Roles Of Protein Phosphatases PP2C2b、MKP1 And Protein Kinase MPK4 In Regulation Of Nicotine Biosynthesis In Tobacco

Nicotine is the major alkaloid in tobacco,accounting for more than 90% of the total alkaloids.Nicotine is synthesized in roots and transported to leaves.Transcriptional regulation of nicotine biosynthesis by transcription factors(TFs)belonging to the APETALA2/ ETHYLENE RESPONSE FACTOR(AP2/ERFs)and basic helix-loop-helix(b HLH)families has been extensively studied.The AP2/ERF TFs in NICOTINE locus 2(NIC2),alone or in combination with b HLH TF MYC2,activate the expression of key genes in the nicotine pathway.While it is known that NIC2 TFs and MYC2 factors are responsive to induction by phytohormones,such as jasmonate(JA),little is known about the regulation of NIC2 TFs and MYC2 at post-translational levels.In this study,we identified a B group protein phosphatase 2C(PP2C),NtPP2C2b in tobacco,We analyzed its expression characteristics and studied the regulatory effect of NtPP2C2b on nicotine biosynthetic pathway.We screened a mitogen activated protein kinase(MAPK)interacting with NtPP2C2b by yeast two-hybrid,designated here as NtMPK4 in tobacco and studied the regulatory effect of NtMPK4 on nicotine biosynthesis in tobacco.In addition,We also studied the regulation of nicotine biosynthesis pathway by another protein phosphatase,NtMKP1.This study revealed a posttranslational regulatory mechanism of nicotine biosynthetic pathway in tobacco.The protein phosphatases and MAPK act upstream of the NICOTINE2(NIC2)locus AP2/ERF TFs to fine-tune nicotine biosynthesis in tobacco.The main results are summarized as follows:(1)Based on phylogenetic analysis,the PP2C gene family in tobacco is divided into 16 subgroups,of which subgroup B contains 10 PP2 C.Amino acid sequence alignment showed thatPP2C2b shares 60%sequence similarity with the subgroup B PP2 C members(AP2C1,AP2C2 and AP2C3)of Arabidopsis thaliana.A MAPK kinase interacting motif(KIM),[(K)X(R)3-4X1-6(L)X(I)],was found in NtPP2C2b.Coexpression analysis and q RT-PCR analysis revealed that NtPP2C2b coexpresses with the key genes in nicotine biosynthetic pathway in a JA-responsive manner,indicating that NtPP2C2b is involved in the regulation of nicotine biosynthesis.(2)Overexpression of NtPP2C2b in transgenic hairy roots and tobacco plants significantly reduced the expression of key nicotine biosynthetic genes such as PMT,QPT,A622,and BBL,as well as the expression of key NIC2 TF,ERF221.In addition,NtPP2C2b overexpression also reduced nicotine accumulation in transgenic plants.Transient expression analysis showed that the co-expression of NtPP2C2b and ERF221 in tobacco cells significantly inhibited the activity of the QPT promoter.These findings suggest that NtPP2C2b plays a role in upstream of ERF221 and negatively regulates nicotine biosynthesis in tobacco.(3)Yeast two-hybrid and protoplast-based two-hybrid experiments showed that NtMPK4 interacts with NtPP2C2b in yeast and plant cells,suggesting that NtMPK4 is a potential substrate of NtPP2C2b.In yeast cell,NtMPK4 interacts with NtPP2C2b through KIM motif.Moreover,compared with overexpressing ERF221 alone,co-expression of NtMPK4 and ERF221 increased the the QPT promoter activation in tobacco cells,indicating that NtMPK4 is a positive regulator of nicotine biosynthesis.(4)Conditional overexpression of NtMPK4 in tobacco hairy roots significantly increased the expression of nicotine pathway genes and regulators.In addition,compared to the control,overexpression of NtMPK4 increased the accumulation of nicotine in hairy roots.These findings further suggest that NtMPK4 positively regulates the nicotine biosynthetic pathway in tobacco.(5)We also studied the regulation of nicotine synthetic pathway by another protein phosphatase,NtMKP1.Similar to NtPP2C2b,NtMKP1 is highly expressed in roots and induced by JA.Overexpression of NtMKP1 in hairy roots and transgenic tobacco plants inhibits the expression of nicotine pathway genes and reduces nicotine accumulation.Compared with overexpressing ERF189 alone,transient co-expression of NtMKP1 and ERF189 in tobacco cells decreased the activation of the PMT promoter.These findings suggest that NtMKP1 may play a role upstream of ERF189 as a negative regulator of nicotine biosynthetic pathway in tobacco.In summary,we reveals the post-translational regulatory mechanism of protein phosphatase NtPP2C2b,NtMKP1,and protein kinase NtMPK4 in regulating the nicotine biosynthesis.These findings provide new ideas and direction for metabolic engineering of nicotine content in tobacco.