Enzymatically driven post-translated modifications (PTMs) usually happen within the intrinsically disordered regions of a target protein and can modulate variety of protein functions. Late embryogenesis abundant (LEA) proteins are a family of the plant intrinsically disordered proteins (IDPs). Despite their important roles in plant stress response, there is currently limited knowledge on the presence and functional and structural effects of phosphorylation on LEA proteins. In this study, we identified three phosphorylation sites (Ser90, Tyr136, and Thr266) in the soybean PM18 protein that belongs to the group-3 LEA proteins. In yeast expression system, PM18 protein increased the salt tolerance of yeast, and the phosphorylation of this protein further enhanced its protective function. Further analysis revealed that Ser90 and Tyr136 are more important than Thr266, and these two sites might work cooperatively in regulating the salt resistance function of PM18. The circular dichroism analysis showed that PM18 protein was disordered in aqueous media, and phosphorylation did not affect the disordered status of this protein. However, phosphorylation promoted formation of more helical structure in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol (TFE). Furthermore, in dedicated in vitro experiments, phosphorylated PM18 protein was able to better protect lactate dehydrogenase (LDH) from the inactivation induced by the freeze-thaw cycles than its un- or dephosphorylated forms. All these data indicate that phosphorylation may have regulatory effects on the stress-tolerance-related function of LEA proteins. Therefore, further studies are needed to shed more light on functional and structural roles of phosphorylation in LEA proteins.
Keywords: Cryoprotective function; Intrinsically disordered protein; Late embryogenesis abundant; Phosphorylation; Salt-tolerance; Yeast.
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