OsAGO1b Regulates Multiple Processes Of Rice Growth And Development

Argonaute(AGO)proteins are the major component of RISC(RNA-induced silencing complex),involving gene silencing pathway mediated by small RNAs,and regulating the expression of target genes of small RNAs.AGO proteins could recruit siRNA(small interfering RNA)and miRNA(microRNA)in plants,and regulate many aspects of plant growth and development.In Arabidopsis,the biochemical functions and regulation mechanisms of AtAGO1 protein in plant development have been widely investigated.Rice(Oryza sativa L.)genome encodes four AGO1 members homologous to AtAGO1,i.e.OsAGO1 a,OsAGO1b,OsAGO1 c and OsAGO1 d.However,their regulatory roles and the underlying mechanisms in rice growth and development are mainly unclear.The key factor of enhancing rice grain yield is to improve the plant architecture.Plant architecture of rice is crucially determined by leaf morphology and effective tiller number.Thus,in order to improve plant architecture,it is necessary to elucidate the molecular regulation networks of leaf and tiller growth and development.Our previous study showed that the OsAGO1b-overexpressed rice plants displayed several abnormal phenotypes,including up-ward rolled leaves and reduced tiller numbers.In addition,OsAGO1 b RNAi plants exhibited significant reduced seed setting percentage and pollen fertility.Based on the background mentioned above,we analyzed the expression patterns of rice four AGO1 members and the phenotypes of their overexpression and RNAi plants.Furthermore,the relationship between auxin and OsAGO1 b,and the molecular mechanisms of leaf rolling and tiller development regulated by OsAGO1 b were explored and discussed in this study.The main research results are as follows:(1)The PAZ and Piwi domains of rice AGO1 members were highly similar to AtAGO1,suggesting the biochemical function of rice AGO1 members might be similar to AtAGO1.The phylogenetic analysis of 19 plant AGO1 proteins showed that the AGO1 proteins from dicots and monocots were clustered into three groups.One of the monocot AGO1 group containing OsAGO1 a and OsAGO1 b was closest to the dicot group containing AtAGO1,and the other monocot AGO1 group containing OsAGO1 c and OsAGO1 d was particularly specific to monocots.(2)The four rice AGO1 genes were all expressed in higher levels in mature tissues and were expressed in lower levels in young tissues during vegetative growth stage.The expression of OsAGO1 b was gradually decreased with the development of spikelets,which is quite different to the other three AGO1 members.The overexpression and RNAi transgenic plants of OsAGO1 a,Os AGO1 c and OsAGO1 d showed no abnormal phenotype compared to ZH11 wild type plants.However,OsAGO1b-overexpressed plants exhibited pleiotropic developmental defects,including up-ward rolled leaves and extremely reduced tiller numbers.Down-regulation the OsAGO1 b resulted in abnormal phenotypes of spikelets,including bigger hulls,enlarged glumes,deformed anthers,reduced pollen fertility and seed setting percentages in OsAGO1 b RNAi plants.(3)The leaves of OsAGO1b-overexpressed plants were significantly narrow and up-ward rolled from 20 DAG.Further observation of the narrow and rolled leaves showed that the number of small vascular bundles was decreased,and the sclerenchyma cells on the abaxial side of vascular bundles were partly defective formed.In situ hybridization of OsAGO1 b in ZH11 leaves indicated that the OsAGO1 b mRNA was transcribed in different cell types of young leaves and leaf sheaths without polarity distribution.(4)The expression levels of SLL1 and SRL2,both of which are involved in regulating the sclerenchyma cell development in leaves,was slightly increased in OsAGO1b-overexpressed plants,suggesting that the expression of SLL1 and SRL2 might not be involved in the regulation of the defective development of sclerenchyma cells in OsAGO1b-overexpressed plants.Several members of OSHB,ARF and YABBY families involved in leaf adaxial-abaxial polarity development were significantly up-regulated in the leaves of OsAGO1b-overexpressed plants,and the enhanced expression of OSHB1 and OSHB3 might account for the defective development of sclerenchyma cells on the abaxial side of leaves.In addition,the expression levels of some mature miRNA(miR166f and miR319b),TAS3-siRNA 5'-6D(+)and TAS3-siRNA 5'-7D(+)were reduced in rolled leaves of OsAGO1b-overexpressed plants.Further study showed that OsAGO1 b did not influence the expression levels of the primary miRNAs of miR166 and miR319.(5)The growth of roots and the development of adventitious roots were obviously inhibited in OsAGO1b-overexpressed plants.Further analysis showed that the auxin distribution was reduced in root tips,and the endogenous IAA contents of leaves were significantly decreased during vegetative growth stage of OsAGO1b-overexpressed plants.OsAGO1b-overexpressed seedlings were sensitive to IAA treatment;on the contrary,OsAGO1 b RNAi seedlings were insensitive to IAA treatment.Subsequent analysis showed that the response speed to IAA treatements was delayed in OsAGO1b-overexpressed seedlings,but the response intensity to IAA was enhanced.The exogenous IAA did not affect the expression of OsAGO1 b in shoots of seedlings,but promoted the expression of OsAGO1 b in roots of seedlings.(6)OsAGO1b-overexpressed plants exhibited reduced tiller numbers,and the plants showed monoclum phenotype during vegetative growth stage.Further observation indicated the formation of tiller buds was delayed and the bud outgrowth was inhibited in OsAGO1 b overexpression plants.In situ hybridization of OsAGO1 b in ZH11 tiller buds showed that OsAGO1 b was mainly expressed in mature vascular bundles and sheaths,but weakly expressed in tiller buds.Moreover,the distribution of endogenous auxin was enriched in tiller buds of OsAGO1 b overexpression plants.(7)Expression profiling of tiller buds using Affymetrix GeneChip microarrays showed that 1001 genes were differentially expressed with more than 2-fold change between ZH11 and OsAGO1 b overexpression plants.Among them,601 genes were up-regulated and 400 genes were down-regulated compared to ZH11.The expression of genes involving transcriptional expression,RNA metabolism,signal transmission and transport between cells were significantly changed in OsAGO1b-overexpressed plants.(8)The expression of OsPIN1 b and OsPIN9 were reduced more than 2 times in OsAGO1b-overexpressed tiller buds compared to ZH11.OsPIN1 b and OsPIN9 were both mainly expressed in young tissues,especially strongly accumulated in initiated tiller buds.Nevertheless,OsPIN1 b and OsPIN9 RNAi plants did not exhibit obvious abnormal phenotypic changes,which suggested that down-regulation of OsPIN1 b and OsPIN9 did not lead to inhibition of bud outgrowth,but might affect the distribution of endogenous auxin in tiller buds.(9)The expression of OsSPL14,D14 and OsTB1 were up-regulated in dormant tiller buds of OsAGO1b-overexpressed plants,which might resulted in the delayed development of leaves and tiller buds,as well as the inhibition of buds outgrowth.Furthermore,the expression levels of miR156 a,miR160a,miR164 a,mi R166 f,miR167a,miR319 b,mi R390,miR396 a and miR444 d were decreased;however,the primary transcripts of miR156,i.e.pri-miR156 a,pri-miR156 g and pri-miR156 j were increased.The results indicated that enhanced OsAGO1 b expression could suppress the accumulation of a few mature miRNAs,and facilitate the transcription of MIR156 genes.(10)Overexpression of OsAGO1 b in japonica rice varieties Nipponbare and Songjing,indica rice varieties Annong and Huanghuazhan background resulted in monoculm and inward rolled leaves,similar to that of overexpressed OsAGO1 b in ZH11,suggesting that the pathway of OsAGO1 b regulating leaf and tiller growth was conserved in indica and japonica rice.