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. 2018 Jun 19:6:e5037.
doi: 10.7717/peerj.5037. eCollection 2018.

Comprehensive analysis of mitogen-activated protein kinase cascades in chrysanthemum

Aiping Song #  1 , Yueheng Hu #  1 , Lian Ding  1 , Xue Zhang  1 , Peiling Li  2 , Ye Liu  1 , Fadi Chen  1
Affiliations

Comprehensive analysis of mitogen-activated protein kinase cascades in chrysanthemum

Aiping Song et al. PeerJ. .

Abstract

Background: Mitogen-activated protein kinase (MAPK) cascades, an important type of pathway in eukaryotic signaling networks, play a key role in plant defense responses, growth and development.

Methods: Phylogenetic analysis and conserved motif analysis of the MKK and MPK families in Arabidopsis thaliana, Helianthus annuus and Chrysanthemum morifolium classified MKK genes and MPK genes. qRT-PCR was used for the expression patterns of CmMPK and CmMKK genes, and yeast two-hybrid assay was applied to clear the interaction between CmMPKs and CmMKKs.

Results: We characterized six MKK genes and 11 MPK genes in chrysanthemum based on transcriptomic sequences and classified these genes into four groups. qRT-PCR analysis demonstrated that CmMKKs and CmMPKs exhibited various expression patterns in different organs of chrysanthemum and in response to abiotic stresses and phytohormone treatments. Furthermore, a yeast two-hybrid assay was applied to analyze the interaction between CmMKKs and CmMPKs and reveal the MAPK cascades in chrysanthemum.

Discussion: Our data led us to propose that CmMKK4-CmMPK13 and CmMKK2-CmMPK4 may be involved in regulating salt resistance and in the relationship between CmMKK9 and CmMPK6 and temperature stress.

Keywords: Chrysanthemum morifolium; Mitogen-activated protein kinase; Phylogenetic analysis; Protein-protein interaction; Stress and development response.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Phylogenetic and domain analyses of MPKs.
(A) Phylogenetic relationships of A. thaliana, H. annuus and C. morifolium MAPK. The phylogenetic tree was constructed by MUSCLE using the MEGA7.0 program. (B) Schematic diagram of the amino acid motifs of A. thaliana, H. annuus and C. morifolium MPKs. Motif analysis was performed using MEME 4.0 software as described in the methods. The black solid line represents the corresponding MPK and its length. The different-colored boxes represent different motifs and their positions in each MPK sequence. The blue circle: A. thaliana; the green square: H. annuus; the yellow triangle: C. morifolium.
Figure 2
Figure 2. Phylogenetic and domain analyses of MKKs.
(A) Phylogenetic relationships of A. thaliana, H. annuus and C. morifolium MKKs. The phylogenetic tree was constructed by MUSCLE using the MEGA7.0 program. (B) Schematic diagram of amino acid motifs of A. thaliana, H. annuus and C. morifolium MKKs. Motif analysis was performed using MEME 4.0 software as described in the methods. The black solid line represents the corresponding MKK and its length. The different-colored boxes represent different motifs and their positions in each MKK sequence. The blue circle: A. thaliana; the green square: H. annuus; the yellow triangle: C. morifolium.
Figure 3
Figure 3. Expression patterns of CmMPKs (A) and CmMKKs (B) in different organs/tissues obtained by qRT-PCR analysis.
L1: unexpanded leaves; L2: mature leaves; L3: senescent leaves; S: stems; R: roots. All samples were run in triplicate, and the data were normalized relative to EF1a.
Figure 4
Figure 4. Expression patterns of CmMPKs (A) and CmMKKs (B) under cold and heat shock treatment in chrysanthemum obtained by qRT-PCR analysis.
CK: control plants; C4: the plants treated with 4 °C; H40: the plants treated with 40 °C. Details of the treatments are reported in “Materials and Methods”. All samples were run in triplicate, and the data were normalized relative to EF1a.
Figure 5
Figure 5. Expression patterns of CmMPKs and CmMKKs under abiotic stress treatments in chrysanthemum obtained by qRT-PCR analysis, and shown as a heatmap.
The details of the treatments are reported in “Materials and Methods”. All samples were run in triplicate, and the data were normalized relative to EF1a.
Figure 6
Figure 6. Interactions of CmMKKs with CmMPKs in yeast.
The CmMKK ORF fragments were cloned into the pGADT7 (AD) vector in-frame with the GAL4 activation domain, while the CmMPK ORF cDNA fragments were cloned into the pGBKT7 (BD) vector in-frame with the GAL4 binding domain. pGADT7-T-CmMKKx was used as bait, and pGBKT7-CmMPKs were used as prey; (A) pGADT7-CmMKK2; (B) pGADT7-CmMKK3; (C) pGADT7-CmMKK4; (D) pGADT7-CmMKK5; (E) pGADT7-CmMKK6; (F) pGADT7-CmMKK9. In addition, pGADT7-T/pGBKT7-53 was used as a positive control, while pGADT7-T/pGBKT7-Lam was used as a negative control.
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