Biological Function And Mechanism Characterization Of AGO18b In Maize

Inflorescence architecture characterizes each species of flowering plant.Inflorescence size reflects the grain-bearing capacity in grass species.Maize possesses a canonical grass architecture and two distinct types of inflorescences,male inflorescence-tassel and later-developed female inflorescence-ear.Both inflorescence types of maize derive from vegetative apical meristems,which develop at roughly the same time when the terminal apical meristem elaborates the tassel while one or more meristems in the axils of vegetative leaves develops as ears.Knowledge regarding the molecular control of the reproductive transition and the determination of meristem identity is abundant.Transcription factors(TFs)controlling the transition have been well studied not only in Arabidopsis,but also in rice(Oryza sativa)and maize(Zea mays).MicroRNAs represent another class of reproductive transition genes,via repressing the expression of key TFs.Argonaute protein(AGO)is one of core components in the small RNA-induced silencing complex(RISC),which involve in the regulation of multiple developmental facets.Argonautes(AGOs)are important players in regulating plant development.Most AGOs strongly expresses in maize inflorescence structures,coincident with the induced expression of phasiRNAs.Here we show that the expression of AGO18 b,a grass-specific AGO,is tightly induced during tassel development,and then characterize its termporal-spatial expression patterns on maize tassel various meristems and development process by qRT-PCR and in situ hybridization.Further to explore AGO18 b biology function,using AGO18 b expression-suppressed materials,mutator-mediated(ago18b::mum)and RNAi ago18 b mutants.Finally,through omics combimation analysis of Transctiptome poly(A)RNA-seq,Small RNA-seq and cRIP-seq,it is challenging to reveal how AGO18 b involved in the molecular regulation mechanism and mode on maize tassel development process.The major achievement of this study is as following: 1.Base on B73 genome sequencing,a total of seventeen Argonaute genes in maize genome had been identified and confirmed,which were classified to three subclass through phylogenetic analysis of Arabidosis,rice and maize.AGO18a/b was a grass-specific subclade which belongs to the AGO1/5/10 clade.We examined AGO18 b expression levels at multiple developmental stages of tassels by quantitative reverse transcription PCR(qRT-PCR).The increase in the expression of AGO18 b became pronounced during cell fate specification of the pre-meiotic tassel length,and the increase was continued to meiotic tassels.The increase became quite sharp between 10-cm and 14-cm stages,during which the cells undergo their transition from differentiation to meiosis.AGO18 b mRNA was highly enriched in a variety of meristems of the immature tassel,with its highest level detected in inflorescence meristems.Enrichment of AGO18 b was also observed in tapetum and germ cell of meiotic anther.2.Both Mutator-mediated(ago18b::mum)and RNAi ago18 b mutants show an increase in plant height and tassel length during reproductive growth,while the tassel tranches,spikelet density,number of leaves and nodes,adaxial/abaxial polarity of leaves,days to silking,ear architecture(ear length,cob diameter)and grain yield-related traits(kernel row number,kernel number per row)showed no significant difference.Intriguingly,11.0% dyad cells at the anaphase II showed non-synchronous sister chromatid segregation(observed cell number,n=900),was significantly higher than that in W22-ref(n=520),about 1.2%(P = 8.82E-05).However,the after effect resulting from the non-synchronous segregation of chromatins was still unknown.3.Sequencing of the small RNA libraries from the 6-cm tassels revealed that 24-nt small RNA was the predominant population(45-48%),21-nt was the second dominant(12-15%),and microRNAs constituted a few percents of all the small RNAs.No obvious difference was observed between wild-type and ago18b::mum plants.Overall,the 21-nt phasiRNA fraction in the 6-cm tassel(~0.3-mm anther)closely resembles that of the anther in its 0.4-mm premeiotic stage.However,the fraction of the 24-nt phasiRNA differed greatly between the anther(~1-2%)and tassel(35%),indicating that 24-nt phasiRNAs were enriched in floral organs other than anther.In the ago18b::mum tassel,the reduced expression of AGO18 b resulted in the increased fraction of 24-nt phasiRNAs,when compared to W22-ref.4.By small RNAs analysis of cRIP-seq,AGO18 b preferentially bind to 21-nt sRNAs in maize premeiotic tassels,and 40-50% of AGO18b-bound 21-nt sRNAs could be mapped to phasiRNAs.However,only 3.8-8.9% of AGO18b-bound 24-nt sRNAs could be mapped to 24-nt phasiRNAs.There was no preference of the 5'-nucleotide in the 21-nt and the 24-nt sRNAs in maize sRNAs.The 5'-U preference sharply increased in the AGO18b-bound 21-nt sRNAs,to a fraction of 62-66% of all 21-nt reads,and the 5'-adenine(A)preference in the AGO18b-bound 24-nt sRNA reached to a fraction of 53% of all 24-nt reads.Interestingly,neither type of phasiRNAs showed a 5'-nucleotide preference.However,the selective binding of the 21-nt 5'-U phasiRNAs and the 24-nt 5'-A phasiRNAs was evident.5.It is known that miR2275 and miR2118 are involved in the production of 24-nt and 21-nt phasiRNAs,respectively,and both miRNAs contain a 5'-U.In the pre-meiotic tassels,the expression level of mi R2275 reached two orders of magnitude higher than that of mi R2118.MiR2118 was also enriched when compared to the extremely low abundance in the input samples.We noticed that the 5p strands of miR2275 family members expressed at a much higher level than the 3p strands.However,their 3p strands contains a 5'-U but the 5p strands harbors a 5'-A,so miR2275-3p strands were enriched in AGO18 b while miR2275-5p strands were excluded.6.The abundance profile of AGO18b-associated miRNAs was very different from the expression profile in maize premeiotic tassel.Although the collective abundance of miR159 family ranked the first,miR166a-3p the most abundant miRNA species associated by AGO18 b in the premeiotic tassels.7.Furthermore,we analyzed the mRNA tag sequences cross-linked with AGO18 b.Many targets of miR166a-3p and miR159a/b/f/j/k-3p were enriched in the AGO18b-bound mRNAs,while less were enriched for those of the miR396 c.We noted that transcripts from four maize genes homologous to the HD-ZIP III family TFs PHV,PHB-1D,REV and ICU/ATHB-5 were significantly bound by AGO18 b.Consistent with the assumption that the AGO18b-miRNA-mRNA complex is functional,most mRNAs associated by AGO18b-miR166 a were up-regulated in the ago18b::mum tassels.In all,AGO18 b strongly enriches miR166 a and its canonical targets,HD-ZIP III transcription factors to exert a negative regulation on maize tassel.