| Rice(Oryza sativa L.)is an important model plant for the study of plant functional genomics,and also the major staple food for more than half of the world's population.At present,the production of rice is still unable to meet the population growth needs of the world.The main factors affecting rice yield are various genetic and environmental factors,such as biological and abiotic stresses.The defect of pollen development is closely related to the decline of rice yield.It is crutial that characterizting the function of genes involved in male reproductive development for improving rice yield.The leaf senescence is an important stage of plant growth and development.The premature senescence of leaves in the late growth stage of rice affects the grain filling seriously and results in the drop of seed setting rate.The Argonaute(AGO)protein family is one of the executor of in the epigenetic regulation of gene expression.Although there are 19 AGO proteins in rice,most of them have not been described functionally.In this study,in order to clarify the function of Os AGO2,we obtained the transgenic rice plants with decreased expression of Os AGO2 by antisense RNA technology and CRISPR/Cas9 technology.Through phenotypic observation,bioinformatics analysis,genetic and cytological analysis and epigenetic analysis,we revealed the function and molecular mechanism of Os AGO2 in regulating rice pollen development and leaf senescence.The main results are as follows:1.Sequence analysis of the PAZ and PIWI domain of AGO2 proteins of rice and other eukaryotes indicated that the PAZ and PIWI domains of AGO2 are highly conserved.And the monocots rice and maize share high sequence homology,indicating that AGO2 s from different species may have conserved functions.2.The expression profile of Os AGO2 was analyzed by q RT-PCR,RNA in situ hybridization and GUS staining.The results showed that Os AGO2 was highly expressed in developing anthers before stage 12 and in flag leaves.3.Using antisense RNA and CRISPR/Cas9 technology we obtained Os AGO2 down-regulated transgenic rice lines and two different lines(ago2-1 and ago2-2;Casago2-1 and Casago2-2)were selected for futher study respectively.Compared with the wild type Zhonghua 11(ZH11),the four rice lines with decreased expression of Os AGO2 showed the significantly decreased seed setting rates,shortened and curly stamens with defective pollen grains.And the transgenic plants also exhibited delayed growth,and early senescence of flag leaves during the flowering stage to grain filling stage.4.Cytological observation showed the vacuolated and abnormally degradated tapetum cells in Os AGO2 down-regulated lines.TUNEL analysis showed that the PCD signal of the tapetum was started earlier and more continuous in Os AGO2 down-regulated plants than that in wild type.The accumulation level of reactive oxygen species(ROS)was quite high,and the dynamic change of ROS was disturbed during the development of anther,which correlated with the dynamic changes of the abnormal PCD signals in tapetum.5.The Os AGO2 down-regulated lines displayed significantly decreased chlorophyll content and defective chloroplast thylakoid structure,such as irregular and loosed stroma lamellae and grana lamellae,which affected the normal development and function of chloroplast.In Os AGO2 down-regulated lines,the signal of DNA fragment rupture appeared during leaf development,and the PCD process was started earlier compared with that in wild type plants.The ROS levels in leaves were also increased significantly,which may have a damaging effect on chloroplast structure;and the chloroplast structure defects may cause the delayed elimination of the extra intracellular ROS.The excessive accumulation of active oxygen also resulted in an early PCD process in leaves,which caused the premature leaves.6.A candidate target gene,i.e.Os HXK1(Os07g0446800)regulated by Os AGO2 was screened out by gene chip analysis.The expression of Os HXK1 was upregulated in Os AGO2 downregulated plants,but not detected in wild type.An overexpression vector of Os HXK1 was constructed and introduced into rice,and the resulting transgenic plants(OEHXK1)showed similar phenotypic changes to that of Os AGO2 down-regulatd lines.And the above results indicated the suppression of Os HXK1 expression is very important for the ROS level control and the the occurrence of tapetum PCD;and the normal development of microspores and leaves was nagetively regulated by Os HXK1.Downregulation of Os HXK1 in ago2-1 resulted in the rice plants with normal phenoyepe similar to wild-type plants,which indicating that Os AGO2 participates in the regulation of Os HXK1 expression.The DNA methylation level of Os HXK1 promoter region was changed compared with that in wild type plants,and chromatin immunoprecipitation assay showed that Os AGO2 could bind to Os HXK1 promoter to regulate Os HXK1 expression through DNA methylation.7.The expression of the characterized key genes in tapetum development were down regulated in ago2-1,Casago2-1 and OEHXK1-1 anthers,and the mutants of these key genes showed delayed tapetum degradation and delayed PCD signal leading to pollen abortion.The above results suggested the downregulation of Os AGO2 may affect these genes of tapetum development in some way.The expression of RBOH family genes associated with ROS generation in plants were increased in Os AGO2 downregulated lines and Os HXK1 overexpression lines,which indicated that the increased expression of RBOH family genes disturbed the dynamic balance of ROS in both anthers and leaves,and the dynamic balance of ROS ensures the timely PCD occurrence in tapetum and leaves.Our results show that Os HXK1 is a key regulator of ROS level and PCD process in rice anther and leaf development.Os AGO2 could suppress Os HXK1 expression through DNA methylation and regulates the dynamic balance of ROS,which ensures normal PCD process of tapetum and leaves.Our results reveal a Os HXK1-dependent patyway to regulate ROS level in rice. |
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