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.
Highlights
The strawberry (Fragaria ananassa Duch.) is an established experimental model for the study of fleshy fruit ripening, a complex process that involves substantial changes in numerous physiological and metabolic pathways
FaMPK6 was fused to green fluorescent protein (GFP) that serves as a tag for both visualization and immunoprecipitation
Given that FaMPK6 was observed to be capable of sensitively responding to jasmonic acid (JA) stimulus, to demonstrate the applicability of the current method, we further investigated whether FaMPK6 played a role in the regulation of strawberry fruit ripening
Summary
The strawberry (Fragaria ananassa Duch.) is an established experimental model for the study of fleshy fruit ripening, a complex process that involves substantial changes in numerous physiological and metabolic pathways. Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is integral to a broad spectrum of cellular signal systems and mitogen-activated protein kinase cascades (MPKs), which are evolutionarily conserved signal transduction modules that are found in all eukaryotes. They play important roles in diverse aspects of plant growth and development, as well as hormone, biotic, and abiotic responses. We previously demonstrated that AtMPK6 is involved in ABA signaling [24,25,26] and there is evidence that ABI1, a key component of the ABA signaling pathway, physically interacts with AtMPK6, thereby inhibiting AtMPK6 activity [27] and providing direct evidence that AtMPK6 can serve as a key signal in ABA signal transduction
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