Abstract
Tobacco (Nicotiana tabacum) is considered as the model plant for alkaloid research, of which nicotine accounts for 90%. Many nicotine biosynthetic genes have been identified and were known to be regulated by jasmonate-responsive transcription factors. As an important regulator in plant physiological processes, whether small RNAs are involved in nicotine biosynthesis is largely unknown. Here, we combine transcriptome, small RNAs and degradome analysis of two native tobacco germplasms YJ1 and ZY100 to investigate small RNA’s function. YJ1 leaves accumulate twofold higher nicotine than ZY100. Transcriptome analysis revealed 3,865 genes which were differently expressed in leaf and root of two germplasms, including some known nicotine and jasmonate pathway genes. By small RNA sequencing, 193 miRNAs were identified to be differentially expressed between YJ1 and ZY100. Using in silico and degradome sequencing approaches, six nicotine biosynthetic genes and seven jasmonate pathway genes were predicted to be targeted by 77 miRNA loci. Three pairs among them were validated by transient expression in vivo. Combined analysis of degradome and transcriptome datasets revealed 51 novel miRNA-mRNA interactions that may regulate nicotine biosynthesis. The comprehensive analysis of our study may provide new insights into the regulatory network of nicotine biosynthesis.
Highlights
Tobacco (Nicotiana tabacum) is considered as the model plant for alkaloid research, of which nicotine accounts for 90%
Most of the enzymes involved in this pathway have been identified and characterized in the course of many decades of research, including ornithine decarboxylase (ODC), putrescine N-methyltransferase (PMT), aspartate oxidase (AO), quinolinate synthase (QS), quinolinate phosphoribosyl transferase (QPT), N-methylputrescine oxidase (MPO), and berberine bridge enzyme-like (BBL), among o thers[4,6,14]
YJ1 accumulated more than two folder higher nicotine in the leaves of different stages than ZY100, suggesting the regulation and biosynthesis of nicotine may differ between these two germplasms (Fig. 1B)
Summary
Tobacco (Nicotiana tabacum) is considered as the model plant for alkaloid research, of which nicotine accounts for 90%. Growth Stage of nicotine biosynthesis, and these researches have highlighted large regulatory impacts from two transcription factors families: APETALA2 (AP2)/ethylene response factors (ERF) and MYC2-like basic helixLoop-helix (bHLH)[15,16,17,18] These studies have collectively established a rich set of resources which could be called “nicotine module” for understanding both the genetic and biochemical basis of nicotine biosynthesis and for modeling the uptake, transportation, and accumulation of this classic plant defense compound[4]. These studies have revealed insights including characterization of five miRNA families that exhibit differential accumulation profiles under drought c onditions[39], and a functional demonstration that miR164 targets mRNA transcripts of NtNAC-R1 and that such targeting increases upon topping and leads to multiple root-related p henotypes[40]
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