Morphological Characteristics And Related Gene Expression Analysis Of Osmads1+Osmads6-5 Spikelet In Rice

Rice floral organ development is directly related to its yield and quality.Therefore,rice floral organ development research has the important significance.Our team found two mutant Osmadsl and Osmads6-5,which are related to floral organ development in rice.Four whorls of Osmadsl flowers have different degrees of phenotypic alterations.Osmadsl are characterized by the elongated thinning leafy lemma and palea,the transformation of increased number of lodicules into glume-like organs,the decreased number of stamens and occasionally extra pistils.Spikelets of Osmads6-5 exhibit normal lemma and apparently enlarged palea,the absolute transformation of lodicules,stamens and pistil into glume-like organs and the indeterminate floral meristem.To further understand these two genes functions in regulation of rice floral organ development and their interrelation,we constructed Osmads1+Osmads6-5 and analyzed its phenotype and expressional regulation of this double mutant.The results are as follows:1.Phenotype characteristics of Osmads1+Osmads6-5 spikeletsPhenotypes of Osmads1+Osmads6-5 mature spikelets have significant variation.Osmads1+Osmads6-5 had the elongated leafy lemma and palea,which was very similar to Osmads1;lodicules in whorl 2 completely degenerated and many elongated glume-like organs grow grew in this region;stamens in whorl 3 and pistil in whorl 4 disappeared entirely,and replaced by "spikelets-in-floret" structure which consisted of obviously elongated pedicel and numerous extra spikelets.All these spikelets had multiple whorls glume-like structures,which is similar with Osmads6-5.In addition,just like Osmads6-5,the double mutant spikelets completely lost the floral meristem determinacy.Paraffin section revealed the number of the vascular bundles greatly increased in the palea and lemma of Osmads1+Osmads6-5 similar with Osmads1.The results of Scanning electron microscopy(SEM)showed that the the development of palea primordium and lemma primordium did not differ significantly between Osmads1+Osmads6-5 and wild-type flowers,but their development dramatically speeded up and eventually developed into leafy organs.Initiation of the lodicules primordium in whorl 2 was the same with those of wild-type,but the number and developmental speed of lodicules primordium increased dramatically and eventually developed into glume-like organs.Stamens primordium and pistil primordium of the double mutant did not differentiate obviously,but the central floral meristem differentiated into multiple branche primordiums which continued to develop into obviously elongated pedicel upwards.Subsequently,the continuous differentiation of these branche primordiums lead to the formation of multiple spikelets primordium which would develop into this structure,which was completely wrapped by glume-like organs and was similar with Osmads6-5.2.Relationship between OsMADSl,OsMADS6 and other floral organ identity genesWe analyzed the expressional levels of a few flower developmental key genes in wild-type,Osmadsl,Osmads6-5 and Osmadsl+Osmads6-5 by Quantitative Real Time PCR.We found the expressional levels of OsMADSI6/3/7 were down-regulated in two single mutants but significantly down-regulated in Osmadsl+Osmads6-5.The expressional levels of DL and Osjag were up-regulated in Osmadsl and down-regulated in Osmads6-5,while that of DL was essentially unchanged and Osjag was down-regulated in Osmadsl +Osmads6-5.These results demonstrated that OsMADSl and OsMADS6 might determine rice flower organ development by regulating expression of these genes.Interestingly,the expression level of OsMADSl gene was significantly up-regulated in Osmads6-5 mutant,whereas the expression level of OsMADS6 gene was not impacted.This indicates that OsMADS6 may suppress the expression of OsMADS1,but OsMADS1 did not directly affect the expression of OsMADS6.This result was further confirmed via in situ hybridization.The results of in situ hybridization showed the expressional region of OsMADSl extended to multiple whorls glume-like organs in the Osmads6-5,resulting in up-regulation of its expressional level.The expressional region and expressional quantity of OsMADS6 remained unchanged in Osmadsl.In conclusion,OsMADS1 and OsMADS6 control rice floral organ and floral meristem identities through regulating the expression of some floral organ identity genes in different stages of floral organ development.It is thus speculated that OsMADS1 and OsMADS6,cooperatively or separately control the development of all four whorl flower organs during the double mutant’s floral organ development,but their function has a chronological order.Among them,the OsMADS1 acted at first and individually regulated the development of palea and lemma.OsMADSl and OsMADS6 co-regulated the development of lodicules.The function of OsMADS6 separately resulted in the degeneration of stamens and pistil,and OsMADS6 specified floral meristem fate.