Explanation Of Mechanism Underlying The Waterlogging Tolerance In Actinidia Valvata And Functional Identification Of Two Ethylene Responsive Factors

Kiwifruit（Actinidia Lindl.）,native to China,is one of the most successful fruit trees in artificial domestication and cultivation of wild germplasm resources in the20^thcentury.Although the commercialization of kiwifruit in China started late,China has now become the largest producer of kiwifruit in the world and is considered as the research center of kiwifruit.However,kiwifruit generally is considered to be sensitive to waterlogging due to its fleshy roots which are prone to decay under waterlogging condition.Waterlogging stress is one of the main factors threatening the growth and development of kiwifruit.To date,most studies on the waterlogging tolerance of kiwifruit have focused on phenotypic evaluation and physiological mechanism exploration,and most materials studied are from A.chinensis and A.deliciosa which are extremely sensitive to waterlogging stress.Therefore,screening the waterlogging-tolerant kiwifruit genotypes,exploring their waterlogging tolerance mechanism,and mining the key resistance genes are meaningful for cultivating waterlogging-tolerant kiwifruit varieties.A.valvata KR5 is an excellent rootstock germplasm resource with strong waterlogging tolerance and can survive under long-term waterlogging stress.In this study,we performed transcriptome sequencing analysis using the roots of KR5 after waterlogging stress.Then,we identified candidate waterlogging-tolerant genes and verified their functions and explored their regulatory mechanism.The main results were as follows:1.We performed RNA-seq and Iso-seq using the roots of KR5 after 0 h,12 h,24 h and 72 h of waterlogging stress,and a total of 130,236 full-length transcripts were obtained.Compared to those before waterlogging treatment,2,514 genes were differentially expressed at each waterlogging stage.KEGG enrichment analysis showed that these co-expressed differential genes mainly responded waterlogging stress though participating in‘starch and sucrose metabolism’, ‘amino acid metabolism’and‘ROS scavenging’pathways.In addition, differentially expressed TFs analysis found that TFs such as ERF,LOB and WRKY were involved in the waterlogging response of A.valvata.We identified a series of key candidate genes by WGCNA,including genes related to glucose metabolism（Av SUS1,Av ADH1,Av ADH2,Av PDC1）,amino acid metabolism（Av Ala AT1）and ROS scavenging（Av POD73）;TFs Av ERF77,Avb HLH128-like, and Av LOB41 are key regulators in this network.In summary,A.valvata responded to waterlogging stress mainly through carbohydrate metabolism,free amino acids metabolism and reactive oxygen species（ROS）scavenging pathways regulated by some TFs such as ERF.2.A total of 131 ERF TFs were identified based on Iso-seq data of A.valvata KR5. Evolutionary analysis showed that 131 ERF proteins were divided into 11 subgroups and were renamed Av ERF1-Av ERF131.Conserved motifs analysis showed that the ERFs from same subgroup had similar motifs.According to the FPKM values of RNA-seq data,the expression profiles of 131 ERF genes under waterlogging stress was analyzed.The results indicated that six members of ERF-VII group,Av ERF73,Av ERF75,Av ERF77,Av ERF78,Av ERF79 and Av ERF80,were significantly induced under waterlogging treatment.After that, the expression levels of the above six genes were verified by RT-q PCR in roots of two waterlogging tolerant kiwifruit genotypes（KR1 and KR5）and two sensitive kiwifruit genotypes（‘Xuxiang’and‘Hayward’）,and finally two waterlogging tolerant candidate ERF genes,Av ERF73 and Av ERF75,were obtained.Transcription activation test showed that both two proteins had transcriptional activation activity.The subcellular localization test found that Av ERF73 protein was located in the nucleus,while Av ERF75 protein was located in both the cytoplasm and nucleus.In summary,there are 131 ERF TFs in A.valvata KR5,and Av ERF73 and Av ERF75 may be the key regulators of waterlogging tolerance mechanism.3.Overexpression of Av ERF73 in Arabidopsis thaliana and A.chinensis cv. Hongyang enhanced waterlogging tolerance of transgenic plants.We performed RNA-seq using the roots of transgenic line OE53 and WT kiwifruit plants waterlogged for 1 day.Compared to WT plants,a total of 2,769 upregulated differentially expressed genes were identified in OE53.GO enrichment analysis showed that these upregulated genes were mainly enriched in the hypoxia response process and the biosynthesis pathway of terpenoid compounds and sterols mediated by mevalonate pathway（MVA）.In addition,we performed DAP-seq using the same samples,and identified 218 direct target genes of Av ERF73.Transcriptome analysis coupled with DAP-seq showed that Av ERF73 might directly activate Ac NAC022 involved in the‘cellular response to hypoxia’ process and Ac HMGS1 involved in the mevalonate pathway to enhance waterlogging tolerance,which was also verified by a dual-luciferase reporter assay.In summary,Av ERF73 positively regulates the waterlogging tolerance of kiwifruit mainly by participating in hypoxia response and MVA pathway.4.Overexpression of Av ERF75 in tobacco and A.chinensis cv.Hongyang enhanced waterlogging tolerance in transgenic plants.In addition,using Av ERF75 protein as bait,we identified 10 TFs interacting with it through yeast-two-hybrid（Y2H） library screening,including 4 LOB,3 ERF,1 b HLH,1 C3H,1 C2H2 and 1 RAV proteins.Among them,Av LOB41 located in nucleus is a candidate gene for waterlogging tolerance identified by transcriptome analysis,which is significantly induced by waterlogging stress and has no self-activation activity. Y2H point-to-point verification and Bi FC assay further verified the interaction between Av ERF75 and Av LOB41.In summary,Av ERF75 positively regulates the waterlogging tolerance of kiwifruit by interacting with Av LOB41 protein.This study systematically studied the molecular mechanism of waterlogging tolerance in A.valvata KR5 through RNA-seq and Iso-seq,and identified the ERF gene family based on transcriptome data.More importantly,two key ERF-VII genes were screened,and their function and regulatory mechanisms were analyzed.This study deepens the understanding of molecular mechanism of kiwifruit waterlogging tolerance,provides genetic resources for genetic engineering breeding of waterlogging-tolerant kiwifruit.