Adaptation Mechanism Of Isoetes Sinensis Between Terrestrial And Submerged Conditons And Functional Analysis Of Leafy

Isoetes L.is an acient genus with a unique position in plant evolutionary process.This genus is one of the earliest basal vascular plants,and the main characteristics in the genus are heterosporous and a strong reduced plant body.Phylogenetic analysis reveals that Isoetes is a group of lycopsids and it maybe can date back to the Devonian period.The species in this genus are the only survival as the closet relatives of the famous tree lysopods.In addition,Isoetes occupy a variety of niches,including oligotrophic soft-water lakes,seasonal pools,intermittent streams,higher altitude wetlands,and terrestrial conditions.Moreover,the species repeatedly adapt to the environmental changes.However,we do not know how Isoetes adapts to the environmental changes.LEAFY is a plant-specific transcription factor.LEAFY plays an essential role in regulating floweing process in flowering plants.This gene can be found from alga to angiosperm.Genearlly,the roles of LEAFY is suggested to regulate cell divison in lower plants and regulate flowering process in higher plants.Moreover,role in flowering process is gradually obtained in the plant evolutionary process.Considering the important roles of Isoetes in evolutionary process,we do not know the biological function of LEFY in this genus up to date.Isoetes sinensis Palmer is a typical amphibious plant.The species can repeatedly adapt to the environmental changes.We used the species as an example to investigate adaptation mechanism of Isoetes under submerged and terrestrial conditions and the functional characteristics of LEAFY.The main results are as follows:1.To investigate the adaptation mechanism of I.sinesis under submerged and terrestrial conditions,cDNA libraries of terrestrial condition(TC)and submerged condition(SC)were sequenced using Illumina RNA-seq technology.In total,about 87 million high-quality reads were obtained and further assembled into 31,619 unigenes based on De novo assembly.Average length of the assembled unigenes was 1618 bp.Moreover,28,208 unigenes were annotated using NCBI,COG,NR,GO,and KEGG databases.Moreover,1740 diferentially expressed genes were identified with 1146 up-regulated genes and 594 down-regulated genes under TC.GO annotation revealed that the stress-relevant categories were significantly enriched under TC.KEGG pathway ananlysis demonstrated that the phytohormoe signaling and carbohydrate metabolism were significantly accumulated.In addition,we identified 1646 transcription factors and further classified them into 54 transcription factor families.Furthermore,180 transcription factors were dynamically expressed between terrestrial and submerged conditions.This study is the first report for Isoetes to obtain substantial data and establish a general understanding for Isoetes to adapt to the changing environments.Moreover,the data provide a foundation for searching novel genes,comparative geonomics,functional genomics and phylogenetic analysis in Isoetes.2.Light-dark cycle plays an important role in plant growth and development.Given that Isoetes is one of the basal vascular plants in plant evolutionary process,we investigated the characteristics of I.sinensis in response to the light-dark shift under TC and SC.We sampled the materials under terrestrial condition in the light(LTC)and in the dark(DTC),as well as the materials under submerged condition in the light(LSC)and in the dark(DSC),respectively.In total,1145 differentially expressed genes(DEGs)were significantly expressed in LTC vs.DTC,and 1211 DEGs were significantly enriched in LSC vs.DSC.Furthermore,there were 102 transcription factors were significantly enriched in LTC vs.DTC,and 103 transcription factors were significantly accumulated in LSC vs.DSC.Co-expression network analysis revealed that the differentially expressed transcription factors under TC and SC were similaily categorized into two parts for the pairwise comparasion in the light and dark.Further analysis demonstrated that NAC transcription factors played an important role in the light-dark shift under TC.Whereas,NAC transcription factors are not significantly enriched in the light-dark shift under SC.3.CAM photosynthesis is widespread in aquatic and terrestrial species and it is plastic in response to the environmental changes.Isoetes is one of the earliest basal vascular plants,and CAM photosynthesis is popular in the genus.I.sinensis is an amphibious plant and frequently alternates between the terrestrial and aquatic environments.In this study,we investigated and further compared the photosynthetic characters of I.sinensis under TC and SC based on physiological,biochemical and molecular technologies.The results demonstrated that the CAM capacity of I.sinensis was stronger under SC than TC.Titratable acidity levels and organic acid concentration were more enriched under SC than TC.Whereas soluble protein,soluble sugar,and starch levels were more enriched under TC than SC.Furthermore,we used RNA-seq technology to search photosynthesis-related genes.In total,we found nine photosynthetic genes.Expression pattern analysis revealed that the CAM-associated genes were highly expressed under SC,and C3-associated genes were highly expressed under TC.In addition,we measured PEPC and Rubisco enzyme activity.The results showed that PEPC acticity was slightly higher over a durial cycle under SC than TC.However,Rubisco activity was greater during the daytime under TC than SC.4.LEAFY plays an important role in floweing process in angiosperms.Isoetes is a group of heterosporous lycopsids with a unique position in plant evolutionary process.We extract cDNA from five Isoetes species in China,including L hypsophila,I.yunguiensis,I.sinensis,I.orientalis,and I.taiwanensis.We used RACE methods and obtained two LEAFY homologs in the five species,respecitively.We further extracted the genomic DNA to investigate the genomic sequences of the two LEAFY homologs in the five species.We referred the two LEAFY homologs as ILFY1 and ILFY2.The results revealed that the full length of ILFY1 was 1449-1456 bp and the open reading frame was 927-936 bp in length.The full length of ILFY2 was 1768 bp and the open reading frame was 726 bp in length.Phylogenetic tree analysis demonstrated that ILFY1 and ILFY2 were separated into two clades,and the two genes in I.hypsophila were separated with the other four species.We employed quantitative real-time PCR(qRT-PCR)and in situ hybridization methods to investigate expression levels of the two genes in I.sinensis.The data revealted that ILFY1 and ILFY2 were similarily accumulated in both vegetative and reproductive tissues,and the two genes were highly expressed in juvenile tissues.In addition,we transferred the two genes into Arabidopsis wild-type plant and lfy mutants,respecitively.ILFY1 and ILFY2 similarily did not promote precocious flowering,and they did not rescue the Ify mutant phenotype.