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Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus : An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress

View Article: PubMed Central - PubMed

ABSTRACT

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

No MeSH data available.


AcLEA subcellular localization using the Nicotiana benthamiana system.(A) Confocal microscopy images of protoplasts from N. benthamiana leaves infiltrated with the expression vector pEarlyGate103 (GFP) and pEarlyGate10-AcLEA-GFP. Images were captured 96 h after infiltration of indicated constructs in bright field and in fluorescence using an excitation laser of 488 and emission filters BA655-755 for chlorophyll and BA505-605 for GFP. A merged image is also shown. (B) Immunoblots of kinetics of expression and accumulation of proteins infiltrated at the indicated times. Below immunodetection autoradiographs, the Ponceau S stained membrane is shown as loading control.
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Figure 7: AcLEA subcellular localization using the Nicotiana benthamiana system.(A) Confocal microscopy images of protoplasts from N. benthamiana leaves infiltrated with the expression vector pEarlyGate103 (GFP) and pEarlyGate10-AcLEA-GFP. Images were captured 96 h after infiltration of indicated constructs in bright field and in fluorescence using an excitation laser of 488 and emission filters BA655-755 for chlorophyll and BA505-605 for GFP. A merged image is also shown. (B) Immunoblots of kinetics of expression and accumulation of proteins infiltrated at the indicated times. Below immunodetection autoradiographs, the Ponceau S stained membrane is shown as loading control.

Mentions: To decipher the subcellular localization of AcLEA protein, the corresponding coding sequence was fused with green fluorescent protein (AcLEA-GFP) in vectors designed for transient transgene expression in N. benthamiana leaf protoplasts. Confocal microscopy images (Figure 7A) of protoplasts from leaves infiltrated with the expression vector pEarlyGate103-AcLEA clearly show that AcLEA protein exhibits a cytosolic localization in these conditions. The accumulation of AcLEA and GFP proteins in infiltrated leaves was confirmed by immunodetection analysis (Figure 7B). It is noted that cytosolic LEA proteins could be involved in stress protection not only within the cytosol itself but also at the level of membranes delimiting the organelles such as mitochondria, chloroplasts, endoplasmic reticulum, and nucleus (Candat et al., 2014).


Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus : An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress
AcLEA subcellular localization using the Nicotiana benthamiana system.(A) Confocal microscopy images of protoplasts from N. benthamiana leaves infiltrated with the expression vector pEarlyGate103 (GFP) and pEarlyGate10-AcLEA-GFP. Images were captured 96 h after infiltration of indicated constructs in bright field and in fluorescence using an excitation laser of 488 and emission filters BA655-755 for chlorophyll and BA505-605 for GFP. A merged image is also shown. (B) Immunoblots of kinetics of expression and accumulation of proteins infiltrated at the indicated times. Below immunodetection autoradiographs, the Ponceau S stained membrane is shown as loading control.
© Copyright Policy
Related In: Results  -  Collection

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Figure 7: AcLEA subcellular localization using the Nicotiana benthamiana system.(A) Confocal microscopy images of protoplasts from N. benthamiana leaves infiltrated with the expression vector pEarlyGate103 (GFP) and pEarlyGate10-AcLEA-GFP. Images were captured 96 h after infiltration of indicated constructs in bright field and in fluorescence using an excitation laser of 488 and emission filters BA655-755 for chlorophyll and BA505-605 for GFP. A merged image is also shown. (B) Immunoblots of kinetics of expression and accumulation of proteins infiltrated at the indicated times. Below immunodetection autoradiographs, the Ponceau S stained membrane is shown as loading control.
Mentions: To decipher the subcellular localization of AcLEA protein, the corresponding coding sequence was fused with green fluorescent protein (AcLEA-GFP) in vectors designed for transient transgene expression in N. benthamiana leaf protoplasts. Confocal microscopy images (Figure 7A) of protoplasts from leaves infiltrated with the expression vector pEarlyGate103-AcLEA clearly show that AcLEA protein exhibits a cytosolic localization in these conditions. The accumulation of AcLEA and GFP proteins in infiltrated leaves was confirmed by immunodetection analysis (Figure 7B). It is noted that cytosolic LEA proteins could be involved in stress protection not only within the cytosol itself but also at the level of membranes delimiting the organelles such as mitochondria, chloroplasts, endoplasmic reticulum, and nucleus (Candat et al., 2014).

View Article: PubMed Central - PubMed

ABSTRACT

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied.

No MeSH data available.