Molecular Mechanism Of ZmMADS8 Regulating Maize Inflrescence And Kernel Development

Meristem is a group of stem cells with the ability to self-renew and to generate lateral organs in a stereotypical pattern.The identity and determinacy of meristem determine the plant architecture and the floret development.In this study,we characterized a maize MADS-box family gene,ZmMADS8.We created loss of function mutants and ZmMADS8 over-expression family lines,and we analyzed the biological functions and molecular pathways of ZmMADS8 in the regulation of the development of inflorescences and florets,and biosynthesis of zein protein and starch.The main results are showed as follows:1.Ectopic expression of ZmMADS8 and Drop leaf2 change the fate of stem cells in meristemUsing the Ubi promoter to drive ZmMADS8 overexpression and dominant mutant Fascicled ear1(Fas1)to study the genetic effects of ectopic expression of ZmMADS8.ZmMADS8 overexpression line has no significant phenotypic variation.However,ZmMADS8 specifically expressed not only in the floret meristem(FM)of the female inflorescence,but also ectopically in the inflorescence meristem and spikelet pair meristems in the Fas1 mutant.Further studies revealed that another candidate gene Drl2 is also ectopically expressed in Fas1.This result indicated that the Fas1 phenotype was co-ectopic expression of ZmMADS8 and Drl2.2.ZmMADS8 regulates the development of maize floret organsZmMADS8 highly expresses in immature kernels and developing inflorescences.ZmMADS8 specifically expresses in FM primordia of inflorescences,the carpel primordia and palea primordia of the upper flower.We created a loss-of-function mutant of ZmMADS8 using CRISPR/Cas9 technology.The mads8-1 mutant showed increased lemmas and not completely degraded paleas which partially wrapped the seed in female floret.These results indicate that ZmMADS8 negatively regulated the development of the palea and lemma of female florets and plays a pivotal role in determining the identity and determinacy of the lemmas in male florets.3.ZmMADS8 regulates identity of FM by interacting with ZmMADS14ZmMADS14 physically interacts with ZmMADS8 and showed a similar expression pattern to ZmMADS8.To reveal the biological function,we created a loss of ZmMADS14 function mutant.We found that female florets of the mads14-1 mutant exhibited extra lemmas but reduced anthers.The extra flower or spikelet or branch-like organs were observed between the upper and lower florets in the tassel-inflorescence of the mads8-1;mads14-1 double mutant.Intriguingly,the floret meristems were repeatedly reversed into branch meristems which were developed into branch-like structures.These results indicate that both ZmMADS8 and ZmMADS14 synergistically regulate determinacy of floret meristem.4.ZmMADS8,ZmMADS47 and O2 synergistically regulate the content of zeinThe mads8-1 kernel showed a reduced hard endosperm and an increased starchy endosperm with a loosely arranged starch granules.The mads8-1 kernels had lower zein protein content,higher non-zein protein content andhigher lysine content,although total protein content showed no significant difference.ZmMADS8 was able to enhance transactivation activity of the ZmMAS47-O2 transcription complex to the 19 k D α-zein genes and regulate biosynthesis of zein protein.5.ZmMADS8 regulates starch content by interacting with DEP1-1The total starch content decreased by 5% in the mads8-1 single mutant,by 6% in the dep1-1 single mutant,and by 12% in the mads8-1;dep1-1 double mutant.ZmMADS8 may cooperate with DEP1-1 to regulate starch synthesis in maize.In summary,ZmMADS8 participated in the regulation of palea and lemma development of maize florets and can also interact with ZmMADS14 to regulate the identity of floret meristem.In addition,ectopic expression of ZmMADS8 and Drl2 can change the fate of stem cells in the inflorescence meristem,resulting in the Fas1 mutant phenotype.Furthermore,ZmMADS8 could interacts with ZmMADS47 to regulate endosperm protein quality by transactivation activity of O2 to 19 k D α-zein genes expression level and ZmMADS8 interacted with DEP1-1 to regulate the starch content of endosperm.