Phenotype Analysis And Map-based Cloning Of Root Architecture Mutant Raal-1Regulated By ABA In Arabidopsis

Plant roots recently become to be a good material for the study of plant developmental plasticity andan new model to study the regulatory mechanism of ABA, with its relatively simple structure andextremely complex configuration. ABA is a lively and complex plant hormone, not only plays crucial rolesin various physiological processes during the plant life cycle, but also widely involved in biotic and abioticstress responses. Compared with auxin, another plant hormone, the study about the regulated of ABA onplant root configuration is relative lack. Although recent genetic and molecular studies started to unravelthe importance of ABA regulation in root meristem formation and root growth, the role of abscisic acid(ABA) in the regulation of root growth is still not completely clear. Therefore, we assume that it is possibleto research the role of ABA in the formation of plant roots use methods of reverse geneticsWe screened a mutant raa1-1by ABA which phenotype is short primary root and little lateral rootsfrom EMS mutagenesis of Arabidopsis. Our study shows that: Exogenous ABA can partially restored thephenotype defects in root configuration of mutant raa1-1, the difference between WT and raa1-1inprimary root length and lateral root number can be reduced by the treatment of ABA. Through the analysisof the impact of other kinds of treatment on the root configuration, we found there is no significantdifference between raa1-1and WT in the change of root conformational under the treatment of droughtstress, oxidative stress and kinds of hormones. The results indicate that the response of raa1-1to ABA isrelatively specific in root configuration. We also found that high concentrations of exogenous sugartreatment can restored the phenotype of raa1-1in the primary root and lateral root. The experiment of ABAclassic reaction about germination, dehydration, and infrared index revealed that the germination of raa1-1was retarded, the dehydration of raa1-1was faster and the temperature of leaves in raa1-1was lower inthe normal state, which suggested that: the physiological process regulated by ABA are differ in the mutantraa1-1. In addition, our study showed that raa1-1were yellow-green and also remained pale greenthroughout its development. All the phenotypes of raa1-1provide a reliable basis for analysis the geneticbackground of mutant and choose the materials for map-based cloning.The experiment of backcross between raa1-1and WT revealed that raa1-1is a single gene recessive mutant. The positional cloning results show that: the phenotype of raa1-1is due to early termination ofRAA1by a base mutation in the coding region of gene RAA1. The mutant gene RAA1encoding apseudouridine synthase, the enzyme located in the chloroplast and other plastid, not only played a role inuridine isomerization but that its physical presence was necessary for proper chloroplast rRNA processing.We identified an Arabidopsis T-DNA insertion homozygous mutant raa1-2and found the phenotype ofraa1-2was accord with raa1-1, this result further illustrated the mutant phenotype was caused by theATRAA1gene mutation. We also constructed the super-expression vector of RAA1, and obtained thetransgenic Arabidopsis seedlings by Agrobacterium-mediated transformation method. The transgenic plantswill provide more information to understand the function of raa1-1.In conclusion, this experiment illustrates that ABA involved in the regulation of the mutant raa1-1root configuration, besides, sugar may take part in the process. The mutant raa1-1is caused by single generecessive mutation, the gene RAA1in the mutant encoded a incomplete protein without biological activity.The lack of pseudouridine synthase will infect the normal metabolize of rRNA, such as processing, splicing,stability and degradation, which may result in various physiological processes regulated by ABAabnormality, thereby bring raa1-1with a series of ABA-related phenotype.