Molecular Mechanism Underlying HEN1-Controlled Ovule Development In Arabidopsis Thaliana

Sexual reproduction is the key step in the whole life cycle of plant,which is essential for plant progeny and generation alternation.In Arabidopsis thaliana,the sexual reproduction can be divided into three stages:the formation of male and female reproductive organs,fertilization,and embryogenesis.As main female reproductive organ of plants,ovule is the place where the fertilization occurs and therefore has received widespread attention.In recent years,a large number of high-throughput data indicate that microRNAs as a key regulatory factor play an important role in ovule development.It has been reported that microRNA production regulators,such as AGO9 and DCL1 regulated functional megaspore formation or integument growth,respectively.However,no genetic pathway from microRNA processing to microRNA to its target has been revealed during ovule development.We report here that HEN1 regulate ovule development through miR167-mediated down regulation of ARF6 and ARF8.Main results and conclusions are as follows:?1?Male and female transmission of hen1-8 is normalPrevious studies have shown that the mutations of HEN1 lead to reduced fertility.However,it was unclear what processes during reproduction were affected in hen1 mutants.Therefore,we first carried out silique analysis and found that the average seed set of hen1-8,a weak hen1 allele,was 25%,while that of hen1-8/+was around 100%,similar to that of wild type.Reciprocal crosses showed that male and female transmission of hen1-8 is normal,indicating that hen1-8 is not defective in gametophytic functionality.?2?hen1-8 is defective in integument growthReciprocal crosses between hen1-8 and wild type showed that the reduced seed set of hen1-8 was due to sporophytic defects in female.Optical section of ovules by CLSM showed that hen1-8 ovules were defective at stage 3-I such that integument growth was impaired.These results showed that HEN1 is important for integument growth.?3?Defective ovules of hen1-8 failed to attract pollen tubes,leading to reduced female fertilityTo determine the cause of failed fertilization when the hen1-8 mutants were used as the female parent,we pollinated wild type or hen1-8 pistil with Pro_Lat52:GUS pollen and performed histochemical GUS staining at 12 HAP.At 12 HAP,81.1%ovules in wild type while 47.4%in hen1-8 were targeted by a pollen tube.Furthermore,we pollinated hen1-8pistil with wild type pollen and analyzed by aniline blue staining at 48 HAP.At 48 HAP,96.6%ovules in wild type while 56.5%in hen1-8 developed.These results suggested that Defective ovules of hen1-8 failed to attract pollen tubes,leading to reduced female fertility.?4?HEN1 mediates the expression of ARF6 and ARF8 through miR167HEN1 is critical for the production of many microRNAs,among which miR167 was reported to mediate ovule development.Ectopic expression of ARF6 and ARF8,the target genes of miR167,in integument resulted in defective ovule development,To test whether ARF6 and ARF8 were ectopically expressed in hen1-8 due to reduced miR167 accumulation,we examined the expression of ARF6 and ARF8 by quantitative PCRs and RNA in situ hybridization.Indeed,both genes were significantly upregulated in hen1-8 ovules compared to that in wild type.In addition,both genes were ectopically present in hen1-8 integuments.These results were consistent with our hypothesis that HEN1 mediates the expression of ARF6 and ARF8 through miR167 in integument cells during ovule development.