Map-Based Cloning And Functional Study Of Albinic Leaf And Retardation Mutant Alr In Rice (Oryza Sativa L.)

Development of living organisms depends on the faithful delivery of genetic information over many generations.A sedentary lifestyle subjects plants to constant exposure to endogenous and exogenous mutagens,including abnormal DNA replication,arrested cell cycle,UV irradiation and reactive oxygen species.Damaged DNA change genetic information,and wrong DNA would lead to mutant,infertility and death.Cells have evolved multiple mechanisms for maintaining high fidelity DNA replication despite constant challenges from endogenous metabolism products and exogenous mutagens.The key elements for genome surveillance are dNTPs:dATP,dGTP,dTTP and dCTP,which are the building blocks of DNA required for DNA replication,recombination and repair events..Hence,the formation and regulation of dNTP pools are crucial for normal cell development.In contrast to yeast and mammalian cell lines,our knowledge of DCD function and regulation in plants is limited.In this study,we identified and characterized a rice dNTP pool-disturbed mutant,albinic leaf and growth retardation(alr),which exhibits arrested chloroplast biogenesis and reduced cell number.We demonstrated that ALR functions as a DCD by in vitro enzyme activity assays,and functional analysis of alr prompted us to understand the genetic effect and molecular mechanism of mutant phenotype.The main results are as follows:1.alr mutant exhibits pleiotropic developmental defects.The alr mutant had near-albinic to virescent leaves,reduced stature,and necrotic spots.The chlorophyll a,chlorophyll b contents and plant height at different stages were less than those in the wild-type.The chloroplasts of alr plants were smaller and undifferentiated similar.The decreased expressions of chloroplast formation and function related genes were effect normal chloroplast development in alr mutant plants.Microscopy revealed that the shorter plant of alr mutant resulted from reduced cell number rather than cell length.The alr mutant showed changes in agronomic traits,indicating that the alr mutation conferred pleiotropic developmental defects.2.ALR gene encodes a typical DCD protein that is expressed in all tissuesGenetic analysis showed that the albinic leaf phenotype of alr mutant is controlled by a single recessive nuclear gene.Using the map-based cloning way,we cloned ALR.Complementation and RNA-interference tests comfirmed that Os01g0765000 is the ALR gene.We demonstrated that ALR functions as an OsDCD by in vitro enzyme activity assays.OsDCD was expressed ubiquitously in all tissues,and OsDCD protein was located to cytoplasm.3.OsDCD regulates the production of dTTP and maintains the balance of dNTP pool.We report that alr plants have typical DCD-mediated imbalanced dNTP pools with decreased dTTP.The dTTP level was reduced by 0.71-fold,whereas dCTP increased 3.31-fold.All genes known to accelerate dTTP formation were up-regulated in the dTTP salvage pathway.Application of exogenous dTTP recovered the wild-type phenotypes.4.Lack of OsDCD induces DNA damage and programed cell death(PCD)symptom.Comet assays and trypan-blue staining showed that OsDCD deficiency causes accumulation of DNA damage in alr mutant with leaf aging,sometimes leading to cell apoptosis.The degree of DNA damage in the top-fourth leaves in the mutant was more severe than that in the wild-type as well as the flag leaf of alr mutant,and many DNA damage checkpoint and repair response genes were also activated in alr plants.The high frequency of HR in alr indicated limited DSB repair,suggesting that alr cells undergo hypersensitive PCD because of the accumulated DNA damage.5.Decreased dTTP level affects nuclear and chloroplastic replication in plant development.The shorter stature of alr plants was due to reduced cell numbers and the mitotic index was decreased.The alr mutant had reduced 4C cell numbers determined by flow cytometry,suggesting less mitotic activity and cell division.The expressions of cell cycle-related genes were changed,suggesting an arrested cell cycle at the G1/S phase in alr mutant.Together with the expressions of nuclear and chloroplastic genome replicate-genes were down-regulated,thus demonstrated both nuclear and chloroplastic replication were blocked.