Physiological And Genetic Mechanism Of Low-Boron Tolerance Mutant Lbt And Cloning Of The Candidate Gene In Arabidopsis Thaliana

Boron（B）is an essential micronutrient for the growth and development of plants,which is widely involved in diverse physiological and biochemical processes.At present,several genes related to B uptake and transport have been identified from many plant species,but the molecular mechanism underlying plant to low-B tolerance（LBT）remains unclear.On the basis of previously identified Arabidopsis lbt mutants,we studied the plant growth,root morphology,B uptake and accumulation,changes of phytohormone,starch and callose accumulated,systematically,Analysis the location and clone of the low-B tolerane genes,and research the function of the candidate gene.The main results are as follows:（1）Physiological mechanism underlying tolerance of the lbt mutant to low-B stressIdentification of phenotype of lbt mutant under B deficiency by solid plates system.Compared with Col-0,shoot fresh weight and primary root length of lbt mutant were significantly higher and longer.In addition,the leaf area of the lbt mutant was remarkable larger than that of Col-0.The primary root length,root tip number and none-root-hair-zone length of lbt mutant is larger than those of Col-0,however,the root hair density of lbt is less than that of Col-0.Under the dark culture condition,the hypocotyl length of lbt was also significantly longer than that of Col-0.There is no remarkable difference in the expression of the genes responsible for efficient B uptake and efficient B concentrations between Col-0 and lbt under both normal low B conditions.The ABA concentration of the lbt mutant root was significant higher than that of Col-0 under B deficiency.lbt has less callose in roots and fewer starch grains in leaves under low-B condition.These results suggested that the mechanism underlying low-B tolerance in the lbt mutant is not related to B uptake and transport,and it may be caused by changes in the metabolism of callose,starch and ABA.（2）Location of QTL for low-B toleranceThe lbt mutant and Cs1909（RILs）are used as the parents to develop a F₂ mapping population.The results indicated that the lbt phenotype under the control of a monogenic recessive gene using the primary root length as phenotype index.A total of102,283 aa×bb-type SNPs between Cs1909 and lbt were obtained using whole genome sequencing（WGS）and determined 620 bin markers for linkage map construction.Further,we constructed a linkage map that included four linkage groups.QTL analysis identified one QTL for root length under low-B condition.This QTL for low-B tolerance was physically located in the region of 10.4-14.8 Mb on the Chr.4,which explained as high as 75.1%phenotypic variations of low-B tolerance.There are 1,324genes in this region,but no reported genes related to B uptake and transport are observed.（3）Cloning of candidate genes underlying low-B toleranceThe candidate genes were predicted with the MutMap method.The F₂ individuals form lbt mutant crossed with Col-0 to construct a LBT（Low-B Tolerance）-bulk and a LBS（Low-B Sensitive）-bulk.Only one SNP（SNP-Index=1）was identified by WGS,and this SNP was located on the QTL for low-B tolerance.And this SNP was physically mapped onto the exon of At4g19180（APY7）,which encodes an Apyrase,and caused a conservative cysteine（C）mutating into arginine（R）.The mutation induced changes of the physical and chemical properties and the tertiary structure of the APY7 protein.（4）Verification of APY7 gene functionVerification of the function of APY7 using the T-DNA insertion mutant of APY7（apy7-1）,p APY7:APY7/lbt lines（restorer lines）,35S:APY7 lines（overexpression lines）and p APY7::GUS lines.Under low-B condition,the phenotype of restorer lines was similar to Col-0,showing greater low-B sensitivity,and the phenotype of apy7-1 mutant were similar to the lbt mutant,showing low-B tolerance.It suggested that the APY7 is the target gene that causes the low-B tolerance of the lbt mutant.The RT-q PCR assay result indicated that APY7 is expressed in the shoot and root,especially in the root tip,and it is not transcriptionally regulated by B condition,which was similar to the change of callose.APY7 has low similarity in amino acid sequences with other APY members.The apy1-1 mutant showed greater sensitivity to B deficiency than Col-0 did.Therefore,the APY family may be related the mechanism of plant response to B deficiency.In this study,we characterized an Arabidopsis mutant lbt specifically tolerant for low-B,indicate that the phenotype of lbt is not dependent on B uptake and transport,which is controlled by a monogenic recessive gene.The low-B tolerance gene APY7encodes an apyrase,the role of which in low-B tolerance may depend on the synthesis of callose.