The N-Terminal Region of Soybean PM1 Protein Protects Liposomes during Freeze-Thaw.

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.

Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding.

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.

Effects of Fe3+ and Zn2+ on the structural and thermodynamic properties of a soybean ASR protein.

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.

Fe binding properties of two soybean (Glycine max L.) LEA4 proteins associated with antioxidant activity.

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 Fe(3+), Ni(2+), Cu(2+) and Zn(2+) were observed to bind these two proteins, while Ca(2+), Mg(2+) or Mn(2+) did not. Results from isothermal titration calorimetry (ITC) indicated that the binding affinity of GmPM1 for Fe(3+) was stronger than that of GmPM9. Hydroxyl radicals generated by the Fe(3+)/H(2)O(2) 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.

Polyproline II structure is critical for the enzyme protective function of soybean Em (LEA1) conserved domains.

Group 1 late embryogenesis-abundant (LEA1) proteins protect enzyme activity from dehydration and are structurally conserved with three different 20 amino acid motifs in the N-terminal, middle and C-terminal domains. Three soybean Em (LEA1) domain peptides (Em-N, Em-2M and Em-C) covering these respective motifs were constructed and had differential protective ability on lactate dehydrogenase against freeze-thaw: Em-C > Em-2M > Em-N. CD spectroscopy revealed that Em-2M and Em-C contained both polyproline II (PII) helical structure and α-helix, while Em-N had a high potential to form α-helix but did not contain PII structure. The PII helical structure between the third and fifth glycine in the middle motif was shown, through site mutation, to be critical for the enzyme protective function of soybean Em (LEA1) conserved domain under freezing stress.

Cardiac VFM visualization and analysis based on YOLO deep learning model and modified 2D continuity equation.

In order to realize the visual analysis of cardiac fluid motion, according to the characteristics of cardiac flow field ultrasound image, a method for the cardiac Vector Flow Mapping (VFM) analysis and evaluation based on the You-Only-Look-Once (YOLO) deep learning model and the improved two-dimensional continuity equation is proposed in this paper. Firstly, based on the ultrasound Doppler data, the radial velocity values of the blood particles are obtained; due to the real-time VFM's high requirement on the computing speed, the YOLO deep learning model is combined with an improved block matching algorithm for the localization and tracking of myocardial wall, and then the azimuth velocity of myocardial wall speckles can be obtained; in addition, it is proposed in this paper to use a nonlinear weight function to fuse the radial velocity of the blood particles and azimuth velocity of myocardial wall speckles nonlinearly, and further the vortex streamline diagram in the cardiac flow field can be obtained. The results of the experiments on the evaluation of the Ultrasonic apical long-axis view show that the proposed method not only improves the accuracy of VFM, but also provides a new evaluation basis for cardiac function impairment.

A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo.

In vitro studies have shown that LEA proteins from plants and invertebrates protect and stabilise other proteins under conditions of water stress, suggesting a role in stress tolerance. However, there is little information on LEA protein function in whole plants or plant cells, particularly with respect to their anti-aggregation activity. To address this, we expressed in tobacco BY-2 suspension cells an aggregation-prone protein based on that responsible for Huntington's disease (HD). In HD, abnormally long stretches of polyglutamine (polyQ) in huntingtin (Htt) protein cause aggregation of Htt fragments within cells. We constructed stably transformed BY-2 cell lines expressing enhanced green fluorescent protein (EGFP)-HttQ23 or EGFP-HttQ52 fusion proteins (encoding 23 or 52 glutamine residues, pertaining to the normal and disease states, respectively), as well as an EGFP control. EGFP-HttQ52 protein aggregated in the cytoplasm of transformed tobacco cells, which showed slow growth kinetics; in contrast, EGFP-HttQ23 or EGFP did not form aggregates and cells expressing these constructs grew normally. To test the effect of LEA proteins on protein aggregation in plant cells, we constructed cell lines expressing both EGFP-HttQ52 and LEA proteins (PM1, PM18, ZLDE-2 or AavLEA1) or a sHSP (PM31). Of these, AavLEA1 and PM31 reduced intracellular EGFP-HttQ52 aggregation and alleviated the associated growth inhibition, while PM18 and ZLDE-2 partially restored growth rates. Treatment of EGFP-HttQ52-expressing BY2 cells with the polyphenol epigallocatechin-3-gallate (EGCG) also reduced EGFP-HttQ52 aggregation and improved cell growth rate. The EGFP-HttQ52 cell line therefore has potential for characterising both macromolecular and small molecule inhibitors of protein aggregation in plant cells.

[Functions of late embryogenesis abundant proteins in desiccation-tolerance of organisms: a review].

Late embryogenesis abundant (LEA) proteins are well associated with the desiccation tolerance in organisms. LEA proteins are categorized into at least seven groups by virtue of similarities in their deduced amino acid sequences. Most of the LEA proteins have the characteristics of high hydrophilicity and thermo-stability. The LEA proteins are in unstructured conformation in aqueous solution. However, they adopted amphiphilic alpha-helix structure during desiccation condition. LEA proteins are localized to the different organelles in the cells, i.e. cytoplasm, endoplasmic reticulum, mitochondria and nucleus. The multi-functional capacity of LEA proteins are suggested, as protein stabilization, protection of enzyme activity, membrane association and stabilization, antioxidant function, metal-ion binding or DNA protection, etc. Here, we review the structural and functional characteristics of LEA proteins to provide a reference platform to understand their protective mechanisms during the adaptive response to desiccation in organisms.