MdBTs Interact With MdEIL1 To Regulate Leaf Senescence In Apple

In Arabidopsis thaliana,EIN3/EILs?ethylene insensitive 3/ethylene insensitive-like 1?is a typical transcription factor,which could bind to the promoter of downstream genes in ethylene signal pathway.It regulates transcription of downstream genes in response to ethylene signal transduction.At the same time,it could regulate the transcriptional level of genes in other pathway to effect plant growth and development.In our research,an ethylene signal relative transcription factor named Md EIL1 was cloned from ‘Gala' apple?Malus × domestic ‘Royal Gala'?.Sequence analysis showed that Md EIL1 contained 1 980 nucleotides,which encoded a protein containing 658 amino acids.Phylogenetic tree analysis showed that Md EIL1 belonged to EIN3/EIL transcription factor family,that has high homology with Pyrus bretschneideri?XP₀09341360.1??Eriobotrya japonica?ACM89299.1??Prunus persica?ABK35086.1?? Prunus mume?XP₀08231880.1?? Morus notabilis?XP₀10106128.1??Vitis vinifera?XP₀02276380.1?.Next,we used bioinformatics methods,finding that included 12 Md EILs in apples.These were named Md EIL1,Md EIL02,Md EIL03,Md EIL04,Md EIL05,Md EIL06,Md EIL07,Md EIL08,Md EIL09,Md EIL10,Md EIL11,Md EIL12,depending on the location and genetic characteristics in the chromosome.Analysis of gene structure showed that they had a different number of exons.Md EIL101,Md EIL02,Md EIL03,Md EIL06,Md EIL09,Md EIL11,Md EIL12 contained only one exon,while Md EIL104,Md EIL05,Md EIL07,Md EIL08,Md EIL10 contained two Exons.Homology analysis of Md EILs showed that EILs were widespread in higher plants,monocots,dicots,mosses and ferns,but there is no EIL in Chlorella.q RT-PCRs analysis indicated that Md EIL1 had different levels of expression in Apple's roots,stems,leaves,flowers and fruits,expression in leaves was generally higher than other organizations.The results showed that Md EILs expression pattern was constitutively expressed,and had a certain organizational differences.q RT-PCR analysis also found that the expression of Md EILs changed regularly in the leaves of different developmental stages.Hinting Md EILs may be involved in the aging process of the leaves.The expression of senescence-related genes?SAGs?was increased in Md EIL1 overexpression transgenic callus.Ectopic expression of Md EIL1 in Arabidopsis displayed premature aging leaves.Further studies showed that,Md EIL1 directly bound to the promoter of mi R164,ORE1,NYC1,NYE1 and PAO genes,thus promote leaves to premature aging.EIN3 / EILs as a transcription factor in plants,not only can bind to different downstream gene promoter regulating the expression of genes,but also regulate itself or other proteins function by interacting with difference proteins.As a result,EIN3 / EILs could regulate plant growth,development and stress response.In this study,it was found that Md EIL1 was one of Md BT2 interaction proteins by the yeast two-hybrid library screening.Yeast two-hybrid,pull-down and Co-IP experiments also proved that Md EIL1 interacted with Md BT1,Md BT2.It was proved that the TAZ domain of Md BT2 and C-terminal domain of Md EIL1 were essential for interactions by yeast two-hybrid experiment.After using in vitro degradation experiments,it was verified that Md BT2 negatively regulate protein accumulation levels of Md EIL1,when added 26 S proteasome inhibitor MG132,Md BT2 negative Md EIL1 ability to regulate protein accumulation decreased was by using degradation experiments in vitro.The transient expression system experiment verified that Md EIL1 was ubiquitinated,and this process is regulated by the Md BT2.In addition,Md EIL1 overexpression transgenic callus were treated with the viral vector of Md BT2,the expression of senescence-related genes were decreased,suggesting that Md BT2 inhibit leaf senescence through Md EIL1.