Map-Based Cloing And Functional Analysis Of A Gene OG1 Regulating Glumes Opening And Closure In Rice Spikelets(Oryza Sativa L.)

Flowering is essencial for reproductive success in angiosperm.Anthesis mechanism is different between dicotyledons and monocotyledons,opening and closure of petals and glumes indicate anthesis in dicotyledons and monocotyledons respectively.Rice is one of the most important cereal crops and a model monocotyledon species.Its yield will be affected severely if anthesis is unnormal.Researchers have been devoted to exploring the mechanism underlying rice anthesis and find lodicules locating at the base of spikelets linking lemma and palea is the critical tissue,whose expansion and withering controlling the opening and closure of glumes,however,the mechanism underlying this process is still unexplored.There are many factors affecting rice anthesis,including environmental factors,such as light,temperature,humidity and mechanical stimulus,otherwise,phytohormones are reported to be involved in flower development.Jasmonic acid(JA)can promote flower opening in rice.In our study,we obtained a mutant named open glume1,which displayed open glumes after flowering.Through map-based cloning stategy and subsequent transgenic analyses,we isolated OG1 gene.We also try to dissect the unerlying mechanism.Some main results are listed as follows:1.We found ogl mutant was comparable with the wild type in the seedling,tillering and heading stages,including plant height,tiller number and spikelet number per panicle.When flowering,most of the wild-type florets opened at the daytime ranging from 10:00 to 12:00 a.m.under the suitable environment,finally,the florets closed and grain filling initiated.However,the initial flowering time of the ogl mutant was delayed with elongated stamen filament and brown anthers but enclosed in the glumes and the glumes kept open after flowering.Moreover,it took about 3 days to finish flowering for one panicle in the wild type,while 7 days in the mutant.Statistical analysis on selected panicles with continuous one week showed that about 85 florets per panicle opened in the normal flowering time of a day ranging from 10:00 to 12:00 a.m in the wild type but no concentrated flowering time could be observed in the ogl mutant with an average number of 10 and 19 florets opening in the day and night,separately.Finally,the florets of ogl could not close normally,resulting in malformed,shriveled and pathogen sensitive seeds with open glumes.The grain weight per panicle of the wild type was about 2.5 fold to that of the ogl mutant at the mature stage.2.Stereoscope observation and Transmission electron microscopy(TEM)observations showed that the lodicules of ogl mutant could not wither after flowering with full inclusions,which resulted in the glumes keeping open after flowering.TUNEL assay also showed almost no death in the ogl lodicule cells.Then,transcriptome analysis and qRT-PCR results showed the expression levels of some senescence associated genes were greatly down regulated in the ogl mutant,which implied the cell degradation process was delayed resulting in the open glumes.3.Two F2 populations from reciprocal crosses between the wild type and ogl mutant were constructed for genetic analysis.The results showed that the plants with normal closed glumes and open glumes segregated at a ratio of 3:1 in both populations,indicating that the mutant phenotype is controlled by a single recessive nuclear gene.Then we isolated the OG1 locus by a map-based cloning approach.An F2 population from a cross between ogl(an indica variety)and Nipponbare(a japonica variety)was generated to fine mapped the target gene,OG1,which encodes a 12-oxophytodienoate reductase 7(OsOPR7),a critical enzyme involved in JA biosynthesis.A 4bp insertion in the first exon caused a frameshift,resulting in premature translational termination and loss of function of OsOPR7.Later RNA interference(RNAi)and complemental transgenic lines both demonstrated that OsOPR7 corresponded to the OG1 gene.4.There are three conservative amino acid residues at the C terminus of OsOPR7 by alignment with other homologous amino acid and the three residues are the locating signal of peroxisome.Later subcellular localization result also confirmed the conclusion by merged with PTS1-RFP(a peroxisome marker).Moreover,GUS staining and qRT-PCR results suggested the ubiquitous expression pattern of OsOPR7 in rice plant,especially in floral organs,which implies the function in floral development.5.Measurement of JA contents in the wild type and ogl mutant suggested almost no JA accumulation in the ogl mutant,which confirmed the critical role of OG1 gene in JA biosynthesis.When treated with exogenous JA,flowers of the ogl mutant could close normally and the lowest concentration needed was 50 ?M.6.To find out the pathway by which JA controlling the flower opening and closure in rice,we measured the osmolality in lodicules of wild type and ogl mutant during the whole anthesis stages and found the high-keeping value after flowering in the ogl mutant.Later measurement of inclusions of lodicules found the soluble sugar content was much higher after flowering in the ogl mutant,which may cause the higher osmolality and unwithered lodicules preventing the closure of glumes.Transcriptome analysis on lodicules after flowering was used to explore how JA regulating sugar metabolism and results showed a class of sugar transporters genes,SWEETs,including OsSWEET4,OsSWEETll,OsSWEET5,OsSWEET14 and OsSWEET15,were greatly down regulated in the ogl mutant after flowering.The variation trend of expression levels of OsSWEET4 is correspond to those of both osmolality and soluble sugars,implying the role of OsSWEET4 involved in JA-regulated sugar metabolism in lodicules during the whole anthesis.