Effect Of Aluminum On The De Novo Root Regeneration In Nodal Cuttings Of Camellia Sinensis(L.) And Isolation Of Key Regulatory Genes

Aluminium ion(Al)in acid soils is very rhizotoxic to most plant species,but is essential for root growth and development in the tea plant(Camellia sinensis(L.)O.Kuntze).However,the molecular basis of Al-mediated signaling pathways in root regeneration of tea plants is largely unclear.In this study,we profiled the physiological phenotype and phytohormones,using stem cuttings of ’Shaancha 1’ tea plant treated with different Al concentrations,and revealed the key genes and regulatory network that Al promotes the formation of adventitious roots in tea nodal cuttings by transcriptome sequencing(RNA-Seq)and weighted gene co-expression analysis(WGCNA).The main results were as follows:1.Aluminum promotes de novo root regeneration from tea nodal cuttingsThe anatomical analysis showed Al-treated stems began to develop adventitious root(AR)primordia within seven day and ARs occurred after 21 days,while the control had a significant delay.ICP-OES was conducted to measure the contents of mineral contents in the tea nodal cuttings.The results showed that Al supply increased tea-cutting Al accumulation,and may also coordinate with S and K to regulate AR regeneration.2.Transcriptome sequencing and identification and analysis of DEGsSamples collected at days 1,3,7,14,21,28,and 43 after different Al treatment(0.02 or 0.3-m M)were subjected to Illumina sequencing platform for RNA-seq.A total of 2543 differentially expressed genes(DEGs)were identified in tea nodal cuttings treated with different Al concentrations.It is worth noting that most DEGs were mainly distributed on days 3,28,and 43.The KEGG enrichment analysis of DEGs showed that these genes were not only enriched in some pathways related to Al resistance or tolerance such as“phenylpropanoid biosynthesis”,but also involved in some pathways related to plant growth and development,including “plant hormones biosynthesis and signal transduction”.We further found expression patterns of many genes involved in biosynthesis of cytokinin(CK),ethylene(ETH)and jasmonic acid(JA)were induced to a higher level on the 3rd day;as well,expression profiles of auxin transporter related genes were markedly increased during the whole rooting process.HPLC-MS/MS was conducted to measure the contents of phytohormones related in the DEGs analysis.The results showed that the accumulation patterns of these hormones were consistent with the expression patterns of their synthetic and transduction genes.3.Construction of regulatory networks and identification of key genesThe transcriptomics and metabolomics data sets were used for WGCNA to constructed a co-expression regulatory network and identification of key genes.CsERFs,CsWRKYs,and CsMYBs transcription factors and genes with possible crucial roles in regulating AR regeneration were uncovered in the network.In summary,our findings suggest that multiple phytohormones and genes related to their biosynthesis form hierarchical transcriptional cascade during Al-induced de novo regeneration in tea nodal cutting.