Mechanisms Of Bullet Formation And Development In Lycoris Based On Aerial Culture System

Lycoris is a genus of bulbous flowers belonging to the Amaryllidaceae that are mainly distributed in Eastern Asia.This genus includes approximately 20 native species and is of important ornamental,medicinal and ecological values.China is the main distribution of Lycoris,from which Zhejiang and Jiangsu province are the resources and diversified distribution centers,accounting for more than 80%of the national resources.Lycoris is rich in color and unique in flower patterns,and has become an excellent plant material for cut flower,potted flower and flowering ground cover.However,hindered by a long sexual reproduction cycle and low regeneration rate under natural conditions,natural regeneration method can not meet the increasing demand for Lycoris bulbs,and the heavy exploitation of natural habitats required to satisfy such demand has seriously diminished wild Lycoris resources in China.The establishment of an artificial breeding system is imminent.In this study,two widely used species,Lycoris sprengeri(Ls)and Lycoris aurea(La)were used as plant materials.Based on the most widely used breeding method for artificial breeding of bulbous flowers,chipping,the characterization of formation and development process of bulblet was carried out on the morphology,cytology,subcellular and physiological levels.The "reference transcriptome" of Lycoris and the complete transcriptome database of bulblet formation and development process were constructed by continuously sampling and combined sequencing on two platforms,Pacbio Iso-seq and Illumina RNA-seq.Based on the platform we established,the differentially expressed genes(DEGs)were screened systematically in both inter-species and intra-species comparison modes.The relationship between phytohormone ethylene biosynthesis and its signal transduction and the ability of bulblet regeneration was revealed.Based on these results we focused on the relationship among ethylene biosynthesis and signal transduction,plant defense responses and wounding-induced regeneration.This study is of great significance for further analysis of the basic molecular mechanism of bulblet formation and development,as well as construction of artificial breeding system and commercialization of bulbous flowers cultivation in China.The main research results are as follows:1.Determination of the bulblets formation and developmental stages of Lycoris under aerial culture conditionsIn order to explore the tissue origin and molecular mechanisms related to bulblet formation and development of Lycoris without adding exogenous PGRs,an aerial culture system was established.This study carried out a comparative study of two Lycoris species with different regeneration abilities on morphology,cytology,subcellular and physiological levels.The propagation process was divided into four main stages based on systematic observations:(1)Competence stage(0-3 d),(2)Axillary bud initiation and elongation(3-9 d),,(3)Bulblet formation(9-15 d)and(4)Bulblet development and expansion(15-45 d).Based on the statistical analysis of the number of regenerated bulblets,the two species were defined as High Regeneration System(Ls)and Low Regeneration System(La),Meanwhile,the formation method of bulblet and formation of tissue resource,and the key formation and development time points provided sufficient data for the selection of samples and tissues for the subsequent sequencing.2.Determination of the "axillary bud" origin of bulbletBased on the established aerial culture system,this study revealed the axillary bud-origin of bulblet in Lycoris.Based on the abaxial side formation characteristics of bulbets,this study also suggested the indispensable role of basal plate during bulblet formation in Lycoris.Different from the adventitious bud formation method of single scale culture in Lilium,the formation method of Lycoris during aerial culture belongs to the axillary bud formation via direct organogenesis.And it requires the tissue combination of both basal plate and scales.Meanwhile,histological observations revealed the spatial order of bulblet formation,from inner scales to outer scales.Active mobilization of starch granules was observed via transmission electron microscopy(TEM),reflecting the rapid response after cutting treatment.Based on the systematic observations of the established system,we focused on the very early stage(6 h)of no visible morphological changes during the bulblets formation and development of Lycoris for the first time.And we also suggested that the molecular regulation during the very early competence stage played an important role in activating the potential of bulblet regeneration.3.Construction of the full length reference transcriptome of Lycoris via combined sequencingSamples were collected at 0 h,6 h,48 h,6 d,15 d,27 d,36 d based on four stages from the established aerial culture system.By incorporating data from both Pacbio Iso-Seq and Illumina RNA-seq,we developed an integrated pipeline and constructed the "Representative transcriptome"of Lycoris.From Illumina platform,352.12 Gb clean data were obtained.After trimming and removal of low quality reads(LQs).a total of 486,423,193 and 526,792,197 cleaned reads were obtained from Ls and La,respectively.The final cleaned reads of Ls were further used to correct the LQs obtained in the third step Cluster from Pacbio Iso-seq.After further removal of redundant and lowly expressed transcripts(rmp<0.1),a total of 258,031 transcripts were yielded for the Final Illumina assembly.A mixed pool of equal amounts of RNA from seven developmental stages in Ls generated 10.39 Gb clean data flrom the Pacbio Iso-Seq platform.Among them,LQs were further corrected by Illumina(company)clean reads,combined with and HQs,37,518 full-length transcript sequences were obtained in total.After the integration and quality control of data from the two platforms,the final transcriptome assembly contained 285,128 full-length transcript sequences,and the number of predicted genes was 204,933.Final cleaned reads from Illumina were mapped to the final transcriptome,and the mean mapping rate was 76.76%(Ls))and 68.35%(La),respectively.We identified 4,074 expressed transcription factors(TFs)and 1,262 transcription regulators(TRs)during this process.Gene annotation were conducted using NR,GO and KEGG database.4.Comparative transcriptomic analysis of two Lycoris species during bulblet formation and developmentA total of 112,779 inter-species DEGs(La vs Ls)were obtained via ImpulseDE2 method,while 67,973 and 36,251 intra-specices DEGs were obtained from Ls and La,respectively.Expression pattern clustering analysis,GO and KEGG functional enrichment analysis were carried out.Global heatmap of both inter-species and intra-species comparisons revealed significant gene expression differences at 6 h after cutting treatment,indicating the changes at the transcription level occurred much earlier than the appearance of morphological changes.Based on the global gene expression analysis,transient 6h-up-regulated genes accounted for the largest proportion in Ls,while transient 6h-down-regulated genes accounted for the largest proportion in La.Metabolic pathways,biosynthesis of amino acids,and cysteine and methionine metabolism obtained via KEGG enrichment analysis showed different expression patterns in two species.Meanwhile,they were all closely related to the biosynthesis of ethylene,indicating a critical role of ethylene during this process.(1)Active ethylene biosynthesis and signal transduction in the effective regeneration system(Ls)This study revealed the promotion role of phytohormone ethylene on the formation of bulblets in Lycoris for the first time.Inter-species comparison of gene expression patterns revealed significant interspecific differences on key factors of ethylene biosynthesis and signal transduction pathways,including ACO,ACS,ERS1,ERS2,ETR1,ETR2,EIN4,CTR1,EIN2,EIN3/EIF1.Most of these genes showed significantly up-regulated expression pattern in the high regeneration system(Ls),which indicates that ethylene might play an important role in regulating plant regeneration induced by wounding.Based on the wounding responsiveness of ethylene.,this study further revealed the relationship between the wounding response ability and the ability of bulblets formation.(2)The dual role of basal plate cutting via chipping methodDifferent from the traditional research,that basal cutting treatment eliminating the apical dominance,which promotes the outgrowth of axillary bud.Based on the rapid response of wounding induced genes ANAC071,RAP2.6L,ACS2,LOX2,etc.in the high regeneration system(Ls),this study revealed the "dual role" of the basal plate cutting during the bulblet formation and development of Lycoris.On the one hand,basal plate cutting eliminated apical dominance by regulating auxin transport,on the other hand,basal plate cutting acted as a wounding signal in activating the formation of bulblets.ERF and AUX/IAA represented the largest proportion of TF and TR,respectively,which were consistent with the results of hormone-related DEGs.These results revealed the key participation of both ethylene and auxin and further suggested that the regulation of auxin on bulblet formation might be mediated by the synthesized ethylene in response to auxin.Also,as a defense hormone,ethylene has an effect on the transport of auxin.The interaction of both auxin and ethylene is involved in the regulating of bulblet formation and development of Lycoris.