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A Method for Assaying of Protein Kinase Activity In Vivo and Its Use in Studies of Signal Transduction in Strawberry Fruit Ripening.

ABSTRACT: Strawberry (Fragaria × ananassa) fruit ripening is regulated by a complex of cellular signal transduction networks, in which protein kinases are key components. Here, we report a relatively simple method for assaying protein kinase activity in vivo and specifically its application to study the kinase, FaMPK6, signaling in strawberry fruit. Green fluorescent protein (GFP)-tagged FaMPK6 was transiently expressed in strawberry fruit and after stimuli were applied to the fruit it was precipitated using an anti-GFP antibody. The precipitated kinase activity was measured in vitro using 32P-ATP and myelin basic protein (MBP) as substrates. We also report that FaMPK6 is not involved in the abscisic acid (ABA) signaling cascade, which is closely associated with FaMPK6 signaling in other plant species. However, methyl jasmonate (MeJA), low temperature, and high salt treatments were all found to activate FaMPK6. Transient manipulation of FaMPK6 expression was observed to cause significant changes in the expression patterns of 2749 genes, of which 264 were associated with MeJA signaling. The data also suggest a role for FaMPK6 in modulating cell wall metabolism during fruit ripening. Taken together, the presented method is powerful and its use will contribute to a profound exploration to the signaling mechanism of strawberry fruit ripening.

SUBMITTER: Wang W

PROVIDER: S-EPMC8508642 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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A Method for Assaying of Protein Kinase Activity In Vivo and Its Use in Studies of Signal Transduction in Strawberry Fruit Ripening.

Wang Wei W Dai Zhengrong Z Li Jie J Ouyang Jinyao J Li Tianyu T Zeng Baozhen B Kang Li L Jia Kenan K Xi Zhiyuan Z Jia Wensuo W

International journal of molecular sciences 20210928 19

Strawberry (<i>Fragaria × ananassa</i>) fruit ripening is regulated by a complex of cellular signal transduction networks, in which protein kinases are key components. Here, we report a relatively simple method for assaying protein kinase activity in vivo and specifically its application to study the kinase, FaMPK6, signaling in strawberry fruit. Green fluorescent protein (GFP)-tagged FaMPK6 was transiently expressed in strawberry fruit and after stimuli were applied to the fruit it was precipit ...[more]

PMID: 34638834

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Project description:Although ethylene is well known as an essential regulator of fruit development, little work has examined the role ethylene plays in the development and maturation of mulberry (Morus L.) fruit. To study the mechanism of ethylene action during fruit development in this species, we measured the ethylene production, fruit firmness, and soluble solids content (SSC) during fruit development and harvest. By comparing the results with those from other climacteric fruit, we concluded that Morus fruit are probably climacteric. Genes associated with the ethylene signal transduction pathway of Morus were characterized from M. notabilis Genome Database, including four ethylene receptor genes, a EIN2-like gene, a CTR1-like gene, four EIN3-like genes, and a RTE1-like gene. The expression patterns of these genes were analyzed in the fruit of M. atropurpurea cv. Jialing No.40. During fruit development, transcript levels of MaETR2, MaERS, MaEIN4, MaRTE, and MaCTR1 were lower at the early stages and higher after 26 days after full bloom (DAF), while MaETR1, MaEIL1, MaEIL2, and MaEIL3 remained constant. In ripening fruit, the transcripts of MaACO1 and MaACS3 increased, while MaACS1 and MaACO2 decreased after harvest. The transcripts of MaACO1, MaACO2, and MaACS3 were inhibited by ethylene, and 1-MCP (1-methylcyclopropene) upregulated MaACS3. The transcripts of the MaETR-like genes, MaRTE, and MaCTR1 were inhibited by ethylene and 1-MCP, suggesting that ethylene may accelerate the decline of MaETRs transcripts. No significant changes in the expression of MaEIN2, MaEIL1, and MaEIL3 were observed during ripening or in response to ethylene, while the expressions of MaEIL2 and MaEIL4 increased rapidly after 24 h after harvest (HAH) and were upregulated by ethylene. The present study provides insights into ethylene biosynthesis and signal transduction in Morus plants and lays a foundation for the further understanding of the mechanisms underlying Morus fruit development and ripening.

Dataset Information

A Method for Assaying of Protein Kinase Activity In Vivo and Its Use in Studies of Signal Transduction in Strawberry Fruit Ripening.

Publications

A Method for Assaying of Protein Kinase Activity In Vivo and Its Use in Studies of Signal Transduction in Strawberry Fruit Ripening.

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