Rice LBD12-1 Suppresses Shoot Apical Meristem Size Through Repressing AGO10-HD-ZIP ? Signaling Cascades

Meristems are the origin of all structures in living organisms,while shoot apical meristem and vascular meristem are important for the aerial organ formation in plants.Delicate controlling system is involved in stem cell and meristem maintenance.Argonaute genes regulate the expression level and pattern of HD-ZIP III genes which are important for plant meristem maintaining and lateral organ development.In this research it is found that the transgenic rice plants overexpressing LBD12-1 exhibited abnormal organ formation and stunned growth.Further research revealed that LBD12-1 directly binds to the promotor region of AGO10,represses the expression of AGO10 and HD–ZIP III genes,and further reduces SAM size under salinity condition.Results from our experiment are summarized as follows:1.Overexpressing LBD12-1 in Kitaake results in stunned growth,twisted leaves,abnormal anther,delayed heading date,reduced setting rate and increased tiller number.These phenotypes are negatively correlated with the expression level of LBD12-1.2.LBD12-1 and LBD11-1 are two Class I LBD family genes with conserved LOB domain,which share the same CDS sequence and consequently protein sequence.LBD12-1 is nuclear located with no transcriptional activity.Phylogenetic analysis indicated that LBD12-1is the homolog of AtLBD12.As the function of Class I LBD genes mainly related with organ formation,we proposed LBD12-1 might function in the same way.3.LBD12-1 is expressed in vascular and meristem region of tissues including root,shoot apical,leaf,stem and young panicle using RT-q PCR.Protein level analysis using western blot and tissue localization analysis indicated its accumulation in vascular and meristem region in shoot apical,buds and young panicle but not in vegetative organs.The discordance of mRNA expression pattern and protein accumulation leads to the possible existence of unknown post-transcription or post-translation regulation of LBD12-1.4.Digital Gene Expression profiling analysis showed that LBD12-1 regulated a serial of genes which mainly participate in oxidation-reduction process,carbohydrate metabolic process and single-organism metabolic process,and regulate plant architecture formation,in which AGO10 might be the direct target of LBD12-1.The expression level of AGO10 and downstream HD-ZIP III genes are negatively related with plant height and the expression level of LBD12-1.In vivo and in vitro experiments(ChIP,DNA pull down,EMSA and tabacoo transient assay)suggested that LBD12-1 directly binds to the 1.4 kb promoter region of AGO10 and represses its expression.LBD12-1 participates in plant organ development andmorphology formation through regulating the AGO10 HD-ZIP III pathway.5.Overexpressing AGO10 in LBD12-1OE background resulted in a wild type plant height together with normal leaves,anther and SAM size.This result indicate that overexpression of AGO10 can rescue the phenotype caused by overexpressing LBD12-1.In addition,ago10 mutants showed phenotype mimic to LBD12-1OE plants.These results suggest that AGO10 is downstream of LBD12-1.6.No significant phenotype was observed in lbd11-1/lbd12-1 double mutant compared to wild type which indicated LBD12-1 might not functions in normal growing conditions.Further analysis revealed that ABA and salt treatment induced the expression of LBD12-1 and down regulation of AGO10 in WT.But in the mutant,neither the shoot apical meristem shrinked nor the expression level of AGO10 and HD-ZIP III down regulated under stress condition.Besides,the response of short term response genes were also reduced in lbd11-1/lbd12-1 double mutants.These results suggest that LBD12-1 represses the expression of AGO10 and salt response genes and reduce the shoot apical meristem under salinity condition.