Genetic Interactions Between DL,OsJAG And OsMADS6 In Floral Organ Development In Rice

Studies have indicated that MADS-box gene OsMADS6,zinc-finger gene OsJAG and YABBY-family gene DL are three atypical ABCDE-class genes,which play pivotal roles in floral organ development in rice.Among them,OsMADS6 define lodicule,stamen and carpel development and floral determinacy;OsJAG controls stamen formation but has little effect on the inner three whorls;DL is mainly involved in pistil development.But how these three genes work together in rice floral organ morphogenesis remains less understood.In this study,null allelic mutants of these three genes were used to construct a double mutant dl-sup7 Osmads6-5 and a triple mutant dl-sup7 Osjag Osmads6-5,with which the genetic interactions between these three genes and with other homeotic genes were investigated.The main results are as follows:1.Phenotypes of the double mutant and triple mutant spikeletsThe flower of mutant dl-sup7 displayed normal outer three whorls,but complete transformation of carpel into stamen-like organ.Osjag flower displayed significant changes of four whorls;the most severe defect is ectopic carpel-like organs in the third whorl at the position of stamens.In Osmads6-5 flower,all floral organs except lemma were homeotically transformed into lemma-like organs.The double mutant flower looked basically similar to that of Osmads6-5,which mainly consisted of glume-like organs.The double mutant and Osmads6-5 had the same whorl 1 and whorl 2 phenotypes;their differences mainly existed at whorls 3 and 4.In the double mutant,stamens were mainly homeotically transformed into glume-like organs at whorl 3,but most of the flowers still kept 1-5 stamens.The double mutant lost pistil identity completely at whorl 4,causing flowers appeared to lose determinacy;instead,an inflorescence-like structure developed at this whorl,which consisted of a variable number of spikelet-like structures.This is obviously different from Osmads6-5.Interestingly,these ectopic spikelet-like structures consisted of glume-like organs,analogous to Osmads6-5 spikelet.The triple mutant exhibited obvious abnormalities of four whorl floral organs.Its floral meristem lost determinacy completely by generating multiple inflorescence-like organs at the floral center.It showed the same whorl 1 characteristics as in Osjag,and the same whorl 2 feature in Osmads6-5;in whorl 3 and whorl 4,the identities of stamens and pistil were disturbed,replaced by elongated floral axis with several glume-like organs and multiple inflorescence-like organs,on which new branches and spikelets were produced.It is worthwhile to note that the morphology of the secondary spikelet-like structures looked similar to Osmads6-5 and osjag,which consisted of fine and hooked glume-like structures.The phenotypes of the double mutant and triple mutant suggest that DL,OsJ AG and OsMADS6 could synergistically specify floral organ identity and floral meristem determination through interacting with each other and regulating the expression of floral homeotic genes.2.Expression regulation effects of DL,OsJAG and OsMADS6 on each other and on Floral Homeotic GenesQ-PCR analyses showed that the expression of floral homeotic genes OsMADS16/3/58/8 were significantly downregulated in both the double and the triple mutants.Only OsMADS1 expression remained unchanged in the triple mutant.The results suggested that these three genes act as upstream regulators of floral identity genes;they synergistically help to specify floral organ identity and the determinacy of the floral meristem through regulating the expression of floral identity genes.To clarify the genetic relationship between DL,OsMADS6 and OsJAG,in situ hybridization was performed.The results showed that OsMADS6 could activate the expression of OsJAG in the inner three whorls and suppress DL expression at the second whorl;OsJAG could promote the expression of OsMADS6 at the third whorl;DL did not significantly affect the expression of OsMADS6 and OsJAG.