Identification Of LpARR Family Genes Involved In Cytokinin Signaling In Perennial Ryegrass And Functional Analysis Of LPARR10

Perennial ryegrass（Lolium perenne）,an important cool-season grass of Poa subfamily,has been widely planted and used in China as an important lawn grass and high-quality forage grass.Perennial ryegrass is suitable for growing in cool and humid environment,but its stress tolerance is not strong,and abiotic stresses such as high temperature,cold,high salt and heavy metals seriously reduce the quality of turf and forage grass.Therefore,it is of great theoretical and practical significance to study the stress tolerance mechanism of perennial ryegrass and improve the quality and quality of perennial ryegrass products.Cytokinin is a positive regulator of plant response to stress.The study on signal transduction pathway of downstream transcription factors and its molecular regulation mechanism is one of the research hotspots in the field of plant molecular biology.In this study,we identified the genes of the LpARR family of cytokinin signaling pathway in perennial ryegrass,and analyzed their expression patterns under different treatments,and initially identified the members of LpARRs that responded to stress tolerance.In this study,we further cloned LpARR10,a member of B-type LpARR in perennial ryegrass,and studied the function of LpARR10 in regulating plant stress tolerance by overexpressing the salt tolerance and cadmium tolerance phenotypes of transgenic Arabidopsis thaliana,and preliminaries explored the regulatory mechanism of LpARR10 in stress tolerance.The experimental results obtained in this study are as follows:（1）According to the ARR family gene member information of Arabidopsis,we identified19 genes in the LpARR family from the transcriptome data of perennial ryegrass previously determined by our team,including 9 A-type LpARRs and 10 B-type LpARRs,which were similar to the number of ARR family members of Arabidopsis thaliana.Referring to the results of cluster analysis,we named A-type LpARRs as LpARR3,LpARR5,LpARR6,LpARR7,LpARR8,LpARR9,LpARR15,LpARR16 and LpARR17;B-type LpARRs as LpARR1,LpARR2,LpARR10,LpARR11,LpARR12,LpARR13,LpARR14,LpARR18,LpARR19 and LpARR20.（2）In the LpARR family of perennial ryegrass,the function of LpARRs of A-type is different from that of B-type LpARRs,but the function is similar between members of class A or B-type.Except for a few genes,the expression levels of LPARR transcription factors in perennial ryegrass were increased in leaves and roots under high temperature（38℃）,salt（Na Cl）,6-BA,abscisic acid（ABA）,Al（Al Cl₃）,cadmium（Cd Cl₂）and Ethephon.In addition,the expression levels of some A-type LpARR transcription factors（such as LpARR6-9）increased under 6-BA treatment,while the expression levels of other A-type LpARR showed A downward trend under these treatments.Under low temperature（4℃）stress,the expression of A-type and B-type LpARR transcription factors of LpARR family was contrary to the above treatment,the expression of A-type LpARR transcription factors in leaves and roots was increased under cold stress,while the expression of B-type LpARR transcription factors was decreased.In addition,the expression levels of three genes（such as LpARR1,LpARR2 and LpARR20）in B-type LpARR were significantly decreased in PEG-simulated drought treatment.（3）Based on the LpARRs gene expression profile under adversity stress,we screened B-type LpARR transcription factor LpARR10 for the test of adverse function tolerance.The length of open reading frame（ORF）of LpARR10 is 1794 bp,with 6 exons encoding 598amino acids.Amino acid sequence analysis showed that LpARR10 had a high degree of recognition with ARRS transcription factors homologous to Arabidopsis,rice and sorghum,and had the highest homology with barley Hv ARR12.LpARR10 possesses the REC domain of B-type ARRs,metal and phosphorylation binding sites,and YXXK functional domain specific to B-type ARR transcription factors.The transient overexpression of LpARR10-GFP fusion protein in the protoplast indicated that the signal of GFP green fluorescent protein was detected in the nucleus,indicating that LpARR10 was localized in the nucleus and in line with the nuclear localization characteristics of transcription factors.（4）We further overexpressed LpARR10 in wild-type（WT）Arabidopsis thaliana using Aturobacterium-mediated inflorescence impregnation.There was no significant difference between WT and transgenic Arabidopsis under normal conditions.Under high salinity and high cadmium stress,the root length and fresh weight of transgenic Arabidopsis thaliana seedlings were greater than WT.In pot experiments,under high salinity and high cadmium stress,the chlorophyll content and photochemical efficiency（Fv/Fm）of WT decreased significantly,cell membrane permeability increased,leaf relative electrical conductivity increased,shoot fresh weight and survival rate decreased,and growth and development were inhibited.However,in transgenic lines overexpressing LpARR10,chlorophyll content,photochemical efficiency（Fv/Fm）,relative electrical conductivity of leaves,shoot fresh weight and survival rate were all better than WT.It was demonstrated that LpARR10 can improve salt tolerance and cadmium tolerance of Arabidopsis thaliana.The expression levels of AtSOS1 and AtSOS3 in overexpressed transgenic lines were significantly higher than those in WT under normal or salt treatment conditions,suggesting that LpARR10 may play a role in improving salt tolerance by up-regulating the expression of AtSOS1 and AtSOS3.