Study On MiR167-dependent Regulatory Mechanism Of Anther Dehiscence And Endosperm Development

MicroRNA167(miR167)targets the auxin response factor(ARF)and plays a key role in plant growth and development and stress responses.The miR167 participates in Arabidopsis flower development through the controlling patterns of ARF6 and ARF8 expression.Mutations in the miR167 target sites of ARF6 or ARF8(mARF6 or mARF8)triggerd ectopic expression of these genes in domains of anthers,and consequently resulted in abnormal enlargement of the anthers with no pollen production.Jasmonate(JA)biosynthesis usually occurs in filaments,and is a key event in inducing anther dehiscence.JA signaling in the upper filament promotes water transport from the anther into the filaments,prompting anther desiccation,epidermal JA perception,thus prompting anther dehiscence.ARF6 and ARF8 are required for production of JA in filaments.The arf6 arf8 mutants exhibited shortened filaments,failed to produce pollens and JA.The anther dehiscence defect in stamens of arf6 arf8 mutants can be rescued by providing JA.Howener,unlike the arf6 arf8 mutant,spraying with JA did not restore mARF6 or mARF8 anther dehiscence.These observations suggest that distinct regulatory mechanisms must exist to account for the anther dehiscence in arf6 arf8 and mARF6(or mARF8)mutants.Moreover,a previous study showed that miR167 had the greatest differential expression during endosperm development,which suggests that it may play an important role in maize endosperm development.However,little is known about the regulatory roles of miR167 in maize endosperm development.Here,we used Arabidopsis and maize to systematically study the effect and regulatory mechanism of miR167 on anther dehiscence and endosperm development respectively.The main results are as follows:1.Here,we have engineered Arabidopsis weak mARF6 and mARF8 lines and mir167mutants.Weak mARF6 and mARF8 lines had delayed anther dehiscence and reduced fecundity.Four MIR167 precursor genes(MIR167a,MIR167b,MIR167c,and MIR167d)encode miR167.To test which member of miR167 could account for these phenotypes,we examined these four MIR167 precursor genes expression patterns in flowers.X-Gluc staining analyses of MIR167 promoter:GUS fusion plants revealed that miR167a was strongly expressed in anther.Thus we generated plants with mutations in MIR167a using CRISPR/Cas9-based genome engineering.Mutations in MIR167a recapitulated weak mARF6and mARF8 phenotypes.The above results indicated that MIR167a was the major mir167precursor gene regulating ARF6 and ARF8 in anthers.In the absence of miR167 regulation,mutants had delayed anther dehiscence.2.Combining tissue architecture and cell morphology,we observed and analyzed the phenotype of Arabidopsis miR167-deficient anthers.Anther growth phenotypes in mARF6,mARF8,and mir167a anthers of Arabidopsis first appeared at flower stage 11,anther cells were larger than wild-type anthers.Various studies have shown that auxin can directly affect cell membrane or membrane components,and thus affect cell enlargement.To test whether the enlarged cells of miR167-deficient anthers are related to the auxin response,we crossed mARF6 and mARF8 pollen to DR5:GUS line(auxin response reporter).X-Gluc staining analyses of DR5:GUS fusion plants revealed that auxin response was increased in mARF6 and mARF8 anthers compared to wild-type.Meanwhile,material exchange is necessary for plant growth.The rate of growth is positively associated with the rate of growth material exchange.To test whether the enlarged cells of miR167-deficient anthers are related to the material exchange,we crossed mARF6 and mARF8 pollen to a phloem unloading tracer,P_SUC2:YPET.In mARF6 and mARF8 anthers,the expression time and area of a phloem unloading tracer,P_SUC2:YPET,were longer than wild-type anthers.Thus,it appears that phloem content was delivered into mARF6 and mARF8 anthers over a larger area and until a later stage than occurs in wild type.In mARF6 and mARF8 anthers,phloroglucinol stained a stripe very similar in appearance and extent to that observed in wild-type anthers.In addition,the lignin deposition in endothecium also affected anther dehiscence.Lignin staining results suggest that patterning of differentiated cell types in these anthers is normal.The above results indicate that the excess growth of anther is displayed by the excess growth of connective cells,and positively associated with auxin response and phloem unloading content.3.Arabidopsis ARF6 and ARF8 can activate gene expression,thus the ectopic expression of ARF6 and ARF8 can cause overexpression of some genes related to anthers excess growth.To reveal genes whose altered expression might contribute to excess growth and/or failure of dehiscence in miR167-deficient anthers,we assayed global gene expression patterns by RNA-Seq in wild-type,mARF8,and P_RPS5a>MIR167a(overexpressing MIR167a)stamens of Arabidopsis.The mARF8 stamens overexpressed genes,102 of which were underexpressed in P_RPS5a>MIR167a stamens.The list of 102 genes were enriched for the GO categories response to jasmonate and cell wall loosening.These genes may contribute to the excess growth seen in mARF8 anthers.To verify whether these genes were overexpressed in anthers,we crossed mARF6 and mARF8 pollen to promoter:GUS fusion plants of these genes.We assessed expression of available GUS fusions to the promoters of these genes.Both _PEXPA8:GUS and _PXTH19:GUS had expanded expression patterns in mARF6 or mARF8 anthers.The above results indicate that the overexpressed genes in mARF6 or mARF8,such as EXPA8and XTH19,may contribute to the excess growth seen in mARF8 anthers.4.To clarify the way which miR167 regulates anther dehiscence in Arabidopsis,we explored the conditions leading to anther dehiscence in miR167-deficient mutants.Experimental desiccation enabled dehiscence of miR167-deficient anthers,whereas high humidity conditions delayed anther dehiscence in wild-type flowers.This observation suggests the model that delayed or failed dehiscence of miR167-deficient anthers might be a consequence of excess water content arising from persistent growth.These results indicate that mir167 action limits anther growth and accelerates dehiscence.To determine whether the jasmonate-MYB21 pathway is required for miR167 production,we assayed P_MIR167a:GUS expression in stamens of jasmonate-related mutant plants and the expression of miR167 in aos-2 flowers or stamens.By inferences the jasmonate-MYB21 pathway is not required for miR167 in anthers.The above results indicate that miR167 limits anther growth to potentiate anther dehiscence and does not require the jasmonate-MYB21 pathway.Anther abnormal enlargement and delayed dehiscence were caused by loss of mir167 regulation.5.To explore the important role and regulatory pathway of miR167 in maize endosperm development,we identified the miR167 expression and its target genes in maize endosperm.The sRNA-Seq results showed that miR167 had differential expression during maize endosperm development.The qPCR results of expression pattern analysis revealed that MIR167a was up-regulated in the developing endosperm.Two targets,ARF9 and ARF18,were determined by degradome library sequencing,transient expression analysis,phylogenetic analysis and expression pattern analysis.Like Arabidopsis ARF6 and ARF8,maize ARF9 and ARF18 can activate gene expression,thus participate in maize endosperm growth.To screen the genes related to ARF9 and ARF18,we obtained ARF18 overexpressed endosperm through transient expression and assayed global gene expression patterns by RNA-Seq.The list of 121 genes were enriched for the GO categories response to hormone,cell wall loosening and water movement.The above results indicate that miR167 participates in maize endosperm development via controlling patterns of ARF9 and ARF18 expression.In this study,we have studied the miR167-dependent regulatory mechanism of anther dehiscence in Arabidopsis by cell and molecular biology techniques.The findings provide a theoretical basis to explore the role of miR167 in maize anther development.Meanwhile,we made a preliminary exploration of the miR167 regulatory pathway in maize endosperm,and this had helped to better understand the mechanisms employed by miR167 regulation during maize endosperm development.The reveals of miR167-dependent regulatory mechanism in anther and endosperm will provide the theoretic basis for further creation of male sterile maize,heterosis breeding,and high-yield breeding.