The Mechanism Analysis Of Differential Expression Of Rice OsAPS2 Between Superior And Inferior Spikelets

Rice spikelets can be classified as superior or inferior according to their location on a branch and time of flowering.Generally,the superior spikelets locate on the apical primary branches,while the inferior spikelets on the proximal secondary branches.And,by comparison,the former flower earlier and fill faster with larger and heavier grains than the latter.In addition,the inferior spikelets are more sensitive to the environmental factors than the superior spikelets.However,to our knowledge,information is not available on the different responses mechanisms of superior and inferior spikelets in response to the environment.In present study,the spatio-temporal expression differences of small subunit of AGPase(APS2)protein between superior and inferior spikelsts were studied and the interaction network of APS2 was built by the co-immunoprecipitation,aiming to reveal the asynchronism filling of superior and inferior spikelets.First of all,the dynamic changes of grain filling between superior and inferior spikelets under the modeate soil drying were investigated,and the results showed that the superior spikelet was stable while the inferior spikelet changed obviously.Then,the RT-PCR technique was used to analyze the changes in the transcriptional level of OsAPS2 between superior and inferior spikelets at different phase of grain-filling,and found that the expression level of OsAPS2 in superior spikelets was greater than the inferior spikelets.At the same time,the gene did not change significantly in the superior spikelets under modeate soil drying,while it appeared significantly in inferior spikelets.Then we made the APS2 protein polyclonal antibody and used the western-bolt technology to evaluate OsAPS2 expression changes in protein level.The results were the same with RT-PCR,confirming that the expression trends of OsAPS2 in superior and inferior spikelets was related to the asynchronous grain filling between superior and inferior spikelets.On this basis,we performed the Co-Immunoprecipitation(CO-IP)and identified 29 protein that specifically bind to the APS2 protein.The identified protein have diverse biochemical functions and cellular roles,including starch synthesis(3),carbohydrate metabolism(2),glycolysis(5),8 protein synthesis and destination(8),TCA cycle(1),amino acid metabolism(3),signal transduction(2),defense response(4),and transporter(1).Further analysis showed that,APS2 can interaction with other starch synthesis related protein then aggregation together form a starch synthesis proteins complex(SSPC),and then regulate the starch synthesis process of rice spikelet.In addition,this study also identified 2 key signal regulator proteins,that is 14-3-3 family proteins(GF14E and GF14F),which were considered to be importantly in control of the forming process of SSPC.In the meantime,the interaction between 14-3-3 protein(GF14E and GF14F)and APS2 protein werer verifyied by the GST pull-down experiment.Finally,the RT-PCR was used to examin the OsGF14E and OsGF14F in superior and inferior spikelets in response to modeate soil drying,and the results show that these two genes had no significant changes in superior spikelets while significantly increase in inferior spikelets in different grain filling stage,which was the same with OsAPS2 variation trend in superior and inferior spikelets.Therefore,the present study proved that the 14-3-3 proteins(GF14E and GF14F)were the important signal to the environment factor which was mediated by the different response mechanism of superior and inferior spikelets starch synthesis.The present study provide a better understanding of the asynchronous filling of superior and inferior spikelets,which also expected to be highly useful for improving the grain filling of rice.