The Molecular Regulatory Mechanism Of Oil Accumulation Mediated By Lgl-miR171a/LgSCL6 In Fruits Of Lindera Glauca

Lindera glauca,with superior adaptability,wide distribution,and rich oil content in the fruits,has emerged as a novel species of woody biodiesel,and the mechanism of oil accumulation in L.glauca fruits is worth further investigated.This study focuses on the molecular regulatory mechanism of oil accumulation mediated by miRNA and its targets in developing fruits of L.glauca.For this purpose,the L.glauca fruits from different development stages were selected as experimental materials to explore dynamic patterns of fruit development and oil accumulation,and then high-throughput sequencing analyses of mRNA and miRNA from different developing fruits were performed to identify some crucial functional genes,transcription factors and miRNAs specifically involved in oil accumulation,all of which may lead to establish regulatory network of oil accumulation in developing L.glauca fruits.In this regard,the analysis of lgl-miR171a was performed as an important attempt to explore the transcriptional expression mechanism of lgl-miR171a in the response of L.glauca to fruit development and oil accumulation,and the interaction and splice site of targeted LgSCL6 were characterized for lgl-miR171a.Also,genetic transformations of lgl-MIR171a and its targeted LgSCL6 were conducted on Arabidopsis,and all the obtained lines（including wild type,overexpression,mutation and its recovery）were used to analyze the changes of phenotype and oil accumulation as well as expression levels of some key genes involved in oil biosynthesis,which should help to gain new insight into molecular regulatory mechanism of oil accumulation mediated by lgl-miR171a-LgSCL6 in L.glauca fruits.The obtained main findings are summarized as follows:1.According to the dynamic changes of fruit morphological characteristics（volume,and fresh and dry weight）and oil accumulation（content and FA compositions）of L.glauca,the period of fruit development can be divided into three stages,including the early stage（25-75 DAF）,middle stage（75-125 DAF）and late stage（125-175 DAF）,among which 25-75 DAF was detected by significant increase in weight and volume of developing fruits,but showing low oil content.As for 75-125 DAF,the growth of developing fruit was relatively slow or retardant,but its oil content increased rapidly from 3.58%（75DAF）to 17.34%（125 DAF）.In 125-175 DAF,designated as maturation stage,the epicarps of developing fruits were gradually pigmented,and oil content（31.25%）was reached the highest value at 150 DAF.Also,a total of 8 fatty acid（FA）compositions were detected for developing L.glauca fruits,including capric acid（C10:0）,lauric acid（C12:0）,palmitic acid（C16:0）,palmitoleic acid（C16:1）,stearic acid（C18:0）,oleic acid（C18:1）,linoleic acid（C18:2）and linolenic acid（C18:3）,among which oleic acid was the main FA component,accounting for the highest content（34.95%）in fully ripened fruits at 175 DAF.All these revealed a specific response of oil accumulation to fruit growth and development of L.glauca.2.Based on the analyses for dynamic patterns of fruit development and oil accumulation,the fruits of L.glauca from three developing stages（50,125 and 150 DAF）were selected for mRNA transcriptome sequencing,and a total of 69,160 unigenes were obtained.Compared with 50 DAF samples,2,854differentially expressed genes were identified in developing fruits.By functional annotation,346 genes encoding for functional genes and transcription factors（such as SUS3,plastidial PDC,ACCase,FATA,DGAT1,ABI3 and WRI1）involved in oil accumulation were identified.And then the fruits of different development stages were used for miRNA transcriptome sequencing,and a total of 312 miRNAs were identified,among which 284 miRNAs,belonging to 39 families,exhibited differential expressions during fruit development of L.glauca.Importantly,the application of an integrated analysis of mRNA and miRNA sequencing and the prediction of miRNA targets has led to the identification of 1,137 targets,and most of which were identified as transcription factors and functional genes implicated potentially in plant hormone signaling（such as miR156/SPL,miR171/SCL6,miR172/AP2,miR166/PHB,miR393/TIR1 and miR160/ARF）.Finally,one regulatory network of oil accumulation for developing fruits of L.glauca mediated by miRNAs was established by a combination of analysis for temporal transcript pattern,protein interaction and q RT-PCR detection,of which lgl-miR171a/LgSCL6 was characterized to locate in the pivotal position of regulatory network,as main focus of study in the following work.3.According to the obtained mRNA and miRNA transcriptome sequencing dates,the precursor sequence of lgl-MIR171a（570 bp）was successfully obtained from developing fruits of L.glauca,which was able to form a stable hairpin structure with the minimum free energy of-34.60 kcal/mol.The results of q RT-PCR showed that lgl-miR171a displayed transcript abundance in the flowers,fruits and leaves of L.glauca,whereas low or no expression was detected in the shoots and roots,and notably,lgl-miR171a transcript was gradually up-regulated during fruit developing,emphasizing that lgl-miR171a transcript was specifically responded to different tissues and developing fruits of L.glauca.4.By genome walking,the full-length sequence（1,432 bp）of promoter for lgl-MIR171a gene was cloned from L.glauca fruits,containing multiple responsive elements for stress（light,salt,drought and wounding）and hormone（Me JA,IAA and GA）.The expression of GUS driven by Pro _MIR171apromoter was analyzed,and the result showed that GUS protein was mainly expressed in the euphylla,mature leaves and inflorescences of transgenic plants,but no expression in the hypocotyls,stems and roots,and notably,the GUS activity was increased gradually with silique development,so it was concluded that the Pr _OMIR171apromoter may be as tissue-specific and development-responsive promoter for L.glauca fruits.To identify the crucial regulatory regions of Pr _OMIR171apromoter,the transgenic tobacco plants with GUS gene driven respectively by the full-length and 5’deleted Pr _OMIR171apromoters were studied.It was observed that GUS expression decreased with the absent lengths of 5’deleted promoters（from 1,432 to569 bp）,from which some cis-acting elements of Pr _OMIR171apromoter were characterized to be involved specifically in the responses of light（located at-1432/-311）,salt（-1432/-1061）and IAA response（-1432/-830）as well as Me JA,drought and wounding response（-1432/-570）.Thus,there existed a complex transcript regulatory mechanism of lgl-miR171a in developing L.glauca fruits.5.According to mRNA transcriptome sequencing dates,the gene of LgSCL6 targeted by lgl-miR171a was cloned from L.glauca fruits with the length of 2,334 bp of ORF,encoding 777 amino acids for the protein with molecular weight of 84.15 k Da.Subcellular localization indicated that LgSCL6 protein was a nuclear transcription factor.Notably,the results from both splicing site analysis by 5’RLM-RACE and transient co-transformation of lgl-MIR171a and LgSCL6 confirmed that the expression of LgSCL6 was negatively regulated by lgl-miR171a.6.The plant expression vectors for lgl-MIR171a and LgSCL6 were respectively constructed to obtain transgenic Arabidopsis lines,and then the phenotype of different genotypic plants(WT,LgSCL6-OX,lgl-MIR171a-OX,Pro _MIR171a::MIR171a,mir171a and lgl-MIR171a/mir171a)were analyzed.Compared with the WT,lgl-MIR171a driven by 35S promoter could result in shorter root length and reduced branches,but giving rise to increase plant height,leaf chlorophyll content and seed weight,all of which exhibited opposite effects on the phenotypes of the lines from overexpression of LgSCL6 or mutation of mir171a.Also,the lines obtained from the recovery of mir171a mutation showed a limited difference of phenotypes as compared with WT,implying that the expression of exogenous lgl-MIR171a could restore the adverse effects caused by miR171a mutation,and thereby revealed that lgl-miR171a could regulate plant growth and seed development by the cleavage of its targeting LgSCL6.Impressively,only chlorophyll content increased in the leaves of Pro _MIR171a::MIR171a lines,indicating the expression of lgl-MIR171a driven by its specific promoter(Pro _MIR171a)could not cause pleiotropic effect on plant development compared with the 35S promoter.7.The changes for oil accumulation（content and FA compositions）of the seeds and transcriptional expressions of some crucial genes involved in oil biosynthesis in Arabidopsis with different genotypes were detected and analyzed.It was found that compared with the WT,the Pro _MIR171a::MIR171a lines could increase oil content,especially for FA compositions of C18:2,C18:3 and C20:1,but the decreased content of oil and its FA compositions with different degree were observed for other genotypic plants,revealing that heterologous expression of lgl-MIR171a driven by its specific promoter(Pro _MIR171a)could effectively promote oil accumulation in transgenic Arabidopsis seeds.Moreover,the results of q RT-PCR showed that the mutation of mir171a or overexpression of LgSCL6 could lead to down-regulation of expression levels for some key genes relevant to oil accumulation,and lgl-MIR171a/mir171a lines could partially restore expression levels of some key genes involved in oil accumulation.It was therefore clear that inhibition LgSCL6 gene by lgl-miR171a via negative regulation may play a vital role for normal expressions of some key genes relevant to oil accumulation.In addition,compared with 35S promoter,lgl-MIR171a expression driven by its specific promoter(Pro _MIR171a)could lead to the increased expressions of some critical genes for oil biosynthesis,but inhibiting SCL6 expression.All these indicated that transcriptional expression of lgl-miR171a in the response to fruit development mediated by its specific promoter(Pro _MIR171a)was linked with the consequence of increasing expressions of oil accumulation-related genes in developing L.glauca fruits via suppressing expression of its targeting LgSCL6.This study was on the multiple levels by a combination of mRNA and miRNA sequencing analysis and functional identification of lgl-MIR171a and its targeting LgSCL6 to identify crucial mRNAs and miRNAs involved in oil accumulation during the development of L.glauca fruits and then further to elucidate the molecular network mechanism that lgl-miR171a negatively regulated the expression of LgSCL6 to increase oil accumulation in developing L.glauca fruits by functional analysis.This study should provide important foundation for researches on molecular regulatory mechanism of fruit oil accumulation mediated by miRNAs and their targets,and for molecular genetic improvement in high oil production for L.glauca and other oil tree species,and thus displayed important academic value and application prospect.