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Molecular cloning of a functional protein phosphatase 2C (FsPP2C2) with unusual features and synergistically up‐regulated by ABA and calcium in dormant seeds of Fagus sylvatica
Author(s) -
Lorenzo Oscar,
Nicolás Carlos,
Nicolás Gregorio,
Rodríguez Dolores
Publication year - 2002
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.2002.1140318.x
Subject(s) - abscisic acid , biochemistry , phosphatase , biology , complementary dna , dephosphorylation , protein phosphorylation , phosphorylation , microbiology and biotechnology , protein kinase a , gene
Phosphorylation/dephosphorylation of proteins is a general mechanism of hormonal signal transduction, including ABA, and serine/threonine protein phosphatases 2C (PP2C, EC 3.1.3.16) have been suggested to play an important role in this process. By means of differential reverse transcriptase‐polymerase chain reaction (RT‐PCR) and further screening of a cDNA library made from mRNA of ABA‐treated Fagus sylvatica L. seeds, a full‐length cDNA clone (FsPP2C2) encoding a putative PP2C was obtained. Comparison to the databases revealed high homology to plant PP2C and most features of these enzymes, but unusual characteristics were found within the catalytic domain and the N‐terminal region of the amino acid sequence. The coding region of FsPP2C2 was expressed in Escherichia coli as histidine tag fusion protein and shows Mg 2+ ‐dependent in vitro phosphatase activity. Transcription of the FsPP2C2 gene is low during seeds stratification at 4°C or under gibberellic acid (GA 3 ) treatment and clearly increases when seeds are treated with ABA and calcium (Ca 2+ ) together, while the addition of calcium chelators (EGTA or TMB‐8) decreases its expression. Furthermore, FsPP2C2 is only expressed in ABA‐treated tissues, preferentially in seeds, which suggests that this PP2C is specifically induced by ABA in dormant seeds, in a Ca 2+ ‐dependent manner, and also in other ABA‐treated tissues.