Comprehensive Analysis Of LRR-RLKs And Functional Elucidation Of SaDRT100 In Hyperacumulator Sedum Alfredii Hance

With the aggravation of heavy-metal contaminated soil in our country,remediation of polluted soil has become a serious concern needed to be solved urgently.Characterization of genes related to heavy-metal resistance is vital for the culture of genetically engineering breeds with novel traits of high tolerance and the enhancement of the phytoremediation efficiency of contaminated soils.Sedum alfredii Hance(Crassulaceae)is a zinc-cadmium(Zn/Cd)hyperaccumulator without any toxicity symptoms native to China hyperaccumulating up to 9,000 ?g/g Cd and 29,000 ?g/g Zn in shoots.The uptake,translocation and storage of Cd have been intensely researched,however,how it senses Cd-stress stimuli and transfers signals within tissues and how it maintains genome stability under Cd stress are still unclear.Relevant studies have shown that the leucine-rich repeats(LRR)are important motifs for the protein-protein interaction and protein-ligand recognition.Those proteins comprising LRRs have been found to play vital roles in fundamental processes.For instance,Leucine Rich Repeat Receptor Like Kinases(LRR-RLKs)have been shown to play a fundamental role in pattern recognition and the initiation of downstream responses.Another LRR-containing protein,DNA-damage repair/tolerance 100(DRT100),has been deemed to be closely related to DNA damage repair.However,in the hyperaccumulator S.alfredii,such a particular species,whether the members of SaLRR-RLKs in S.alfredii mediate the response to Cd stress and do the SaDRT100 participate in the Cd tolerance are still unclear and needed to be further studied.In our study,for the first time,the LRR-RLKs in S.alfredii Hance were comprehensively analyzed,and identified three key genes Sa0 F.522,Sa0 F.1036 and Sa28 F.115 that might be involved in Cd signal transduction.Sa0 F.522,as a largest regulatory gene,its function in yeast also has been identified.For the SaDRT100,we focused on the study of SaDRT100 function characterization and cadium-tolerant mechanisms including the expression pattern,subcellular localization and heterogous expression in the yeast and Arabidopsis.Our results elucidated the functional characterisitcs of SaDRT100 in S.alfredii and further confirmed the participation of SaDRT100 in the cadium-tolerant process.The main results are as follows:A total of 60 LRR-RLK genes in S.alfredii were identified based on transcriptome analysis,which were classfied into 10 subfamilies,and most of them had highly conserved protein structures and motif compositions.The expression profiles after the Cd treatment for 24 h showed that there were 17 genes upregulated in roots,mainly distributed in subfamilies III and XII,26 genes elevated in the stems(subfamily XII)and 26 genes enhaced in the leaves(subfamilies III,VII and XII).Co-expression network analysis showed that the most highly connected hubs,Sa0 F.522,Sa0 F.1036,Sa28 F.115 and Sa1 F.472,were closely related with other genes involved in metal transport,stimulation response and transcription regulations.Response patterns of the ten hub genes were different under the short-time Cd strss,Sa0 F.522,Sa0 F.1036 and Sa28 F.115 were dramatically up-regulated in the roots,stems and leave of S.alfredii.The heterogenous expression of Sa0 F.522 in a Cd-sensitive yeast increased the Cd sensitivity,indicating that Sa0 F.522 may function in Cd signal perception.To elucidate the functions of SaDRT100,studies on tissue-expression,Cd responsive profiles,subcellular localization and heterogous expressions were performed.The results showed that SaDRT100 exhibited abundant expression in shoots,and could be induced by Cd stress in all the three tissues(root,stem and leaf).SaDRT100 was located in the nucleus of cells.The heterologous expression of SaDRT100 rescued the growth of transformed yeast.Furthermore,Cd tolerance was enhanced in SaDRT100 transgenic Arabidopsis supported by quantification of the fresh weight,root length of seedlings and physiological indexes of adult plants.Physiological indicators under Cd stress showed that SaDRT100 transgenic Arabidopsis reduced ROS production and restrained the uptake of Cd by roots.Our data showed that the heterogenous expression of SaDRT100 can enhance the Cd tolerance in yeast and Arabidopsis thaliana.The assessment of the Cd tolerance of SaDRT100 in S.alfredii would provide valuable data for the application of SaDRT100 in producing new heavy-metal tolerant breed.