Molecular Dissection Of At AtMAPKKK18 In Regulation Of Drought Stress Tolerance

Due to the sessile nature,plants have to endure various environmental stresses during their lifecycles,such as drought,high salinity,temperature extremes,herbivores and pathogens.Among them,drought stress was the most devastating,which significantly restricted plants' growth and geographic distribution.And it has became the major factor that restricting crops' production.On the other hand,plants have evolved sensitive and intricate mechanisms to cope with arid climates.The regulatory circuits include drought stress sensor(s),signal transduction networks,transcriptome modulation and finally the output of proteins and metabolites which function synergistically to survive the harsh conditions.By far,at least three different signal transduction pathways: two ABA-dependent and one ABA-independent,which function in drought stress responses,have been well-illustrated.However,our understanding on how plants cope with drought stress is still fragmental,especially the upstream signaling events.Mitogen-activated protein kinase(MAPK)cascades are universal and vital signaling modules found in all eukaryotes,which are minimally composed of three essential kinases: MAPK kinase kinase(MAPKKK),MAPK kinase(MAPKK)and MAPK.Complete sequencing of the Arabidopsis genome revealed the presence of approximately 80 MAPKKKs,10 MAPKKs and 20 MAPKs,which offer a wide spectrum of signal transduction and cross-talk.In plants,MAPK signaling networks play crucial roles in numerous biological processes including cell division,hormone response,ROS homeostasis,development,senescence,as well as biotic and abiotic stresses.In our study,the MEKK gene MAPKKK18 plays an important role in response to Drought stress.The main results are in details.(1)MAPKKK18 belongs to the MEKK family in Arabidopsis.The T-DNA knockout mutant plants of MAPKKK18 were more sensitive to drought stress,whereas overexpression of MAPKKK18 conferred significantly enhanced drought stress resistance to transgenic Arabidopsis.(2)The mapkkk18 mutant and OE line leaves showed a faster and slower water loss rate compared with the WT,respectively.The leaf water evaporation was mainly governed by two factors,stomatal density and stomatal aperture.By analyzing the stomata numbers of abaxial epidermis from rosette leaves,no obvious difference in stomatal density was observed among WT,mutant and OE line plants.However,the stomata of OE leaves closed much faster than that of WT,while the mapkkk18 mutant displayed slower ABA-induced stomatal closure.This demonstrated that MAPKKK18 regulated stomatal aperture.(3)Consistant with the roles of MAPKKK18 in drought stress resistance,qRT-PCR and GUS staining assay proved that MAPKKK18 was markedly induced by osmotic stress and ABA.And the PEG triggered expression of MAPKKK18 was almost abolished in aba2 or pyr1pyl1pyl2pyl4 mutants and suppressed in abi1,abi2,abi4 and abi5.This demonstrated that the induced expression of MAPKKK18 was ABA and ABA core signaling pathway dependent.The expressions of MAPKKK18 in root and flower were relative higher than other tussiues.(4)To identify the potential target(s)of MAPKKK18,protein interactions between MAPKKK18 and all 10 MAPKKs(MAPKK1-MAPKK10)in Arabidopsis were tested by yeast two-hybrid assay.Co-transformation with MAPKKK18 and MAPKK3 was the sole combination able to grown on selective media.Co-immunoprecipitation detection suggested that MAPKKK18 physically interacts with MAPKK3 in planta.And an in vitro phosphorylation assay using MAPKK3 as substrates indicated that MAPKKK18 could directly phosphorylate MAPKK3.Taken together,MAPKKK18 may modulate drought stress responses by specifically interacting with and phosphorylating MAPKK3 in Arabidopsis.(5)qRT-PCR proved that MAPKK3 displayed almost the same spatial expression patterns with MAPKKK18 in different tissues.However,MAPKK3 was constitutively expressed and not obviously influenced by ABA,mannitol or PEG treatment.Drought resistance assay showed that mapkk3 plants exhibited obviously lower survival rate compared with WT.These data indicated that MAPKK3 acted as a positive regulator in drought stress resistance.(6)To identify the potential target(s)of MAPKK3.By using a yeast two-hybrid system,the interactions were found between MAPKK3 and MAPK1/2/7/13/14.Thus far,an entire MAPK signaling cascade composed of MAPKKK18,MAPKK3 and MAPK1/2/7/13/14,which participates in drought stress tolerance,was established.(7)As the PEG induced upregulation of MAPKKK18 was apparently compromised in abi4 or abi5 mutant,we reasoned that ABI4 and ABI5 might be the direct upstream transcription activator of MAPKKK18.Transient expression in tobacco leaves and chromatin immunoprecipitation assay supported the idea that both ABI4 and ABI5 function as the upstream transcriptional activators of MAPKKK18.