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Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum.

Liu Z, Shi L, Liu Y, Tang Q, Shen L, Yang S, Cai J, Yu H, Wang R, Wen J, Lin Y, Hu J, Liu C, Zhang Y, Mou S, He S - Front Plant Sci (2015)

Bottom Line: However, significant divergences were also found.Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper.These results will facilitate future functional characterization of MAPK cascades in pepper.

View Article: PubMed Central - PubMed

Affiliation: National Education Minster Key Laboratory of Plant Genetic Improvement and Comprehensive Utilization, Fujian Agriculture and Forestry University Fuzhou, China ; College of Plant Protection, Fujian Agriculture and Forestry University Fuzhou, China.

ABSTRACT
The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

No MeSH data available.


Related in: MedlinePlus

Yeast two-hybrid (Y2H) analysis of MAPK-MAPKK interactions. Positive interactions are indicated by yeast growth on SD-His/-Ade selection media. The indicated concentrations of 3-amino-1,2,4-triazole (3-AT), were used to suppress the auto-activation background in the Y2H system. The pepper MAPKs are divided into 4 subgroups (A–D) on the basis of their phylogenetic tree (see Figure 3). The interaction of pDEST22 and pDEST32 was used as negative control.
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Figure 11: Yeast two-hybrid (Y2H) analysis of MAPK-MAPKK interactions. Positive interactions are indicated by yeast growth on SD-His/-Ade selection media. The indicated concentrations of 3-amino-1,2,4-triazole (3-AT), were used to suppress the auto-activation background in the Y2H system. The pepper MAPKs are divided into 4 subgroups (A–D) on the basis of their phylogenetic tree (see Figure 3). The interaction of pDEST22 and pDEST32 was used as negative control.

Mentions: It's well-known that the hierarchically organized MAP Kinase kinase kinase (MAPKKK)-MAP Kinase kinase (MAPKK)-MAP kinase, all encoded by multiple genes (Smékalová et al., 2014) constitute MAPK signaling cascades, in which MAPKs serve as substrates to MAPKK, and MAPKKs serve as substrates to MAPKKKs. The MAPK cascades play important roles in different plant biological processes ubiquitously. To determine MAPKK-MAPK patterns in pepper, we systematically assessed the interactions between all the MAPKs and MAPKKs using yeast two hybrid assay except for CaMPK17-2 and CaMPK19-2 that failed to be cloned possibly due to their low transcript levels (Figure 11). The positive interactions were first selected in the synthetic SD medium lacking leucine and tryptophan and the culture was transferred to the selection medium without Leucine, tryptophan, histidine and adenine with appropriate 3-amino-1,2,4-triazole. By this method, we got interacting MAPKs of only three of the five MAPKKs, (CaMKK3, CaMKK5, and CaMKK6) while no interacting MAPK was found using CaMKK2 and CaMKK9 as baits. Among three MAPKKs that we got interacting proteins, CaMKK3 interacted with CaMPK3; CaMKK5 interacted with CaMPK3, CaMPK6-1, CaMPK6-2, CaMPK4-1, CaMPK4-2, CaMPK4-3, CaMPK1, CaMPK7, CaMPK20-1, and CaMPK20-2; CaMKK6. To test if CaMKK5 can be autoactivated by itself, we performed another round of yeast two hybrid assay with CaMKK5 inserted into both of the prey and the bait vectors, the result showed that no positive clone grew on selection SD medium (Figure S9), suggesting that CaMKK5 can not be autoactivated by itself. The interaction of PcINF1 and SRC2-1 was used as a positive control (Liu et al., 2015).


Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum.

Liu Z, Shi L, Liu Y, Tang Q, Shen L, Yang S, Cai J, Yu H, Wang R, Wen J, Lin Y, Hu J, Liu C, Zhang Y, Mou S, He S - Front Plant Sci (2015)

Yeast two-hybrid (Y2H) analysis of MAPK-MAPKK interactions. Positive interactions are indicated by yeast growth on SD-His/-Ade selection media. The indicated concentrations of 3-amino-1,2,4-triazole (3-AT), were used to suppress the auto-activation background in the Y2H system. The pepper MAPKs are divided into 4 subgroups (A–D) on the basis of their phylogenetic tree (see Figure 3). The interaction of pDEST22 and pDEST32 was used as negative control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585111&req=5

Figure 11: Yeast two-hybrid (Y2H) analysis of MAPK-MAPKK interactions. Positive interactions are indicated by yeast growth on SD-His/-Ade selection media. The indicated concentrations of 3-amino-1,2,4-triazole (3-AT), were used to suppress the auto-activation background in the Y2H system. The pepper MAPKs are divided into 4 subgroups (A–D) on the basis of their phylogenetic tree (see Figure 3). The interaction of pDEST22 and pDEST32 was used as negative control.
Mentions: It's well-known that the hierarchically organized MAP Kinase kinase kinase (MAPKKK)-MAP Kinase kinase (MAPKK)-MAP kinase, all encoded by multiple genes (Smékalová et al., 2014) constitute MAPK signaling cascades, in which MAPKs serve as substrates to MAPKK, and MAPKKs serve as substrates to MAPKKKs. The MAPK cascades play important roles in different plant biological processes ubiquitously. To determine MAPKK-MAPK patterns in pepper, we systematically assessed the interactions between all the MAPKs and MAPKKs using yeast two hybrid assay except for CaMPK17-2 and CaMPK19-2 that failed to be cloned possibly due to their low transcript levels (Figure 11). The positive interactions were first selected in the synthetic SD medium lacking leucine and tryptophan and the culture was transferred to the selection medium without Leucine, tryptophan, histidine and adenine with appropriate 3-amino-1,2,4-triazole. By this method, we got interacting MAPKs of only three of the five MAPKKs, (CaMKK3, CaMKK5, and CaMKK6) while no interacting MAPK was found using CaMKK2 and CaMKK9 as baits. Among three MAPKKs that we got interacting proteins, CaMKK3 interacted with CaMPK3; CaMKK5 interacted with CaMPK3, CaMPK6-1, CaMPK6-2, CaMPK4-1, CaMPK4-2, CaMPK4-3, CaMPK1, CaMPK7, CaMPK20-1, and CaMPK20-2; CaMKK6. To test if CaMKK5 can be autoactivated by itself, we performed another round of yeast two hybrid assay with CaMKK5 inserted into both of the prey and the bait vectors, the result showed that no positive clone grew on selection SD medium (Figure S9), suggesting that CaMKK5 can not be autoactivated by itself. The interaction of PcINF1 and SRC2-1 was used as a positive control (Liu et al., 2015).

Bottom Line: However, significant divergences were also found.Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper.These results will facilitate future functional characterization of MAPK cascades in pepper.

View Article: PubMed Central - PubMed

Affiliation: National Education Minster Key Laboratory of Plant Genetic Improvement and Comprehensive Utilization, Fujian Agriculture and Forestry University Fuzhou, China ; College of Plant Protection, Fujian Agriculture and Forestry University Fuzhou, China.

ABSTRACT
The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

No MeSH data available.


Related in: MedlinePlus