The Study Of Arabidopsis GOLDEN 2-LIKE Transcription Factors Regulating Abscisic Acid-responsive Gene Expression

Abscisic acid(ABA)is one of the most important phytohormones.It is involved in maintaining a lot of agronomically important processes such as plant growth and development processes as well as dormancy,seed maturation,stress tolerances,and water relations.It is known that ABA plays essential roles in different aspects,for instance,abiotic stress responses,seed germination,and plant development.The previous report noted that about 1500 transcription factors are present in the Arabidopsis thaliana genome,which involved in stress-responsive gene expression.It is observed that in the case of plant genomes,transmits about 7% of their coding sequence to TFs,revealed the complex nature of transcriptional regulation.A report noted that due to the interaction between transcription factors and cis-elements in the promoter regions of many stress-related genes and by doing so up-regulate the expression level of downstream genes leading to inducing abiotic stress tolerance.Here we discuss the role of transcription factors Golden 2 like(GLK)role in dehydration stress and ABA response,still,completely remain unknown.Moreover,to know,what is the direct targets of GLK1/2 in the ABA responses? To understand their molecular regulatory mechanism.The maize Golden2-like genes coding proteins belong to the GARP class domain.GARP domain act as transcriptional activators have a key role in various biological processes,for example,GLKs has an important role in chloroplast development,ARR-Bs involved cytokinin signaling,PSR1 participating in phosphorus metabolic process,KANADI have a function to control the organ polarity in b-ZIP G-box binding.It has also noted that GLK1,2 involved in photosynthetic adaptation,which has tolerance against cold stress.According to the literature review,we concluded the novel point,thus far,whether and how Arabidopsis GARP domain especially Golden 2,ARR-B,Psr1 family,TFs GLK1/2 regulates ABA response remains unknown.The present study revealed that double mutants glk1 glk2 revealed ABA hypersensitive phenotype under seed germination and seedling development,on the other hand,the transgenic plants over-expressing independently GLK1 or GLK2 revealed more ABA-sensitive phenotype as a compared to WT in different concentration of ABA treatment in both seed germination and seedling development.Thus,we concluded that Arabidopsis GLKs are involved in ABA-mediated processes.Moreover,to know,the function of GLK1/2 in seed dormancy,the seedling was germination on MS medium in a different condition such as light or dark and exposed in the presence or absence of pre-chill condition for 3 days at 4 °C incubation.The results indicating that glk1 glk2 double mutants induced seed dormancy.Moreover,that under salt and osmotic stress,glk1 glk2 showed relatively longer roots compared to those of WT as well as glk1 or glk2 with 0 mM or 100 mM or 150 mM mannitol and NaCl concentration.The independent overexpression transgenic lines of both GLK1 OX and GLK2 OX observed osmotic and salt stress sensitive phenotypes compared to WT and glk1,glk2,respectively.Further,we found that glk1 glk2 double mutant might have higher reactive oxygen species(ROS)detoxification capability compared to that of WT,concluded that GLK1/2 are participating in controlling genes expression in response to ROS,which generated under osmotic stress.Drought stress experimental result revealed that glk1 glk2 survival rates were significantly higher compared to WT as well as glk1,glk2,respectively.Therefore it is concluded that GLK1/2 participating negatively redundant function in both osmotic and dehydration stress responses.In addition,the protoplast transfection system was used.The results revealed that compared to negative controls,co-transfection of GD-GLK1 or GD-GLK2 together with a reporter,the expression of the reporter gene is completely activated.It is indicating that both GLK1 and GLK2 harbor the transcriptional activation activities.We further examined the expression of WRKY40,RbohB,COR15 A,and COR15 B at a particular condition.The result noted that the above genes were induced quickly,when we temporally induced GLK1 and GLK2,while we could not detect any changing in GFP induction,which concluded,that GLK1/2 is responsible for activation of these genes expression level.Genome-wide RNA-sequencing analysis revealed that double mutants GLK1/2 regulate several essential ABA-response genes.At 0-h-ABA treatment;there were 394 Up-regulatory gens(URGs)and 844 Down-regulatory genes(DRGs)in glk1 glk2 compared with wild type.On the other hand,after 1-h,with 10 ?M ABA treatments,there were 877 URGs and 841 DRGs in glk1 glk2 compared to WT.The hierarchical clustering examination revealed that GLK1 and GLK2 consistently regulate transcriptional orchestration in response to ABA.We noted that after 1h of ABA treatment,GLKs have a positive rule in control of some biological processes;for example,oxidation-reduction response,transcription,hormone-mediated signaling,and drought stress,etc.For further verification of the RNA-sequencing data,we choose some ABA and abiotic responsive genes(ABI5,PYL11,ABI3,and DIN11),(WRKY4,RbohB,COR15 A,and COR15B),respectively.After ABA treatment the qRT-PCR results revealed that GLK1/2 regulates the expression level of numerous important ABA-inducible transcripts which include WRKY40 that acting as a negative regulator.Further,we identified the ABA-responsive GLK1/2 transcriptional target(s).Therefore the promoter sequences of DRGs that were categorized into GO with ABA treatment.We found that after inducing the GLK1 or GLK2 using ?-estradiol,they were dramatically activated WRKY40pro: LUC activities;while,a mutation of the consensus GLK1/2 binding motif blocked the induction of LUC activities,which revealed that high consensus GLK1/2 detection site is necessary for GLK1/2-regulated transcriptional activation.Additionally,according to electrophoretic mobility shift assays(EMSAs),the results revealed that when we tagged GLK1/2 with GST,it was able to bind the probes(P2),while there were no probes alone in GST.When the P2 probe harboring a nucleic acid mutation(CCAATC)was used to conduct EMSA,it was diminished the binding activities.Subsequently,we concluded that GLK1/2 bind to the WRKY40 promoter through the consensus sequence.To identify the involvement of GLK1/2 and WRKY40 control the same target genes in response to ABA.The stringent statistical and filtering criteria result revealed that substantial numbers of URGs and DRGs were shared having statistical significance in different time points.The hierarchical clustering evaluation noted that GLK1/2 and WRKY40 consistence regulate transcriptional orchestration in response to ABA.Further,scatter plotting examination noted a positive correlation between URGs and DRGs in both mutants at different time points.The qRT-PCR analysis observed that repeatedly detected up-regulation of ABI5 and DIN11,while down-regulation of RbohB and COR15 A in glk1 glk2 and wrky40,which are similar with expression level in the triple mutant(glk1 glk2 wrky40).Thus,we concluded that in response to ABA,GLK1/2 and WRKY40 regulates a large set of common downstream target genes.Further,RNA-sequencing result showed that ABA phenotypes of glk1 glk2 and pyr1 pyl1 pyl2 pyl4 showed opposite manners in both cases seed germination and seedling growth stages.The substantial number of genes was overlapped,having a statistical significance.It is possible that it might possible that GLK1/2 is considered a negative regulator in PYL/PYR ABA receptor-mediated ABA signaling pathway,it is also noted that the transcript levels of GLK1/2 are negatively controlled by PYL/PYR-PP2C-SnRKs.Further,the genetic interaction experiments observed that under seed germination and seedling development stages,compared to WT and glk1,glk2 single mutants,wrky40,and glk1 glk2 revealed many sensitive phenotypes in response to ABA.Intriguingly,glk1 wrky40,glk2 wrky40 as well as glk1 glk2 wrky40 showed same ABA-sensitivities compared to that of wrky40,this result revealed that WRKY40 might function genetically downstream of GLK1/2 in term of seed germination and seed development processes.We generate over-expressed WRKY40 in the background of glk1 glk2.We observed that when we over-expressed WRKY40,glk1 glk2(ABA hypersensitive phenotype)is repressed.Thus,due to this experiment,we concluded that under germination and seedling development stages,WRKY40 has essential role genetically downstream of GLK1/2.We also observed in RNA-sequencing analysis that after ABA treatment,the expression of ABI5 was significantly induced in the case of glk1 glk2.There are strongly genetically interlinked between GLK1 and ABI5,that why we tried to fail to create an abi5-7 glk1 glk2 wrky40 mutant.Thus,in an alternative way,we apply CRISPR-Cas9 technology.Additionally,genetic interaction experiments raveled that triple mutant glk1 glk2 wrky40 showed the same ABA-hypersensitivity to that of a wrky4 and glk1 glk2 mutants;on the other hand,a quadruple mutant glk1 glk2 wrky40 abi5-c(ABI5 CRISPR-Cas9 mutant)revealed the same ABA-hyposensitivity to that of abi5-7.Moreover,we identified that glk1 glk2,wrky40,and glk1 glk2 wrky40 showed more ABA-sensitive phenotypes compared to WT,while all three crisper editing lines glk1 glk2 wrky40 abi5-cr-1,2,and 3 revealed less ABA-sensitive phenotypes than WT in term of seed germination and seedling development stages.Concluded,that ABI5 is an important downstream member of GLK1/2 as well as WRKY40.Overall results support that transcription module GLK1/2-WRKY40 act as a negatively regulatory function in ABA response,our experimental results revealed that GLKs mutant plants showed more tolerance again osmotic and salt stress condition compared to wild type.The above-mentioned detail molecular knowledge provided a clue for the rate of surviving plants during unfavorable environmental stress conditions.Further,these mechanisms are implicated in crops plant for the purpose of more yielding per acres.