Map-based Cloning And Functional Analyses Of LEAFY HULL STERILE 1-3 (LHS1-3) And PISTILLOID-STAMEN (PS) Genes In Rice (Oryza Sativa L. Ssp. Indica)

Rice(Oryza sativa L.)is one of the most important cereal crops in the world.The floral induction, inflorescence and floral patterning,and floral organ identity have an important effect on the traits such as yield and quality in rice.Therefore,understanding flower development is an area of importance for agriculture.Moreover,as a model crop of grass,the progress of flower development knowledge in rice is relatively closely related to all the other agronomically important cereal grasses including wheat,maize,barley,rye,oat,and sorghum which makes it a reference plant for the world's most important crop plants.The insights gained mainly from the studies of 3 eudicot species,Antirrhinum majus,Arabidopsis thaliana and Petunia hybrida,are painting an increasingly clear picture of the molecular mechanisms of flower development.However,the lineages that led to the eudicot and the monocot separated about 150 million years ago,which is quite early during flowering plant evolution.So, understanding of flower development in monocot species,such as rice,can not depend on excessively the conclusion from eudicot species,such as Arabidopsis.In the recent years,more and more flower development genes have been identified in rice,with complete genomic sequence and powerful tools for functional analysis.In this study,we characterized two genes involved in the flower development in rice,LEAFY HULL STERILE 1(LHS1-3)and PISTILLOID-STAMEN(PS), and wish to help improve the understanding of flower development in rice.The result as follows:1.Map-based cloning and functional analyses of LHS1-3 gene1.1.LHS1-3 specifies spikelet determinacyIn the wild-type rice,the spikelet produced a terminate floret on the top of spikelet after a pair of sterile lemmas.Therefore,the rice spikelet is determinate.In the lhs1-3 mutant,we found the spikelet lost the determinacy and produced two or more lateral florets in alternate phyllotaxy after sterile lemmas instead of a terminate floret on the top of spikelet.This strongly suggested that LHS1-3 specify spikelet determinacy in rice.1.2.LHS1-3 regulate floral meristem and floral organs developmentBesides the lose of spikelet determinacy,lhsl-3 mutant also showed severe floral organ defects, including the elongated leafy lemma,bipartite palea,decreased or absent lodicules,decreased stamens,and occasionally abnormal pistil.The absence or decrease of floral organs suggested that LHS1-3 might be partly responsible for initiating primordial development of the inner three organs and palea in floral meristem at the early stage of flower development.Moreover,the defects of the lemma and palea suggested that LHS1-3 specified lemma and palea identity at the late stage of flower development.1.3.LHS1-3 specifies the different cell types in lemma versus paleaIn the lhsl-3 mutant,the morphological and histological defects in the lemma were different from those in the palea.The lemma showed the leafy and over-elongated phenotypes,but these were not presented in the palea,whose main phenotype is bipartition.Furthermore,the cell characteristics of the lemma were obviously different from those of the palea.These results suggested that OsMADS1 specified the different cell types in the lemma versus the palea.1.4.lhs1-3 is most probably a complete loss-of-function mutation of OsMADS1The lhs1-3 locus was located at a 34-kb region,containing OsMADSI gene and the other two putative genes.We found a 5166-bp transposon Pong insertion in the OsMADS1 gene and the transcription of the OsMADS1 gene was knocked out completely in the lhsl-3 mutant.Additionally, the loss-of-function of ZmMADS8,an orthologs of OsMADS1 in maize,resulted in the indeterminate spikelet,resembling the lhs1-3 mutant.These results strongly suggested that lhs1-3 was a loss-of-function mutation of OsMADS1.2.Map-based cloning and Functional Analyses of PS Genes2.1.PS specifies the stamen identityThe ps mutant fails to initiate the two stamen promordiums located at axis of lemma/palea instead of producing pistil promordiums.Moreover,the other stamen promordiums can be initiated,but they all contain the pistil identity or strongly degenerate during later development stage.The results suggested that PS gene is indispensable for specification of stamen identity.2.2.PS regulates the expression of floral organ identity genesAt the stage of stamen promordiums initiation and early development,the expression of B class genes OsMADS4/16 and C class genes OsMADS3/OSMADS58 which are required to specify the stamen organ identity,were dramatically down-regulated,while the pistil identity gene DL was dramatically up-regulated in ps flowers as revealed by the QPCR analysis.The results suggested that PS gene regulates expression of floral organ homeotic genes and then maintains the staman identity in rice.2.3.PS regulates the cell proliferation of floral organs The ps mutant has the defects in all floral organs.The lemma and palea is shortened and narrowed, as a result they fail to close the flower.The lodicules are elongated or enlarged occasionally.In the third whorl,all the organs including the pistil-like organs and the degenerate stamens have no normal shapes,and some amorphous tissues and indeterminate-number stigmas are often found in these organs.The pistil development was apparently not affected,but often shows the extra stigma and sterile.The results suggested PS regulates cell proliferation in floral organs.2.4.Conservation and diversity of PS genePS encodes a member of zinc finger protein of C2H2 type and is the ortholog of JAG/NUB in rice. JAG together with its closely related gene NUB,mainly control the leaf and floral organ shapes by promoting cell proliferation in Arabidopsis.In addition,PS also control the floral organ shapes in rice.This indicates that the PS and JAG/NUB play a conservative role in regulating floral organs shapes.However,JAG and NUB are believed to play no role in specifying floral organ identity because their mutant shows no homeotic transformation.In contrast,PS seems playing no roles in leaf development because no obvious defects on leaf morphology were observed in ps plants.This indicates that the rice PS and the Arabidopisis JAG and NUB gained their unique functions during their respective evolutions after the lineage split between eudicots and monocots.