Comparative Study On The Transcriptional Activity Of Phenylalanine Ammonia Lyase (PAL) Multigenes Between Different Allelopathic Rice Accessions And Exploration Of The Regulation Mechanism

Barnyardgrass is one of the noxious weeds in the paddy field,which has a severely impact on rice yield.And massive amounts of herbicides have been used to control these weeds during the process of rice cultivation,which results in more and more problems of ecological safety.In order to defend the weeds,some rice varieties enable to secret sufficiently concentrations of secondary metabolites into the surrounding environment to supress weed growth,and the characteristics are known as rice allelopathy.The secondary metabolites with allelopathic capacity on weeds are recognized as rice allelochemicals;to date,compouds of phenolic acids,terpenoids,flavonoids and their derivatives have been widely documented.Among these compounds,phenolic acids are important allelochemicals in rice,which are mainly derived from phenylpropane metabolism pathway,and phenylalanine ammonia-lyase(PAL,EC 4.3.1.5)is the key enzyme of this metabolic pathway.The content of phenolic acid compounds in rice is depended on the activity of PAL,while PAL activity and gene expression level of PAL in allelopathic rice are higher than that in non allelopathic rice,and the increase folds of PAL gene expression in allelopathic rice is significantly higher than that in non allelopathic rice,when these two rice accession undergoing stressful conditions.PAL in rice is a family with multigenes,in order to reveal the underlying mechanism of difference on gene expression levels of PAL multigenes from allelopathic rice PI312777 and non-allelopathic rice Lemont,the promoters of PAL multigenes from PI312777 and Lemont were amplified respectively,and the transcriptional activity of these promoters was compared.The promoters with obviously different activities were selected to futher comparative study the types and expression level of the transcriptional regulators binding on them,as well as their downstream PAL gene members in the regulation of rice allelpathy.And exploration of the protein interaction network of PAL.The corresponding results are as follows:1? The promoters of the PAL member genes(Os02g0626100,Os PAL2;1;Os02g0626400,Os PAL2;2;Os02g0626600,Os PAL2;3;Os02g0627100,Os PAL2;4;Os04g0518100,Os PAL4;1;Os04g0518400,Os PAL4;2;Os11g0708900,Os PAL11)from allelopathic rice PI312777 and non-allelopathic rice Lemont were cloned and sequenced respectively.The results showed that the promoter sequence of the same PAL member gene from PI312777 and Lemont were different,and Os PAL2;3 and Os PAL2;4 gene promoter sequences have the largest difference.Further using the tobacco transient expression system to compare the promoter activity of the same PAL member genes of PI312777 and Lemont,the results showed that the activity of the Os PAL2;3 gene promoter from PI3127777 was higher than the Os PAL2;3 gene promoter from Lemont,and the revserse was true of the Os PAL2;4 gene promoter.The Os PAL2;3 and Os PAL2;4 gene promoters were fused with green fluorescent protein gene(GFP)and transformed into rice protoplast cells.Determination of the fluorescent intensity from these protoplast cells by using laser confocal microscopy,showing that the fluorescence intensity of GFP driven by the Os PAL2;3 gene promoter of PI3127777 is higher than that of the Os PAL2;3 gene promoter of Lemont,and the results of Os PAL2;4 gene promoters was opppsite.The transcription factors from Os PAL2;3 gene promoter were further studied.2? Os PAL2;3 gene promoters from PI312777 and Lemont's were biotinylated,and promoter DNA pull-down technology was used to obtain the proteins binding on the promoter.It was found that proteins include AT hook motif domain-containing protein,AT hook motif family protein,AT hook motif domain-containing protein,Transcription regulator,Histone H1,Histone H4 and other DNA binding proteins were binded on Os PAL2;3 gene promoters from the two rice accessions,whereas two Whirly transcription factor domain-containing proteins(LOC?Os02g06370,LOC?Os06g05350)were identified from the binding proteins of Lemont Os PAL2;3 gene promoter.Transcriptional level of Whirly transcription factor domain-containing protein(Os WHY,LOC?Os02g06370,LOC?Os06g05350)and histone H4 in the PI312777 and Lemont were detected by using q PCR.The results showed that gene expression of histone H4,LOC?Os02g06370,LOC?Os06g05350 was significantly up-regulated in Lemont co-cultured barnyardgrass,in comparison with the control group with monoculture Lemont rice,whereas no obviously change was detected in the PI312777 under the same treatment.The results indicated that the above three transcriptional regulators act negative role in regulation of Os PAL2;3 gene expression.3?The CDS of Os PAL2;3 gene was cloned and fused with e YFP to construct a recombinant expression vector to genetically transform in PI312777 and Lemont respectively.Evaluation on the allelopathic activity of transgenic rice showed that overexpression of Os PAL2;3 in rice resulted in increased allelopathic inhibition on barnyardgrass,and the inhibitory ratios was increased by 11.11% in PI312777 and 5.56% in Lemont.Determiantion of the content of phenolic acid compounds in transgenic rice and wild-type plant showed that overexpression of Os PAL2;3 in rice resulted in increased the contents of protocatechuic acid and vanillic acid,and the contents of total phenolic acids was also increased.q PCR was used to detect the genes expression of Os PAL,Os C4 H,Os CCA,Os COL,and Os OMT from phenylalanine metabolic pathwa,the results showed that expreesion of these genes were up-regulated in the Os PAL2;3 overexpressed transgenic rice lines in comparison with the wild-type of it.4?Proteins interacting with Os PAL2;3 were obtained by using Co-IP basing on GFP-Trap.The results of mass spectrometry showed that transketolase,carbonic anhydrase,fructose 1,6-diphosphate aldolase,ATP synthase subunit alpha and ATP synthase subunit beta were interacted with Os PAL2;3 protein in vivo.The positive interaction of Os PAL2;3 and the above proteins was validated by using bimolecular fluorescence complexation(Bi FC)combined with laser confocal microscopy observation.In conclusion,the expression sensitivity of PAL gene between allelopathic rice PI312777 and non-allelopathic rice Lemont was different,in which the transcriptional level of Os PAL2;3 in PI312777 was higher than that in Lemont.The different transcriptional activity of Os PAL2;3 from PI312777 and Lemont is correlative with the negative regulation of the transcription factor Os WHY.In addition,overexpression of Os PAL2;3 resulted in increased phenolic acids contents and allelopathic inhibition capacity on barnyardgrass,and Os PAL2;3 interacts with transketolase,carbonic anhydrase,fructose-1,6-diphosphate aldolase,ATPase ? subunit,ATPase ? subunit and other proteins to jointly regulate phenylalanine metabolism in rice.The results showed that the transcriptional activity of Os PAL2;3 contribures to the alleloapthic activity between PI312777 and lemont,and Os PAL2;3 could be considered as a candidate gene to improve the allelopathy of rice in molecular genetic breeding.