Map-based Cloning And Functional Analysis Of The Ca²⁺/H⁺ Exchanger Gene CAX1a In Rice

The panicle apical abortion（PAA）mutant reduced the yield by affecting the development of the apical portion of panicle in rice.In recent years,many genes related to apical spikelet degeneration have been cloned in rice,however the mechanism is not clear.In this study,virescent and panicke abortion 1（vpa1）was isolated from IR64-EMS mutant bank.Normal leaf color mutants but with degenerated phenotype at the top of panicle,selected from hybrid progenies of vpa1 named panicle apical abortion10（paa10）was used.We performed phenotype characterization,physio-biochemical and genetic analysis of paa10.We also cloned the target gene OsCAX1a which controlling paa10 mutation and revealed that OsCAX1a is involved in ion transport.The main conclusions are as follows:1.Under field conditions,paa10 showed obvious leaf-tip necrosis during seedling stage.In heading stage,apical portion of panicle from paa10 was obviously aborted accompanied by a significant decrease in plant height,panicle length,number of grain per panicle and 1000-grain weight.By observing young spikelet at different stages,it was found that the top spikelet began to appear abortion at 8cm.With the ROS content increased and the ROS scavenging enzyme activity decreased significantly,a large number of cell death were found on the tip of leaves and degenerated spikelet in paa10 by trypan blue staining and comet assay the.2.By map-based cloning,the PAA10 was located in the 190kb between RM11157 and RM5972 on the short arm of chromosome 1.By sequence analysis between paa10 and wild type,it was found that the 60th base of exon 4 of LOC＿Os01g37690 was replaced by G→A,which led to the substitution of alanine to threonine.Both complementary and knockout experiments confirmed that the phenotype of leaf-tip necrosis and PAA was caused by the mutation of LOC＿Os01g37690 which is predicted to encode Ca²⁺/H⁺exchangerCAX1a and is preferentially expressed in rice leaves and panicle.OsCAX1a is located in both cell membrane and vacuole membrane.3.The contents of Ca,Mn,Zn,Cu and Fe in leaves and seeds of WT as well as paa10 were measured.The results showed that only the element content in the upper leaves and whole panicle from paa10 were significantly lower compared with wild type indicating that the phenotype of leaf-tip necrosis and PAA may be caused by ion deficiency;we finished the hydroponic experiment during seedling stage and the results showed that the leaf-tip necrosis from paa10 growing in the nutrient solution was obviously alleviated,and chlorophyll and active oxygen index restored to normal;At the same time,we also transferred the wild type and paa10 cultivated in soil for about 30 days to hydroponic culture,and found that the phenotype of leaf-tip necrosis and PAA of paa10 were greatly alleviated on the 60th day and after heading4.OsCAX1a can be induced by exogenous Ca²⁺.During the seedling stage,the Ca²⁺gradient experiment showed that the expression level of OsCAX1a from WT increased obviously with increasing exogenous Ca²⁺,but compared with WT,paa10 showed obvious sensitivity when there was high concentration of Ca²⁺.