Study On Molecular Mechanisms Of Resisting Senescence In Old Platvcladus Orientalis

Old trees can have extremely long life span with several thounsands of years, whosemechnisms about how to regulate or resist aging at physiological and molecular levels hasbecome one of the hot focuses in plant resisting senesences. Furthermore, reactive oxygenspecies (ROS) is considered to be one of the impoprtant factors affecting plant senescences.Subsequently, based on the analysis of the production and elimination of ROS between20-and3000-year-old Platycladus orientalis from Yellow Emperor mausoleum in Shanxi in2010, thesequencings of transcriptome and digital gene expression (DGE) were porformed by usingSolexa technique in these two-age stage of P. orientalis, the differentially expressed genesrelated to ages were mined; and then, the genes related to ROS homeostasis were focused tostudy their expression patterns under the ABA and NaCl stresses. The main conclusions are asfollows:(1) Analysis of ROS and its elimination system between20yr and3000yr P. orientalisshowed that the content of H₂O₂3000-year-old one was higher than20-year-old one in August,while lower in November; but GR and GSH were higher than20-years-old trees in August,wheras SOD and AsA had no significant changes in this season. Similarly,3000-year-old P.orientalis in November also had the higher ability to eliminate ROS. Additonally, thegerminated seeds of3000-year-old P. orientalis still had the higher ability of ROS eliminationcompared to that of20-year-old one. The results suggested that3000-years-old P. orientalis,had the wonderful systems of ROS elimination protecting the cells from being damaged, whichmay be one of the importantly physiological mechnisms of resisting aging in P. orientalis.(2) The investigation of the transcriptome of P. orientalis leaves using the Solexa/IlluminaRNA-seq technologies demonstrated that more than13million sequencing reads weregenerated,363,885contigs and68,219Scaffolds were yielded, respectively, by a combinationof reference-based assemblies, and finally51,664Unigenes were yielded after further assemblywith an average length of475bp. In addition,742SSRs were mined in703Unigenes with the occurrence frequency1.36%by high-through exploiting SSR sites in the transcriptome of P.orientalis leaves. These Unigenes are important genetic resources of P. orientalis in theregulation of resistance ageing.(3) Ten reference gene candidates from transcriptome data of P.orientalis, were selectedand examined by qRT-PCR using29different samples of P. orientalis collected from plants ofdifferent ages, different tissues, and the seedlings subjected to different treatments (cold, heat,salinity, polyethylene glycol, and abscisic acid), and the changes of expression patterns in these10genes were assessed by using three analytical software packages (geNorm, NormFinder, andBestkeeper). The results showed that ubiquitin-conjugating enzyme E2(UBC) andalpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stagesand under all stress conditions.(4) Based on the sequences of transcriptome, the DGE profiles between20-and3000-year-old P. orientalis leaves were established by using Solex technique. After strictlyscreening based on FDR≤0.001and|log2Ratio|≥1,1202genes expressed differentially wereobtained including578up-regulated genes and624down-regulated genes in the3000yr VS20yr P. orientalis. And then, the expression patterns of12genes were further selected to beevaluated by qRT-PCR, which had the similar trends with the DGE profiles. Thesedifferentially expressed genes were classified as signal transduction, hormone, transcriptionfactor, protein degradation detoxification, senescence related gene, stress response and defense,secondary metabolism, others metabolism, and unkown protein that vital for3000-year-old P.orientalis to fight with various stresses. Which maybe regulate physiological and biochemicalresponses to adapt adverse conditions under various stresses in3000-year-old P. orientalis.(5) Analysis of the expression patterns of ROS-homeostasis genes, NAC, NQO, POD,DXS, PAL, GAD, SSADH, GABA-T, TDB, and TRF1, etc., under ABA and NaCl stress showedthat the expressions of SSADH under NaCl and TRF1under ABA were firstly inhibited andthen had some gradual inreases with the extension of stress times, while the rest ones werefirstly promoted by exogenous ABA and NaCl, and then decreased or kept the higher levels, which had the positve correlations with the content of H₂O₂. The results showed these genesmaybe have important roles in regulating ROS homeostasis under ABA and NaCl stress, whichmight be beneficial to the elimination of ROS in the old P. orientalis.In total, based on the analysis of ROS eliminated system in different ages of P. orientalis,the transcriptome and DGE profiles in20-and3000-year-old P. orientalis leaves wereperformed. Large-scale expression analysis showed that many genes which were grouped intosignal transduction, transcription factor, reactive oxygen scavengers, stress response anddefense related gene, secondary metabolism, etc., had the significantly higher expression levelsin3000-year-old P. orientalis compared to20-year-old one, which might be benefit to resistaging of the3000-year-old of P. orientalis. Furthermore, the expression patterns of the genesrelated to ROS homeostasis were discussed under the similated treatments, which might be oneof important mechnisms of resisting aging in old P. orientalis. In a word, our study maked itwell suited for the dissection of ageing-resistance and it's a milestone to carry out the molecularstudy of ageing-resistance and discover useful genes in P. orientalis.