The Preliminary Study On The Role Of ARPK1 In Seed Germination And Post-germination Growth Of Arabidopsis

The abscisic acid（ABA）plays a critical role in plant growth and development.Although the core signal transduction pathway of ABA in Arabidopsis has been illustrated,the interactive regulatory network underlying various ABA-responses is far from fully understood.Herein,we analyzed the fundamental function of ARPK1（ABA Responsive Protein Kinase 1）in ABA-mediated seed germination and post-germination growth of Arabidopsis.We found that,with comparing to those of wild-type plants（Col-0）,the loss of function arpk1 mutants showed increased-sensitivity,however the overexpression lines showed decreased-sensitivity,to ABA during seed germination and root elongation;likewise,enhancement in responding to abiotic stresses（such as NaCl,mannitol and glucose）was observed in arpk1 mutants.Moreover,ABA-induced excessive accumulation of reactive oxygen species（ROS）in arpk1 mutant roots was observed.In addition,we analyzed the correlation of ABI4（Abscisic Acid Insensitive 4）and ARPK1,and the results indicated that ABI4 promotes ARPK1’s expression,suggesting a notion of that ABI4may be epistatic to ARPK1.The main results are summarized in below section:1.When applied exogenous ABA,the loss of function mutants arpk1-1 and arpk1-2 showed reduction of seed germination rates and primary root elongation whereas the overexpression lines ARPK1OE#1 and ARPK1OE#2showed increase in seed germination rates and primary root growth,with compared to those of Col-0.There were no obvious differences between Col-0 and complementary lines ARPK1-com.Thus,these results suggested that altered expression level of ARPK1 could affect seed germination and post-germination growth in response to ABA.2.By using the approach of DAB（3,3’-Diaminobenzidine）and NBT （Nitrotetrazolium blue chloride）staining,in the treatment of ABA,deeper stain was observed in the primary roots of arpk1-1 and arpk1-2 while compared to Col-0,suggesting that increase of ROS level in these mutants was occurred under ABA-treatment.3.By analyzing the GUS distribution and qRT-PCR assay,the results indicated that ARPK1’s expression was significant enhanced in the treatments of ABA, NaCl and mannitol.4.Under treatments of NaCl,mannitol and glucose,decreased seed germination rates and primary root length showed in arpk1-1 and arpk1-2 mutants. However,increased seed germination rates and primary root length showed in ARPK1OE#1 and ARPK1OE#2 lines.These phenomena indicated that changing ARPK1’s expression level could affect seed germination and post-germination growth of Arabidopsis in response to abiotic stresses.5.In the condition of NaCl,deeper DAB and NBT staining in roots of arpk1-1 and arpk1-2 was observed,in roots of ARPK1OE#1 and ARPK1OE#2, however,the staining showed lighter,indicating that,under NaCl-treatment, overexpression of ARPK1 reduced the level of ROS in roots,contrarily,loss of ARPK1 function caused accumulation of ROS.When treated with mannitol or glucose,in roots of arpk1-1 and arpk1-2 deeper NBT staining was observed.It is most likely that loss of ARPK1 function may promote induction of O₂^-under osmotic or sugar stress.6.We generated transgenic line carrying the plasmid pBI101-pARPK1-GUS in abi4 mutants and found that,ABA-induced expression of ARPK1 was suppressed in abi4 background.The qRT-PCR analysis confirmed although the expression of ARPK1 was significantly increased in Col-0,it was’t enhanced in the background of abi4.Together,these data suggested the expression of ARPK1 depends on ABI4 in response to ABA.7.Several genetic lines such as abi4/arpk1,abi4/ARPK1OE,ABI4OE/arpk1 and ABI4/ARPK1 were generated and analyzed.Phenotypic analysis showed that seed germination,post-gemination growth and accumulation of ROS in abi4,abi4/arpk1,abi4/ARPK1OE and ARPK1OE were less sensitive to ABA. On the contrary,arpk1,ABI4OE,ABI4OE/arpk1 and ABI4/ARPK1 were more sensitive to ABA during seed germination and seedling growth.Under NaCl-treatment,the seed germination and root elongation of those lines showed similar phenotypes to those in ABA-treatment.