Functional Analysis Of The Mediator Complex Subunit OsMED16 Gene In Rice

Rice is an important food crop in our country.Photosynthesis is the basis of rice survival,providing it with the material and energy it needs.The development of leaves directly affects the rate of photosynthesis and the growth of plants.Therefore,we must realize that leaves are important for rice development.The Mediator is an evolutionarily highly conserved protein complex,which is also an important cofactor.It mainly connects transcription factors and RNA polymerase II and acts as an important bridge of information transmission and participates in regulating the expression of important genes during plant growth.The Mediator complex consists of four parts: head,middle,tail and kinase.The head and middle module mainly bind RNA polymerase II.The kinase module plays positive and negative regulatory roles in transcription and the tail module mainly binds to specific transcription factors.MED16 belongs to the tail of the intermediate complex and plays an important role in plant growth and development and abiotic stress.In the dicotyledon model plant Arabidopsis thaliana,MED16/SFR6 is a cold-sensitive gene,which not only participates in plant growth and development,but also controls the signaling pathways mediated by salicylic acid and jasmonic acid,which mainly affects the expression of defense genes related to these two signaling pathways.However,there are few studies on OsMED16 in monocotyledon rice.Therefore,it is of great practical significance to clarify the expression pattern and biological function of OsMED16 gene in rice.In this study,OsMED16 is the tail structure of the Mediator complex subunit in rice.We constructed osmed16 knockout mutant by CRISPR/Cas9 gene editing techniques and OsMED16 overexpression plants,and utilized these transgenic and wild-type lines to analyze the OsMED16 expression pattern,the subcellular localization,plant physiological phenotypes and the structure of the blade to illustrate OsMED16 biological function in the rice.The main experimental results of this study are as follows:(1)OsMED16 was expressed in both roots and stems during the seedling stage and the expression level in roots was relatively higher.OsMED16 was expressed in roots,stems,leaves,panicles and leaf sheaths at booting stage and the relative expression levels were higher in stems and panicles.GUS staining analysis showed that OsMED16 was mainly expressed in roots and not detected in stems at the 7th and 15 th days of seedling stage.OsMED16 was mainly expressed in leaves in the48 th day..(2)The transient expression of OsMED16 in rice protoplasts showed that the green fluorescence of OsMED16-GFP could be completely fused with the red fluorescence of nuclear marker protein,indicating that OsMED16 was located in the nucleus.(3)The homozygous osmed16 mutation resulted in the death of rice seedlings,so further studies could not be carried out and OsMED16 overexpression plants were constructed.Phenotypes analysis showed that OsMED16 overexpression plants showed brown spots on leaves and the related agronomic traits such as tiller numbers,seed setting rate,primary and secondary branches and stems were significantly reduced,which ultimately resulted in a very significant decrease in rice yield.(4)This study also analyzed the transcriptome information of OsMED16 overexpression plants and wild-type Nipponbare before and after brown spots appeared in leaves,and the results showed that OsMED16 overexpression resulted in changes in the expression of a large number of genes,especially those related to defense-related genes.(5)The leaves of wild-type,osmed16 mutant and overexpression plants were observed by fluorescence microscopy and transmission electron microscopy.The result found that the leaf lesions of osmed16 mutant and overexpression plants could lead to premature senescence of leaves.(6)Histochemical staining analysis showed that there were a lot of reactive oxygen species in osmed16 mutant plants and the overexpression plants.That is to say,the leaves of transgenic plants showed premature senescence.(7)Different concentrations of Fe were applied to OsMED16 overexpression plants and wild-type plants cultured in hydroponic culture.It was found that brown spots on leaf surface decreased with the increase of Fe concentration,indicating that Fe concentration had a certain influence on the degree of leaf damage.(8)Both yeast two-hybrid and bimolecular fluorescence complementation(Bi FC)assays revealed that OsMED16 and OsMED25 can interact with each other,and OsMED25 plays an important role in the regulation of iron homeostasis,These results suggest that OsMED16 may also be involved in iron homeostasis regulation through interaction with OsMED25.In conclusion,the growth of osmed16 mutant plants were severely inhibited and could not complete the life cycle.Overexpression of OsMED16 plants can also lead to inhibited growth,spontaneous cell death and low yield of rice.At the same time OsMED16 overexpression altered the expression of a large number of genes involved in multiple biological processes,which has a profound impact on the growth,development and defense of rice.