CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids

Abstract

Background: Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress.

Results: CrMPK3 expressed in bacterial system is an active kinase as it showed auto-phosphorylation and phosphorylation of Myelin Basic Protein. CrMPK3 though localized in cytoplasm, moves to nucleus upon wounding. Wounding, UV treatment and MeJA application on C. roseus leaves resulted in the transcript accumulation of CrMPK3 as well as activation of MAPK in C. roseus leaves. Immuno-precipitation followed by immunoblot analysis revealed that wounding, UV treatment and methyl jasmonate (MeJA) activate CrMPK3. Transient over-expression of CrMPK3 in C. roseus leaf tissue showed enhanced expression of key MIA biosynthesis pathway genes and also accumulation of specific MIAs.

Conclusion: Results from our study suggest a possible involvement of CrMPK3 in abiotic stress signal transduction towards regulation of transcripts of key MIA biosynthetic pathway genes, regulators and accumulation of major MIAs.

Figure 1

Activation of MAP Kinases by wounding, UV and MeJA treatment in C. roseus. 6–8 weeks old C. roseus plants were subjected to various stress treatments. Activation of MAPK in response to wounding, UV treatment, and MeJA treatment was determined by in-gel kinase assay using MBP as a substrate polymerized in SDS 10%(w/v) polyacrylamide gel. Autoradiograms represent in-gel phosphorylation of MBP. The experiments have been repeated three times with similar result.

Figure 2

Multiple sequence alignment of CrMPK3 and related MAPK sequences using ClustalW(1.82). The NCBI accession numbers are Lycopersicon esculentum LeMPK3 (AY261514), Solanum tuberosum MAP Kinase, (AB206552), Nicotiana attenuata WIPK (DQ991136) and A. thaliana AtMPK3 (NM114433).

Figure 3

CrMPK3 shows autophosphorylation as well as MBP phosphorylation. (A) CrMPK3 expressed in E. coli, BL21 strain as GST fusion was analyzed for autophosphorylation, as well as phosphorylation of MBP in an in-solution kinase assay using radiolabled ATP. GST alone was used in the assay and showed no auto- and substrate phosphorylation. (B). An in-gel kinase assay with decreasing concentrations of GST-CrMPK3 using MBP as substrate. The amount of recombinant proteins used was 1 μg; 0.1 μg; 0.01 μg; 0.001 μg from lane1 to 4. The phosphorylated MBP was visualized by autoradiography. The experiments have been repeated three times with similar result.

Figure 4

CrMPK3 migrates towards nucleus upon wounding. C. roseus leaf discs transiently transformed with CrMPK3-GFP/CrMPK3^K69R in pCAMBIA1303 vector or only pCAMBIA1303 vector and observed under confocal microscope without wounding and after wounding at the mentioned time points. The experiment was repeated twice with similar results.

Figure 5

Coregulation of CrMPK3expression, kinase activity and expression of MIA genes in C. roseusleaves upon wounding, UV, and MeJA treatment. 6–8 weeks old C. roseus plants were subjected to wounding, UV treatment and MeJA application and leaves harvested at indicated time intervals. (A) Northern blot analysis of CrMPK3. Lowermost panel shows 28 S rRNA as loading control. (B) CrMPK3 activity assay in response to wounding, UV, MeJA treatments. Total protein extracts (200 μg) was subjected to immunoprecipitation with the 4 G10, phosphotyrosine antibody. The immunoprecipitated complex was electrophoresed on SDS 10%(w/v) polyacrylamide gel and immunoblot was performed using anti-CrMPK3. MeJA mock treatment was performed by applying solvent only (ethanol) in the similar fasion to that of MeJA. ‘CBB’ shows representative equal protein loading controls. (C) Quantitative RT-PCR analysis of key MIA pathway genes, Tdc, Str, D4h and Dat. Expression levels were normalized against expression of C. roseus Actin gene as an internal control and are shown relative to untreated control plants. The relative level of each gene in control plants at time 0 was standardized as 1. Values are presented as the mean and the errors bars indicate standard deviation of triplicate samples. The experiments were repeated three times with similar results.

Figure 6

Transient transformation of CrMPK3inC. roseusleaves increases expression of MIA pathway genes. C. roseus leaves were transiently transformed with pCAMBIA 1303-CrMPK3/CrMPK3 ^K69R construct or pCAMBIA 1303 (Empty Vector) alone by A. tumifaciens mediated transformation. Leaf samples were harvested 72 h post infiltration for RNA and protein extraction. A- PCR using genomic DNA of CrMPK3/CrMPK.^3K69R construct and vector transformed leaves (upper panel). A combination of vector and gene specific primers was used to check the presence of transgene. Middle panel shows expression of CrMPK3 in CrMPK3/CrMPK3^K69R construct and vector transformed leaves by sqRT-PCR using gene specific primer pairs. Lower panel shows expressionof Actin gene as equal cDNA loading control. (B) Effect of CrMPK3/CrMPK3^K69R transient transformation on transcripts accumulation of key MIA pathway genes and regulators. qRT-PCR analysis was performed to check expression of CrMPK3, Tdc, Str, D4H, Dat and Zct1, Zct2, Zct3 and Orca3. Expression levels were normalized against expression of C. roseus Actin gene as an internal control and are shown relative to empty vector transformed leaf. The relative level of each gene in empty vector transformed leaf sample was standardized as 1. Values are presented as the mean and the errors bars indicate standard deviation of triplicate samples. The experiments were repeated three times with similar results.

Figure 7

Transient transformation of CrMPK3 in C. roseusleaves increases accumulation of specific MIAs. Specific MIAs, serpentine, vindoline, vincristine and catharanthine accumulation in vector control or CrMPK3 overexpressed leaves were measured. Values are presented as the mean and standard deviation of triplicate samples.