The Mechanisms Of Increased Drought Tolerance Caused By Idr1 Mutation In Iapar9 (Oryza Sativa L.)

Rice is one of the most important crops in the world.As a crop that highly depends on water,rice yield and quality are greatly affected by soil moisture conditions.However,drought happens more frequently with the growing global warming,which have a significant impact on rice production.Cultivating new drought-resistant rice varieties is considered to be a quite effective way to deal with the drought disaster.The advances in molecular biology in recent years promoted a change of rice breeding from traditional hybrid breeding to a combination of the traditional hybrid breeding with molecular biology-assisted rice breeding,which involves cloning of drought-resistant genes and makes them used for transgene,gene editing and marker-assisted selection in order to accelerate the rice breeding.This approach relies on the development of molecular markers,as well as the understanding of drought-resistant genes or QTLs.At present,there are two ways to clone drought-related genes:(1)clone drought-resistant or drought-sensitive genes from different cultivars in natural,and(2)clone drought-resistant or drought-sensitive genes from mutants generated through mutagenesis approach.Our research group obtained an drought-resistant mutant from an upland rice(IAPAR9)seeds population which was created by Co60 mutagenesis.Through years of drought-resistant experiments in concrete tanks,this mutant consistently displayed similar drought-resistant phenotype among different years and was therefore named as idr1-1(for Increased drought resistance 1-1).By using the F2 mapping population created by crossing idr1-1 to HD 119,the idr1-1 mutation was narrowed down to a region containing a base deletion which leads to production of a stop coden.As the earlier research did not reveal the mechanisms of how IDR1 regulates drought resistance clearly,our current investigations into the mechanism underlying the drought resistance in idr1-1 through different approaches were shown as follows:(1)idr1 mutation enhanced drought resistance of IAPAR9.By analysing drought-resistant phenotype of IAPAR9 and idr1-1,the result showed that mutation of IDR1 enhanced resistance of IAPAR9 to drought stress.On the contrary,IDR1 overexpression lines {idr1-1 backgroound)showed reduced drought resistance.Further,the expression of IDR1 was downregulated by drought treatment.Taken together,we concluded that IDR1 negatively regulates drought resistance of IAPAR9.(2)IDR1 regulates drought resistant of IAPAR9,which may be by affecting multiple signalling transductional pathways,especially ROS signalling pathway.By analysing transcriptional data on idr1-1,we found that the expression of POX22.3,GSTUs,genes involved in calcium signalling pathway were upregulated,AB11,AB12,ABI5 and SAPK1-SAPK9 were down regulated,which suggesting that mutation of IDR1 affects the pathways of ROS,calcium and ABA.After drought treatment,OsLG3 and DST that involved in regulating drought resistance were upregulated,which indicated that IDR1 is involved in the regulation of gene expression in response to drought stress.(3)IDR1 regulates drought resistant of IAPAR9 by regulating leaf thickness and proline content but not root morphological characteristics and stomatal movement.Root morphological characteristics and stomatal are two important factors influencing drought resistance of plants.By observing root morphological characteristics and stomatal,we did not find any significant differences between idr1-1 and IAPAR9.Another two factors influencing drought resistance of plants were leaf thickness and proline content.By detecting leaf thickness and proline content,we found that the leaf of idr1-1 was thicker than IAPAR9.Besides,the proline content of idr1-1 was also more than IAPAR9.The above results indicated that IDR1 affects drought resistance of IAPAR9 by regulating leaf thickness and proline content rather than root morphology and stomatal movement.(4)IDR1 regulates drought resistance of IAPAR9 by affecting the scavenging capacity of ROS.RNA-Seq data showed that the expression of POX22.3,a gene related to ROS scavenging in idr1-1 was up-regulated.Then we found that the activity of POD in idr1-1 was stronger than IAPAR9.DAB and NBT staining results showed that idr1-1 accumulates more ROS than IAPAR9 after drought treatment.Treatments with MV(Methylviologen)provided a demonstration that idr1-1 has less ROS accumulation than IAPAR9,which implied that idr1-1 obtains stronger ability of ROS scavenging than IAPAR9.These data collectively suggest that idr1-1 has enhanced capability of counteracting ROS overaccumulation.(5)IDR1 regulates drought resistance of IAPAR9 by interacting with TUD1.IDR1 physically interactes with TUD1 by in vitro and in vivo experiments such as yeast-two-hybride and split-luc.The previous research showed that TUD1 was involved in brassinolide signalling transduction.By physiological research on idr1-1 showed that IDR1 mutation inhibited the sensibility of IAPAR9 to brassinolide,which suggesting that IDR1 was involved in brassinolide signalling pathway.Drought resistance identification result of tud1 showed that TUD1 mutation enhanced drought resistance of tud1.These results indicated that IDR1 functions in the same pathway with TUD1 to regulate drought resistance of IAPAR9.Taken together,this study probes the molecular mechanisms of IDR1's involvement in drought resistance through,a series of investigations.Our results demonstrate that mutation of IDR1 increase drought resistance in several ways:by increasing biosynthesis of proline and increase blade thickness to reduce water loss;by enhancing ability to scavenge ROS to decrease its accumulation to reduce cellular damage;by regulating expression of a collection of drought-related genes to modulate drought resistance of rice plants.