Study On The Molecular Mechanism Of Florets Opening Based On The Transcriptome Changes Of Lodicules Before Floret Opening In Rice(Oryzasativa L.) And Sorghum(Sorghum Bicolor L.)

The floret opening is not only a great moment in the life of grasses,but also an important physiological process which affect the quantity and quality of the crops.The floret opening will be driven by the rapid expanding of the pair of lodicules,which base in the floret,absorbing abundant water and then levering away the rigid lemma.However,little is known about the molecular mechanism of the lodicules regulating the time of floret opening.Therefore,it is important to study the machnism of lodicules expanding in theory and practice.With the development of real-time quantitative PCR and RNA sequencing technology,a reliable and effective way is provided to study the mechanism of the responses of lodicules to floret opening time at the whole level of cell.In this study,the conventional indica rice germplasm Zhongzao 25?which floret opening is diurnal?and the conventional sorghum germplasm 625R?which floret opening is nocturnal?were used as the materials.Firstly,the expression patterns of those genes,which participated in some biological processes involved in lodicules absorbing abundant water and expanding,at four stages before floret opening were studied by using RT-qPCR technique.Secondly,the two cDNA libraries of lodicules at 12 h and 1 h before floret opening were sequenced using Illumina sequencing platform.The transcriptomes changes were compared according to the analysis methods of sequencing datas on the base of reference genome.The aim of this study is to preliminaryly discuss the molecular mechanism of lodicules absorbing abundant water and expanding in rice and sorghum.The main results are as followed:?1?The expression difference of genes involving in starch degradation,invertase and sugar transport of lodicules at four stages before floret opening was compared by using RT-qPCR technique in rice and sorghum.Relative to the lodicules at 12 h before floret opening,the expression of several member of AMYs and BAMs were intensely induced at 2h before floret opening;OsCWIN1and OsVIN2 coding for acid invertase in rice and two VIN2s and two CWIN1s coding for acid invertase in sorghum were induced on different levels at blooming day,sooner or later;and the expression of most genes of them reached the highest level at 4 h before floret opening;OsHXK and SbHXK were also intensely induced at 4 h before floret opening;OsSWEET4 and OsSWEET14 involved in sugar transport in rice and two SbSWEET4s-like,SbSWEET11-like,SbSWEET14-like and SbSWEET2a-like in sorghum were induced significantly at blooming day.Therefore,it was proposed that these mentioned genes involved in the metabolisms of non-structure carbohydrates and transport played an important role in the accumulation of the soluble sugar and sugar signal transduction required to lodicules absorbing abundant water and expanding in rice and sorghum.These results demonstrated that the accumulation and translocation of soluble sugar played an important role in lodicules absorbing water and expanding and floret opening in rice and sorghum at the gene expression level.?2?The expression difference of potassium ion transporting related genes of lodicules at four stages before floret opening was studied by using RT-qPCR technique in rice and sorghum.Relative to the lodicules at 12 h before floret opening,the expression of OsSKOR related to potassium ion channel was intensely induced at 2 h before floret opening in rice,SbSKOR and SbAKT2 related to potassium ion channel were intensely induced at 2 h before floret opening in sorghum;moreover,the expression of OsHAK9 and OsHAK18related to potassium ion transporter were intensely induced at blooming day in rice,SbHAK5,SbKT2,and SbKUP11 related to potassium ion transporter were intensely induced at 4 h before floret opening in sorghum.So it was infered that potassium ion are also osmotic adjustment substances and participated in osmotic pressure adjustment in the process of lodicules absorbing abundant water and expanding.?3?Aquaporins?AQPs?can promote the transport of free water across the plasma membrane quickly.The expression difference of AQPs-related genes located in different subcellular of lodicules at four stages before floret opening was studied by using RT-qPCR technique in rice and sorghum.Relative to the lodicules at 12 h before floret opening,the expression of three PIP1s?OsPIP1;1,OsPIP1;2,OsPIP1;3?and four PIP2s?OsPIP2;1?OsPIP2;4?OsPIP2;5 and OsPIP2;9?were intensely induced on different levels at blooming day,sooner or later.And in sorghum,SbPIP1;3 and two PIP2s?SbPIP2;2-like and SbPIP2;3-like?were intensely induced on different levels at blooming day sooner or later;moreover,the expression of OsTIP1;1,OsNIP2;1and SbTIP1;1?SbTIP2;3,Sb TIP4;2and SbNIP2;1 were also intensely induced at blooming day in rice or sorghum.Above of these results suggested that AQPs might participate in lodicules absorbing water and expanding in rice and sorghum.?4?The expression difference of those genes related to calcium binding proteins of lodicules at four stages before floret opening was studied by using RT-qPCR technique in rice and sorghum.Relative to the lodicules at 12 h before floret opening,the expression of two OsCMLs?OsCML15 and OsCML27?in rice and two SbCMLs?SbCML10 and SbCML32?in sorghum were up-regulated on different levels at blooming day.These results demonstrated that the calcium signaling pathway participated in regulating the process of lodicules absorbing abundant water and expanding before the floret opening of rice and sorghum.?5?The transcriptomes of lodicules at 12 h and 1 h before floret opening were sequenced using RNA sequencing technology in rice.The expressed genes from the two transcriptomes of lodicules at 12 h and 1 h before floret opening were 26369 and 26157respectively,and 3924 differentially expressed genes?DEGs?were screened between the two transcriptomes under the screening condition of FDR?0.001and|log2Ratio|?1.2623DEGs were down-regulated and 1301 DEGs were up-regulated genes.Moreover,change folds of 105 DEGs were more than 100 folds?|log₂Ratio|?6.7?of lodicules during the two stages before floret opening.Gene ontology?GO?enrichment analysis showed that 1624DEGs could be mapped as functional molecule,21.7%DEGs of which had ion-binding activity,7.5%DEGs of which had oxidoreductase activity and 5.7%DEGs of which had transporter activity;meanwhile,1313 DEGs were involved in biological processes,15.0%DEGs participated in localization processes,12.4%DEGs participated in transport process,4.7%DEGs participated in carbohydrate metabolic process and 4.5%DEGs participated in lipid metabolic process.KEGG pathway enrichment analysis revealed that 2229 DEGs were involved in 123 different metabolic pathways,which enriched larger numbers of DEGs including biosynthesis of secondary metabolites?339 genes?,plant hormone signal transduction?152 genes?,nucleotide metabolism?146 genes?,and starch and sucrose metabolism?64 genes?.The functions of those genes,which were specifically expressed at1 h before floret opening and the unique match reads number of which exceeded 30,were mainly concerned in the regulation of physiological processes of cytoskeleton remodeling,membrane stability,energy metabolism,transcription regulation,and signal transduction.Jasmonic acid and analogs have a strong inductive effect on floret opening in rice.16DEGs were involved in jasmonic acid biosynthesis pathway and 11 DEGs were involved in jasmonic acid signal transduction pathway,whose expression levels increased significantly as the floret opening time approched.This result indicated that endogenous JA played an important role in controlling the floret opening of rice and sorghum.?6?The transcriptomes of lodicules at 12 h and 1 h before floret opening were sequenced using RNA sequencing technology in sorghum.The expressed genes from the two transcriptomes of lodicules at 12 h and 1h before floret opening were 22870 and 22880respectively,and obtained 4651 DEGs under the screening condition of FDR?0.001 and|log2Ratio|?1,of which 1445 DEGs were up-regulated and 3206 DEGs were down-regulated and 105 stage-specific expressed genes were included.Through GO enrichment analysis of DEGs,all of these DEGs were classified into 66 GO terms,of which significantly enriched involved in cellular component of cell and cell part,contained function domains with catalytic activity,hydrolase activity and binding capacity,and participated in macromolecule metabolic process and nitrogen compound metabolic process.The KEGG pathway enrichment analysis indicated that these DEGs showed the diverse functions and could be assigned to 122 predicted metabolic pathways,most of which involved in metabolisms process,secondary metabolites,plant hormone metabolism and signal transduction.However,under the screening condition of Q-value?0.05,those DGEs referred to protein and nucleic acid biosynthesis and photosynthesis pathways were down-regulation with the approach of the flowering.The functions of those genes,which were specifically expressed at 1 h before floret opening,were involved into glyco-metabolism,material transport,signal transduction,hormonal regulation,active oxygen metabolism,gene transcription regulation and secondary metabolism;and they maybe took part in the physiological and biochemical processes of lodicule cells absorbing water and expanding.?7?We analyzed the transcriptomes datas of lodicules further and found that there were 3631 and 5904 novel transcripts in rice and sorghum respectively,of which with coding function was less than 30%.Morever,the phenomenon of alternative splicing often occured in the genes of lodicules in rice and sorghum and the main way was intron retention.But the number of genes which have alternative splicing event and the number of alternative splicing event were different.Using RNA sequencing technology,the gene expression profiling data of lodicules from rice and sorghum were obtained for the first time,which provided with important information of genes for study of the molecular developmental biology in the development of lodicules of rice and sorghum.Combined with RT-qPCR and RNA sequencing technology,it was clear that the expression patterns of those genes involved in energy metabolism,carbohydrates metabolism and translocation,cell walls structure modification and hormones metabolism and signaling pathway in lodicules and closely related to water absorption and expansion of lodicules and regulating of the floret opening time.To some extent,this work enriched the physiological and biochemical mechanism of lodicules absorbing water and expanding and revealed the molecular mechanism of regulating the floret opening time of rice and sorghum at the gene transcription level.