The Role And Regulatory Mechanism Of Cytokinins In Wheat Stay-green Mutant Tasg1

Wheat is cultivated as one of the most significant food crop in China.The yield of wheat has had great increase in recent decades,but the agricultural land is occupied by industry,and the global population is increasing rapidly.Therefore,the increase of productivity under constrained agricultural land condition has become an important way to solve the food crisis.Premature senescence has become an important factor affecting wheat yield,so delaying or preventing premature senescence is one of the important ways to increase yield.The wheat stay-green mutant can’t degrade or degrade chlorophyll very slowly at the late growth stage,and still maintain high photosynthesis capacity.Therefore,delaying senescence is a useful way to increase photosynthetic time,which is beneficial to the improvement of wheat yield.The wheat stay-green mutant,tasg1,was previously generated via mutation breeding by applying the ethyl methane sulphonate（EMS）mutagen to HS2,a common wheat cultivated as the WT.Previous studies revealed that tasg1 was a functional stay-green mutant with delayed senescence.Moreover,the stability of proteins in thylakoid membranes,photosynthesis,antioxidant capacities of tasg1 were consistently higher than that of the WT during delayed senescence under drought stress.At the same time,the increase of tillering ability of tasg1 suggested that cytokinin（CK）metabolism was altered of the mutant tasg1plants.In this paper,we used WT and tasg1,as material to study the role and regulatory mechanism of cytokinin in wheat stay-green mutant.The main results are as follows:（1）The relationship between stay-green phenotype of wheat mutant tasg1 and CK metabolism.In the early seedling stage,the number of lateral roots in tasg1 was lower than that of WT seedlings.However,the length of the lateral roots in tasg1 was longer.These results suggested the changed CK metabolism in tasg1.Compared with WT,the content and active CK levels were significantly higher in tasg1 plants.At the same time,the expression levels of cytokinin synthesis-related genes（TaIPTs）were significantly increased,but cytokinin oxidase genes（TaCKs）were significantly decreased in tasg1.Furthermore,WT and tasg1 was treated with lovastatin,a kind of CK inhibitor.It was further proved that tasg1 was more sensitive to the treatment of lovastatin.These results suggested that the metabolism of CK was changed in tasg1.The stabilities of chlorophyll and thylakoid membrane protein complexes in tasg1 were also severely damaged by lovastatin compared to WT.The expression levels of TaSAGs further supported this hypothesis.At the same time,the expression levels of senescence related genes（TaSAGs）were significantly up-regulated,the protein carbonylation was increased,and the antioxidase activities were significantly decreased in tasg1.These results suggested that the altered CK metabolic played an important role in the delayed senescence of tasg1.（2）The relationship between grain development and sucrose metabolism in tasg1.In the present study,it was found that delayed senescence was accompanied by significantly higher grain volume in tasg1 at the early filling stage.However,at the late filling stage,the grain volume of tasg1 was lower than that of WT.Compared with WT,the photosynthesis capacity,and soluble sugar accumulation in flag leaves of tasg1 were significantly increased at the late filling stage.The levels of CK were higher in the grains of tasg1 plants at the early filling stage.At the same time,the expression levels of putative wheat orthologs of cell cycle genes during mitosis were significantly higher in tasg1 plants.Moreover,the CW-invertases,SS activity,and soluble sugar accumulation in grain of tasg1 were significantly increased at the late filling stage.These results suggested that CK could be involved in the larger grain volume of tasg1 by regulating mitosis process at the early filling stage.At the late filling stage,Suc content in the leaves of tasg1 was higher than that in WT because of its greater photosynthesis capacity,However,excess Suc was decomposed into Glu and Fru by invertase,following which the soluble carbohydrate was retained in different tissues,rather than in the grains at the late filling stage.This resulted in the stay-green phenotype and higher biomass but it was responsible for smaller grain volume in tasg1.The invertase activity regulated by CK was directly or indirectly involved in Suc remobilization and the stay-green phenotype of tasg1.The ineffective conversion from soluble sugars to starch might be involved in the smaller grain size in tasg1.（3）The relationship between stay-green phenotype and CK,N metabolism in tasg1.In the present study,it was found that tasg1 had obvious delayed-senescence phenotype at the late filling stage.At the same time,the contents of CKs and total N in the tasg1 were significantly higher than those in the WT plants.RNA-seq further demonstrated that,less genes participated in CK and N degradation metabolic pathway in tasg1 at the late filling stage.This was the important reason for the increases of CK and N in tasg1 at the late filling stage.Both WT and tasg1 treated with NH₄NO₃ showed a delayed senescence phenotype,and not only the content of total N,but also the CK had significantly increase in WT and tasg1.Meanwhile,after treated with lovastatin,a cytokinin inhibitor,tasg1 showed a premature senescence phenotype,and both CK and total N contents were significantly decreased in tasg1 plants.These results suggested that CK interact with N in the process of delayed flag leaf senescence of tasg1（4）The relationship between stay-green phenotype and cisZOGT1 in tasg1.RNA-seq and qRT-PCR data showed the expression of cisZOGT1 in tasg1 was significantly lower than that of WT（about 14%）.cisZOGT1（Traes₂AL₂1065D298）encoded a cis-zeatin O-glucosyltransferase 1.The results of qRT-PCR further proved the lower expression of cisZOGT1 in tasg1 than its WT.A knock-out of the cisZOGT1-B locus was screened from a durum wheat mutant pool‘Kronos’.Therein,mutant 2885 lines exhibited delayed senescence at the late filling stage.Moreover,the contents of total ZR,DHZR,and N in the mutant 2885plants were also higher than that of its WT.Then,cisZOGT1 gene was overexpressed in Brachypodium plants to examine its functions.The transgenic lines overexpressing cisZOGT1also exhibited delayed senescence phenotype at the late filling stage.However,the contents of ZR and DHZR in OE lines were lower than that of its WT.Cotton seedling and cotyledon were used to estimate the active CKs levels in Brachypodium OE lines and WT plants.Compared with WT,the overexpression of cisZOGT1 significantly decreased the active CKs levels.These results suggested CKs and N levels regulated by cisZOGT1 were important to the delayed senescence of plants.Active CKs,as well as the senescence,was perturbed in over-expressing cisZOGT1 plants,but the mechanisms underlying were intricate.In conclusion,the altered CK metabolic played an important role in the delayed senescence of tasg1.At the same time,CK metabolism could regulate delayed senescence of tasg1 at the late filling stage through interacting with N metabolism and regulating sucrose metabolism.