Studies On The TCTP And NFYA Cloning And Their Expression Mechanisms Under ABA Regulation During Somatic Embryogenesis In Larix Spp

Japanese larch (Larix leptolepis) is an important conifer species, which has significanteconomic and ecological value. Somatic embryogenesis provides a useful experimental systemto investigate the regulatory mechanisms of plant development, which can be applied toimprove genetic traits and large-scale breeding. However, a number of challenges are presentin conifer somatic embryogenesis, such as difficulties with embryonal-suspensor mass (ESM)induction, a high frequency of abnormal embryos developing during the maturation stage, anddifficulties with in vitro synchronization. These obstacles create a bottleneck in the productionof somatic embryos. Therefore, understanding the molecular mechanisms underlying thedifferent developmental stages of somatic embryogenesis is important to overcome theseproblems. Several studies have suggested that abscisic acid (ABA)plays an importantregulatory role in plant embryogenesis. In somatic embryogenesis in conifers, exogenouslyapplied ABA not only can block the proliferation of ESMs and induce further development ofearly proembryos, it can also stimulate the deposition of storage compounds within the somaticembryo, induce and maintain embryo dormancy. Based on the somatic embryogenesis of larch,we had studied the effects of exogenously applied ABA, established somatic embryogenesismodel of mock exogenous ABA presence or absence and analyzed the sequence characteristicsand expression patterns of TCTP and four NFYA members. Furthermore, RNA-seq was appliedto the analysis of expression profiles between morphological changes’ materials, together withthe analysis of physiological and biochemical, we initially explored the ABA regulatorymechanism during somatic embryogenesis in larch. The following results were obtained:(1) To study the effects of ABA during somatic embryogenesis, ABA was removed fromthe differentiation and maturation medium. ESMs were cultured on subculture medium for14days, transferred to differentiation and maturation medium either with ABA (normal somaticembryogenesis) or without ABA, established somatic embryogenesis model of mockexogenous ABA presence or absence. As a results, ABA can inhibit cell division and induce the early embryo formation, and promote further development and maturation of embryo. WhenABA was removed from the medium, H2O2levels will increased, ESMs underwent browningon the8day and gradually died. Eventually, only a few partially malformed embryosdeveloped at42days and did not have the ability to germinate.(2) cDNA and genomic sequences of a TCTP gene were cloned from L. leptolepis,designated LaTCTP, which contained two signature sequences TCTP1and TCTP2. Theexpression profile of LaTCTP during somatic embryogenesis was determined by qRT-PCR. Asa results, the expression of LaTCTP decreased gradually during the first5days after transfer ofESMs to differentiation and maturation medium, which may be caused by the absence of auxin.At this point, H2O2level reaches the maximum, which can induce the occurrence of PCD topromote early embryos formation; In somatic embryos rapid development stage, LaTCTPexpression remained high expression levels, which have an important role to promote thefurther development of somatic embryos; while upregulation of LaTCTP in browning,whichhas higher H2O2levels, may be associated with stress-induced. In summary, LaTCTP probablyparticipates in the proliferation of ESMs, formation and further development of early embryos.(3) By comparing the amplification of LaTCTP’s5’flanking sequence using TAIL-PCRand FPNI-PCR, the PCR conditions and parameters were optimization. On the basis of this,new method has created by reducing the length of the linker sequenceand integrated into theprinciple of suppression PCR, designatedsuppression and nested mediated PCR (SNM-PCR);This method increases the binding efficiency of random primers with genomic DNA andamplification specificity, which improved the amplification efficiency of LaTCTP5’flankingsequences and successfully obtained LaNFYAs5’flanking sequences. SNM-PCR has laid thefoundationfor fast and effectively cloning promoter sequences and further analysis of genefunction.(4) Four NFYA homologs were isolated and characterized from L. leptolepis, designatedLaNFYA1, LaNFYA2, LaNFYA3and LaNFYA4. Multiple-sequence alignments showed that thefour putative proteins harbored two functional domains: a subunit association domain (SAD)and a DNA-binding homeo domain (HD). Further analysis showed that miR169target sequences were present in the mRNAsequences of all four genes, which suggested thatLaNFYAs may be regulated by miR169. Promoter analyses of the LaNFYAs indicated that theymay be stress-and hormone-responsive genes. The expression profile of LaNFYAs duringsomatic embryogenesis was determined by qRT-PCR. The level of LaNFYAs transcriptsincreased gradually during the first7days and reached their highest levels at7days.However,the removal of ABA resulted in no significant changes in the levels of LaNFYA1,LaNFYA2and LaNFYA3transcripts in the first14days, which was suggested that theexpressions of LaNFYA1, LaNFYA2and LaNFYA3were induced by ABA. In addition, it wasfound that the mRNA transcript levels of LaNFYA4increased significantly and had almost thesame mRNA accumulation pattern as in normal embryos during the first7days of culturing,and the mRNA transcript abundance of LaNFYA4increased continuously between7and14days on No-ABA treatment, which suggested that LaNFYA4may notonly be closely associatedwith the ABA response, but also involved in the stress response.(5)The RNA-seq was applied to the analysis of expression profiles betweenmorphological changes’ materials. As a results, When ESMstransferred todifferentiationandmaturation medium, the removal of auxin andcytokininresulted in376genesassociated withcell growth downregulated; In theinductionofABAandPEG4000, yellow embryo “head”appeared at5to7days,there are302genesupregulated to promoting the formationofearlyembryos; while the removal of ABA, PEG4000makeESMscells in astate ofosmotic stress,embryoniccellsgradually underwentbrowning at7to10days, in the meantimethereare333stress-related genesupregulatedin order to resistormitigate thedamage caused bystress.In conclusion, thisstudy showedthat during the first two daysafterESMstransferredtodifferentiation andmaturation medium(ABA and No-ABA), due to the absenceofauxinandcytokinin, the conditions of maintainvigorouscelldivision does not exist, whichinducea number ofgenes associatedwithcell growthdownregulated, leading to the rate ofcellgrowth anddivision decreased;At this stage, the ESMstreated by ABA and No-ABA, havesimilarphysiological status and substantially identicaldown-regulated genes, which indicatingthat the role ofABA is not obvious.while in the caseofonlyPEG4000, ESMs was in astate ofosmotic stress for long time, thecellsstarteda lot ofstress-relatedgenes to resistormitigatethedamage caused bystress. However, it still can notstop thecellsunderwentbrowningand diegradually; In theinductionofABAandPEG4000, ABA-responsive genes (LaNFYAs) wereupregulated after twodays, which might promotethe synthesis ofendogenousauxin;Auxin-responsive genes (LaTCTP) were induced after fivedays to promoteformationandfurtherdevelopment ofearly embryo. This study will benefit future studies on ABAregulatory mechanisms during somatic embryogenesis in Larix spp.