The Roles Of Aldehyde Oxidase Family Members In Abscisic Acid Synthesis And Drought Tolerance Regulation In Oryza Sativa

Abscisic Acid(ABA)is an important signaling molecule in plants,which is involved in the responses to many biotic and abiotic stresses.The study on ABA is mainly divided into three parts: ABA synthesis,degradation and ABA signaling pathway.In Arabidopsis thaliana,the ABA synthesis pathway has been well studied: ABA synthesis requires series of catalytic enzymes such as ZEP(Zeaxanthin epoxidase),ABA4(Abscisic acid deficient4),NCED(9-cisepoxycarotenoid dioxygenase),SDR(Short-chain dehydrogenase/reductase)and AAO3(Arabidopsis aldehyde oxidase 3),and among which AAO3 is a member of the Arabidopsis aldehyde oxidase family(AAOs).Here,we focused on Oryza sativa ssp.japonica cv.Nipponbare aldehyde oxidase family(OsAOs),aiming to determine whether OsAOs are also involved in ABA synthesis in rice.To this end,q RT-PCR,subcellular localization,genetically modified rice,liquid chromatography-mass spectrometry,yeast Two-Hybrid,tobacco firefly luciferase complementary imaging techniques were used to analysis the function of OsAO1,OsAO2,OsAO3,OsAO4.1 and OsAO4.2 in the synthesis of free ABA in rice.The main results are as follows:1.Four aldehyde oxidase genes were cloned from the rice(Oryza sativa cv.Nipponbare L.).Among them,the protein encoded by OsAO4.1 and OsAO4.2 lacked most of the molybdenum cofactor binding domain.The four aldehyde oxidases were divided into two groups by phylogenetic tree analysis: OsAO1 and OsAO2,OsAO3,OsAO4.1 and OsAO4.2.q RT-PCR analysis showed that the relative expression of OsAO2,OsAO3,and OsAO4.1 was up-regulated after ABA treatment.OsAO1,OsAO2,OsAO3 and OsAO4.1 were induced by mannitol treatment.Four OsAOs were divided into two subgroups based on tissue expression pattern,in which OsAO3 and OsAO4.1 were not only expressed in seedlings,but also at maturity,whereas OsAO1 and OsAO2 were expressed mainly in seedlings.Subcellular localization showed that all proteins were localized in the cytoplasm.2.By using Agrobacterium mediated transformation,we obtained OsAO3 and OsAO4 CRISPR/Cas9 mutants and over-expression(OE)lines.Upon drought treatment,it was showed that both cr-osao3 and cr-osao4-1 and cr-osao4-2 simaltaneously were sensitive in osmotic stress,and their ABA content in vivo was lower than wild-type(WT)and both OsAO3-OE and OsAO4-OE plants were resistant to osmotic stress,and their ABA content in vivo were significantly higher than WT.Their leaf water loss,seed germination rate,shoot and root growth were also changed.OsAO3 and OsAO4 mutation and overexpression affected several agronomic traits including the number of panicles per plant,the number of spikelets per panicle,and spikelet fertility,but in the opposite directions.3.To explain that OsAO4.1 could function normally in ABA synthesis despite lacking domain,Yeast Two-Hybrid was used to find that OsAO4.1 only interacted with OsAO2.The interaction between OsAO2 and OsAO4.1 was further verified by Firefly luciferase complementation imaging assay(LCI).Further experiments showed the ABA content in OsAO2-RNAi plants significantly reduce compared to WT as cr-osao4-1 and cr-osao4-2.A method to analysis the activity of OsAOs was established based on the color change of yeast and the content measurement of ICOOH(Indole-3-carboxylic acid)catalyzed from ICHO(Indole-3-carboxaldehyde).It was found that OsAO2 and OsAO3 were active while OsAO4.1 and OsAO4.2 were inactive or the activity were very low.The activity of OsAO4.1 or OsAO4.2 can be recovered after the completion of its C-terminal structure.In summary,we found that OsAO2,OsAO3 and OsAO4 are involved in ABA biosynthesis,the growth of rice seedlings and the response to drought stress by using genetic and physiological methods.The function of OsAO4.1 is executed realized through the interaction with OsAO2.