Cytokinin Oxidase/Dehydrogenase 11 Biological Function Resarch In Rice

Cytokinin（CK）is an important hormone regulating the normal growth and development of plants,which plays an active role in delaying leaf senescence and regulating crop yield.Cytokinin oxidases/dehydrogenase（CKX）is a specific degradation enzyme in the oxidative pathway of cytokinin in plants.Regulating the expression of CKX gene can significantly regulate the level of endogenous CK in plants,thus affecting the normal growth and development of plants.There are at least 11 members of the rice（Oryza sativa）cytokinin oxidase OsCKXs family,but the OsCKX gene related to senescence has not been reported.In this study,based on the obtained rice OsCKXs family mutants,the rice mutant osckx11 was screened by reverse genetics,which is closely related to leaf senescence and rice yield.Its gene function was studied by means of molecular biology,physiological and biochemical techniques.The specific research results are as follows:（1）OsCKX11 has the function of regulating leaf senescence.The leaves of all the mutants of OsCKXs family members of rice were treated in vitro and the plant phenotypes were observed.It was found that the leaves of osckx11 mutant had the delay leaf senescence phenotype,and the chlorophyll content,Fv/Fm value and net photosynthetic rate of the mutant leaves were significantly higher than those of the wild type plants in the same period.In addition,the results of DAB and NBT staining showed that the content of reactive oxygen species in the leaves of the mutant was lower than that of the wild type.（2）OsCKX11 has the function of regulating the grain number.The panicle of osckx11mutant growing normally in field were larger.The grain number per panicle increased by14.1-28.7%,the grain number per plant increased by 20.4-26.1%,and the number of primary branches increased by 19.1-22.5%.Meanwhile,the in situ hybridization experiment also showed that the expression of the gene was high in the primary branch primordia and floret primodia.Although the seed setting rate of the mutant decreased slightly,the yield per plant of the mutant increased about 10.6-15.9%compared with that of the wild type.（3）Expression pattern of OsCKX11 and protein subcellular localization.In order to further clarify the function of Os CKX11 gene,the expression pattern of OsCKX11 gene was analyzed.The results showed that the expression of Os CKX11 gene was the highest in the early senescent leaves and 15 cm panicle,followed by the late senescent leaves and roots,and the lower in the non senescent leaves and stems.The expression of OsCKX11gene was the highest in the middle of the whole flag leaf of rice.In addition,OsCKX11 is the only member of the family without signal peptide.Rice protoplast experiments show that it is located in the cytoplasm.（4）In vitro enzyme activity of OsCKX11.OsCKX11 protein was recombinant expressed and purified in vitro.Seven different forms of CKs were used as substrates for enzyme activity in vitro.The amount of substrate consumed before and after enzyme activity was detected by HPLC,and the rate of enzyme activity was calculated.The results showed that OsCKX11 was active to seven forms of CKs,but highest activity to cis-Zeatin（cZ）and trans-Zeatin（tZ）.The results showed that the Km value of tZ was 36.07±2.855μm,the K_catat value was 0.64 s^-1,the K_m value of cZ was 28.35±5.701μm and the K_catat value was 0.51 s^-1.Therefore,OsCKX11 has higher affinity to cZ than tZ,but higher conversion rate to tZ than cZ.（5）Determination of cytokinin content in osckx11 mutant.The content of CK in the flag leaf of osckx11 mutant was detected by liquid chromatography-mass spectrometry.The content of cZ,tZ and isopentenyl adenine（iP）in the flag leaf of osckx11 mutant was significantly higher than that of the wild type,and the seven forms of CKs in the early leaf of osckx11 mutant were all increased,especially tZ,cZ and iP.（6）Function of OsCKX11 in delaying leaf senescence is closely related to abscisic acid（ABA）.Hormone treatment experiments showed that OsCKX11 was induced by ABA.The content of ABA in the leaves of osckx11 mutant and wild type plants was determined.The ABA content in the leaves of the mutant was significantly lower than that of the wild type plants.The results of ABA metabolism related gene expression showed that the expression of OsNCED gene about rice ABA synthesis in the mutant was significantly lower than that in the wild type,while the expression of OsABA8ox gene about rice ABA degradation in the mutant was decreased.In summary,OsCKX11 not only acts on the"source"leaves of rice,lack of function delays leaf senescence,prolongs photosynthetic time and increases photosynthetic rate of rice,but also enhances the"sink"capacity of rice,and significantly increases the grain number and the yield per plant of rice.In addition,We also found that ABA is involved in OsCKX11 regulation of leaf senescence.The gene itself is induced by ABA,and it can also regulate the endogenous ABA level of rice.Therefore,the study on the biological function of OsCKX11 will help to further reveal the mechanism of CK delaying leaf senescence,and provide the theoretical basis for the cultivation of high-yield rice in the future.