Late embryogenesis abundant (LEA) group 4 (LEA4) proteins play an important role in the water stress tolerance of plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, the protective functions and mechanisms of LEA4 proteins are still not clear. In this study, the metal binding properties of two related soybean LEA4 proteins, GmPM1 and GmPM9, were tested using immobilized metal ion affinity chromatography (IMAC). The metal ions Fe³⁺, Ni²⁺, Cu²⁺ and Zn²⁺ were observed to bind these two proteins, while Ca²⁺, Mg²⁺ or Mn²⁺ did not. Results from isothermal titration calorimetry (ITC) indicated that the binding affinity of GmPM1 for Fe³⁺ was stronger than that of GmPM9. Hydroxyl radicals generated by the Fe³⁺/H₂O₂ system were scavenged by both GmPM1 and GmPM9 in the absence or the presence of high ionic conditions (100 mM NaCl), although the scavenging activity of GmPM1 was significantly greater than that of GmPM9. These results suggest that GmPM1 and GmPM9 are metal-binding proteins which may function in reducing oxidative damage induced by abiotic stress in plants.

Abscisic acid-, stress-, and ripening-induced (ASR) protein play important roles in protecting plants from abiotic stress. The functions of some ASR proteins are known to be modulated by binding to metal ions. In this study, we demonstrated that the non-tagged full-length soybean (Glycine max) ASR protein (GmASR) can bind Fe(3+), Ni(2+), Cu(2+), and Zn(2+). The direct binding properties of GmASR to Fe(3+) and Zn(2+) were further confirmed by intrinsic fluorescence assays. The GmASR protein was found to have three Fe(3+) binding sites but only two Zn(2+) binding sites. Natively disordered in aqueous solution, GmASR remained disordered in the presence of Fe(3+), but was found to aggregate in the presence of Zn(2+). The aggregated GmASR protein was partially resolubilized after Zn(2+) was chelated by EDTA. GmASR exhibited Fe(3+)-binding-dependent antioxidant activity in vitro. We speculate that GmASR thus protects against oxidation damage by buffering metal ions, thus alleviating metal toxicity in plant cells under stressed conditions.

Late embryogenesis abundant (LEA) proteins are widely distributed among plant species, where they contribute to abiotic stress tolerance. LEA proteins can be classified into seven groups according to conserved sequence motifs. The PM1 protein from soybean, which belongs to the Pfam LEA_1 group, has been shown previously to be at least partially natively unfolded, to bind metal ions and potentially to stabilize proteins and membranes. Here, we investigated the role of the PM1 C-terminal domain and in particular the multiple histidine residues in this half of the protein. We constructed recombinant plasmids expressing full-length PM1 and two truncated forms, PM1-N and PM1-C, which represent the N- and C-terminal halves of the protein, respectively. Immunoblotting and cross-linking experiments showed that full-length PM1 forms oligomers and high molecular weight (HMW) complexes in vitro and in vivo, while PM1-C, but not PM1-N, also formed oligomers and HMW complexes in vitro. When the histidine residues in PM1 and PM1-C were chemically modified, oligomerization was abolished, suggesting that histidines play a key role in this process. Furthermore, we demonstrated that high Cu²⁺ concentrations promote oligomerization and induce PM1 and PM1-C to form HMW complexes. Therefore, we speculate that PM1 proteins not only maintain ion homeostasis in the cytoplasm, but also potentially stabilize and protect other proteins during abiotic stress by forming a large, oligomeric molecular shield around biological targets.

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.