaking,(3)ROS production and mitochodrion membrane potential turnover,(4)cytochome C release and PCD downstream events execution.8.In the comparitive transcriptome analysis,92,155 unigenes were assembled.GO analysis on them suggest that they should be assigned to some groups as“extracellular compartment”and“transcriptional factors" etc.Also,GO analysis elucidate the funcational groups of“cell killing",“translational factors" and“growth”participate in the transformation period of desiccation loss.KEGG pathway investigation indicated that carbon metabolism should be the important contributor of desiccation tolerance,for instance,the pentose phosphate pathway is the essential part for this trait.And it suggest that pathways of phenylpropanoids biosynthesis,cystein and methionine synthesis,plant-pathogen response and phagocytosis related maybe also take part in the establishment of deisccation tolerance.9.In the digging of differential expression genes(DEGs),genes of LEAs,Sugar transportors(SUTs),antioxdative enzymes and heat shock proteins(HSPs)were selected.The expression of LEAs demonstrated that they are tightly relative to the timecourse of imbibition and dehydration,that is to say they are the core executors in the molecular mechanism of desiccation tolerance.The overexpression of SUTs in dehydration indicated that carbohydrate transportation should be an important compartment of the mechanism.The expression variation of Cu-Zn SOD and peroxidases' genes verified that the main purpose of antioxidative system were changed superoxide toH2O2.And expression changes of HSPs verified their dual functional role:act as regulatory factors for HSP20 is highly induced in early imbibition;and act as molecular chaperon for HSP70 is overexpression in late imbibition.10.Some differential expression of transcript factors were screen in DEGs.Bioinformatic analysis elucidate that some regulatory pathway linked these transcript factors to desiccation and downstream effectors.AP2,coulping with SUTs,impact desiccation tolerance by the variation of carbohydrate metabolism.ABI5?MYB?HSP20 etc.impact desiccation tolerance by regulating LEA and CP47 coulping with ABA.NAC,coulping with intracellular metabolism,influence desiccation tolerance by changing the cell wall struture.And,bZIP and MYB etc.influence desiccation tolerance by antioxidative protection coulping with secondary metabolism of polyphenol and oxidase.11.On the DEGs screening of metacaspase,endonuclease and cytochrome C related genes,results obviously advised the main factors of mitochondrial pathway PCD involve in the desiccation loss clearly.On the one hand,abundancy of metacaspase and nuclease increased in late imbibition.On the other hand,the expression model of cytochrome C related genes were changed from normal electron transportation to biosynthesis and transportation of itself.In other words,mitchondrail pathway PCD is the primary form of desiccation injury in imbibition axes.Based on all of these results,it can be concluded that PCD,especially mitochondrial pathway PCD should probably the main reason of seed viability loss after the loss of desiccation tolerance during imbibition.That is to say,in fact,desiccation sensitivity is the irreversible injury causing by mitochondrial pathway PCD that is induced by dehydration in the axes cambium tissue which lost or absolutely do not possess the massive basis(glasses)of desiccation tolerance.