Protein phosphatase NtPP2C2b and MAP kinase NtMPK4 act in concert to modulate nicotine biosynthesis

Abstract

Protein phosphatases (PPs) and protein kinases (PKs) regulate numerous developmental, defense, and phytohormone signaling processes in plants. However, the underlying regulatory mechanism governing biosynthesis of specialized metabolites, such as alkaloids, by the combined effects of PPs and PKs, is insufficiently understood. Here, we report the characterization of a group B protein phosphatase type 2C, NtPP2C2b, that likely acts upstream of the NICOTINE2 locus APETALA 2/Ethylene Response Factors (AP2/ERFs), to regulate nicotine biosynthesis in tobacco. Similar to the nicotine pathway genes, NtPP2C2b is highly expressed in roots and induced by jasmonic acid (JA). Overexpression of NtPP2C2b in transgenic hairy roots or stable transgenic tobacco plants repressed nicotine pathway gene expression and reduced nicotine accumulation. Additionally, transient overexpression of NtPP2C2b, together with the NtERF221, repressed transactivation of the quinolinate phosphoribosyltransferase promoter in tobacco cells. We further demonstrate that the JA-responsive tobacco mitogen-activated protein kinase (MAPK) 4 interacts with NtPP2C2b in yeast and plant cells. Conditional overexpression of NtMPK4 in tobacco hairy roots up-regulated nicotine pathway gene expression and increased nicotine accumulation. Our findings suggest that a previously uncharacterized PP-PK module acts to modulate alkaloid biosynthesis, highlighting the importance of post-translational control in the biosynthesis of specialized plant metabolites.

Keywords: Alkaloid biosynthesis; MAP kinase; gene regulation; hairy roots; nicotine; protein phosphatase 2C; secondary metabolism; tobacco.

Fig. 1.

Co-expression analysis and JA-induced expression of NtPP2C2b, NtMPK4 and nicotine pathway genes. (A) Schematic diagram of nicotine biosynthetic pathway in tobacco. A622, isoflavone reductase-like protein; ADC, arginine decarboxylase; AO, aspartate oxidase; BBL, berberine bridge enzyme-like; ODC, ornithine decarboxylase; MPO, N-methylputrescine oxidase; NND, nicotine N-demethylase QPT, quinolinate phosphoribosyltransferase; QS, quinolinate synthase; PMT, putrescine N-methyltransferase. (B) Co-expression analysis of NtPP2C, NtMPK4 and nicotine pathway genes was performed using leaf, root, stem and flower transcriptomes (accession no. PRJNA208209). Correlation heatmap showing NtPP2C2 and NtMPK4 co-expressing with nicotine pathway genes. (C) Relative expression of NtPP2C2b, NtMPK4 and other nicotine pathway genes in control (CN) and JA-treated roots. Relative expression was measured using real-time quantitative PCR (RT–qPCR). Tobacco EF1œ was used as an internal control. Data represent mean ±SD of three biological samples. Statistical significance was calculated using the Student’s t-test: *, P<0.05; **, P<0.01.

Fig. 2.

Expression analysis of nicotine pathway genes and nicotine accumulation in NtPP2C2b-overexpressing hairy roots. (A) Relative expression of regulatory (MYC2, ERF221and ERF189 ) and enzyme-encoding (PMT, QPT, A622, BBL and MATE) genes in the nicotine pathway in empty vector (EV) and NtPP2C2b-overexpressing hairy roots (OE1 and OE2). Relative expression was measured using RT–qPCR. Tobacco EF1α was used as an internal control. (B) Nicotine content in EV and NtPP2C2b-overexpressing hairy roots (OE1 and OE2) was measured using gas chromatography-flame ionization detector (GC-FID) and presented as mg g^-1 dry weight (DW). Data represent mean ±SD of three biological samples. Statistical significance was calculated using the Student’s t-test: *, P<0.05; **, P<0.01. ***, P<0.001.

Fig. 3.

Expression analysis of nicotine pathway genes and nicotine accumulation in NtPP2C2b-overexpressing transgenic tobacco plants, and promoter assay in tobacco cells. (A) Relative expression of regulatory (MYC2, ERF221and ERF189 ) and enzyme-encoding (PMT, QPT, A622, BBL and MATE) genes in the nicotine pathway in empty vector (EV) and transgenic tobacco plants overexpressing NtPP2C2b (OX1 and OX2). Relative expression was measured using RT–qPCR. Tobacco EF1α was used as an internal control. (B) Nicotine content in leaves of EV and transgenic tobacco plants overexpressing NtPP2C2b (OX1 and OX2) measured using GC-FID and presented as mg g^-1 dry weight (DW). (C) Co-expression of NtPP2C2b with NtERF221 repressed the NtQPT promoter (proQPT) activity in tobacco cells. proQPT fused to the firefly luciferase (LUC) reporter was co-electroporated into tobacco cells alone or with effector plasmids expressing NtERF221 and NtPP2C2b in different combinations. A plasmid containing CaMV35S-GUS was used as an internal control. LUC activity was normalized against the GUS activity. Schematic diagrams of reporter, effector and internal control plasmids are shown. Data represent mean ±SD of three biological samples. Statistical significance was calculated using the Student’s t-test: *, P<0.05; **, P<0.01. ***, P<0.001.

Fig. 4.

NtPP2C2b interacts with NtMPK4 in yeast and plant cells. (A) NtPP2C2b interacts with NtMPK4 in yeast cells. Mutation in NtPP2Cb KIM (K89A R90Q; mNtPP2Cb) abolished interaction with NtMPK4. NtPP2C2b fused to GAL4 DNA binding domain and NtMPK4 fused to GAL4 activation domain were transformed into yeast strain AH109. Protein-protein interaction was detected by the growth of the cells on triple (-his-leu-trp) selection medium. (B) Protoplast-based assay shows that NtPP2C2b interacts with NtMPK4 in tobacco cells. NtMPK4 fused to the GAL4 DNA binding domain (BD-NtMPK4) was electroporated into tobacco protoplasts alone or in combination with NtPP2C2a or NtPP2C2b, and luciferase reporter. The CaMV35-GUS plasmid was used as an internal control. Luciferase activity was normalized against GUS activity. Each experiment was repeated three times. Statistical significance was calculated using the Student’s t-test: *, P<0.05. (C) NtMPK4-FLAG alone or in combination with NtPP2C2b-eGFP was transiently expressed in N. benthamiana leaves. The proteins were detected using anti-GFP or anti-FLAG antibodies in western blot. (D) The transiently produced proteins were immunoprecipitated, separated on a PHOS-tag gel, and detected using anti-FLAG antibodies. The phosphorylated (higher band) and dephosphorylated (lower band) NtMPK4 showed differential migration on the PHOS-tag gel. The Rubisco large sub-unit (LSU), stained by PonceauS, is shown as a loading control.

Fig. 5.

Expression analysis of nicotine pathway genes and nicotine accumulation in NtMPK4-overexpressing hairy roots and promoter assay in tobacco cells. (A) Relative expression of regulatory and enzyme-encoding genes in the nicotine pathway in control (CN) and dexmethoasone-treated (DEX) NtMPK4-overexpressing hairy roots (LINE1 and LINE2). Relative expression was measured using RT–qPCR. Tobacco EF1α was used as an internal control. (B) Nicotine content in control (CN) and dexmethasone-treated (DEX) NtMPK4-overexpressing hairy roots (LINE1 and LINE2) measured using GC-FID and presented as mg g^-1 dry weight (DW) basis. (C) Co-expression of NtPP2C2b with NtMPK4 increased the NtQPT promoter (proQPT) activity in tobacco cells. proQPT fused to the firefly luciferase (LUC) reporter was co-electroporated into tobacco cells alone or with the effector plasmids expressing NtERF221, and NtMPK4 in different combinations. A plasmid containing CaMV35S-GUS was used as an internal control. LUC activity was normalized against the GUS activity. Data represent mean ±SD of three biological samples. Statistical significance was calculated using the Student’s t-test: *, P<0.05; **, P<0.01.

Fig. 6.

A simple model depicting the regulation of the nicotine biosynthetic pathway by NtPP2C2b and NtMPK4 in tobacco. Expression of both NtMPK4 and NtPP2C2b is induced by JA. NtMPK4 is phosphorylated and activated by an unidentified MAPKK (NtMPKK, indicated by dotted arrow). Activated NtMPK4 likely phosphorylates the NIC2 locus ERFs (NtERF221 and possibly other ERFs in the locus), which, along with NtMYC2 regulates the expression of nicotine pathway genes (such as NtPMT, NtQPT). NtPP2C2b interacts with, and likely dephosphorylates NtMPK4 to maintain the homeostasis between phosphorylated and dephosphorylated forms of NtMPK4, thereby fine-tuning the activation of genes regulating nicotine accumulation.