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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.


(A) MUSCLE multiple sequence alignment of AcLEA isolated from immature seeds of Amaranthus cruentus. The red boxes show the category and position of the conserved motifs. (B) Phylogenetic tree constructed using the neighbor-joining method based on the multiple sequences alignment. Accessions numbers of published sequences in the GenBank are as follows: Amaranthus cruentus (AcLEA, KX852451), Beta vulgaris LEA_Dc3 (XP_010691209.1), Vitis amurensis LEA5 (ADY17817.1), Brassica napus LEA76, Vitis vinifera (XP_002285360.1), Arabidopsis thaliana LEA7 (AT1G00010), Arabidopsis thaliana LEA 1X08_A (AT1G01470), Arabidopsis lyrata subsp. lyrata (ARALDYDRAFT_47395), Cicer arietinum LEA3 (XP_004506901.1), Medicago truncatula LEA3 (XP_013454682.1), Lupinus angustifolius LEA3 (XP_019454903.1), Arachis hypogaea LEA3 (ADQ91835.1), Oryza sativa Indica LEA3 (CAA92106.1), Oryza sativa Japonica LEA3 (ABS44867.1), Triticum aestivum (AHZ35571.1), PVLEA4-25 and PvLEA18 from Phaseolus vulgaris (AAC49862.1 and AAC49859.1, respectively), Zea mays (NP_001150813.1), Catharanthus roseus (AAY84145.1), Solanum tuberosum LEA 2-like (XP_006364193.1), Solanum lycopersicum (NP_001238798.1), Capsicum annuum DC3 (XP_016562822.1), Nicotiana sylvestris Dc3-like (XP_009770536.1), Nicotiana tabacum Dc3-like (XP_016459037.1).
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Figure 1: (A) MUSCLE multiple sequence alignment of AcLEA isolated from immature seeds of Amaranthus cruentus. The red boxes show the category and position of the conserved motifs. (B) Phylogenetic tree constructed using the neighbor-joining method based on the multiple sequences alignment. Accessions numbers of published sequences in the GenBank are as follows: Amaranthus cruentus (AcLEA, KX852451), Beta vulgaris LEA_Dc3 (XP_010691209.1), Vitis amurensis LEA5 (ADY17817.1), Brassica napus LEA76, Vitis vinifera (XP_002285360.1), Arabidopsis thaliana LEA7 (AT1G00010), Arabidopsis thaliana LEA 1X08_A (AT1G01470), Arabidopsis lyrata subsp. lyrata (ARALDYDRAFT_47395), Cicer arietinum LEA3 (XP_004506901.1), Medicago truncatula LEA3 (XP_013454682.1), Lupinus angustifolius LEA3 (XP_019454903.1), Arachis hypogaea LEA3 (ADQ91835.1), Oryza sativa Indica LEA3 (CAA92106.1), Oryza sativa Japonica LEA3 (ABS44867.1), Triticum aestivum (AHZ35571.1), PVLEA4-25 and PvLEA18 from Phaseolus vulgaris (AAC49862.1 and AAC49859.1, respectively), Zea mays (NP_001150813.1), Catharanthus roseus (AAY84145.1), Solanum tuberosum LEA 2-like (XP_006364193.1), Solanum lycopersicum (NP_001238798.1), Capsicum annuum DC3 (XP_016562822.1), Nicotiana sylvestris Dc3-like (XP_009770536.1), Nicotiana tabacum Dc3-like (XP_016459037.1).

Mentions: AcLEA cDNA contains an ORF of 516 bp that codifies for a 172 amino acids protein with a molecular mass calculated of 18.34 kDa and a theoretical pI of 8.58, values that corresponded to experimental data previously reported (Maldonado-Cervantes et al., 2014). The sequence (Supplementary Figure S2A) was deposited in the GenBank with access code KX852451. In order to identify AcLEA similar proteins and consensus sequences, a search was performed using protein BLAST algorithm and multiple alignment was carried out with the sequences of the highest similarity matches (Figure 1A). A search of related sequences in the LEAPdb database (Hunault and Jaspard, 2010) confirmed that all these sequences are grouped in the LEA_4 Pfam (PF02987). According to the classification proposed by Battaglia et al. (2008), in this family are included LEA proteins from Group 3, such as the cotton protein D-7 (Dure, 1993). Group 3 LEA proteins are characterized by a repetitive motif of 11 amino acids TAQAAKDKTSE (motif 3) in the middle of the sequence that is preceded or followed by ATEAAKQKASE (motif 5); in the N-terminal region is usually conserved the SYKAGETKGRKT (motif 4); meanwhile GGVLQQTGEQV (motif 1) and AADAVKHTLGM (motif 2) are frequently observed in the C-terminal. In many proteins motifs 3 and 5 are present more than once. Motifs 1 to 5 were detected in AcLEA wherein the motif arrangement is M4-M5-M3-M1-M2 with only one complete motif of each type (Figure 1A). On the other hand, the motifs arrangement for LEA group 6 is in the order M3-M1-M2-M4 (Rivera-Najera 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
(A) MUSCLE multiple sequence alignment of AcLEA isolated from immature seeds of Amaranthus cruentus. The red boxes show the category and position of the conserved motifs. (B) Phylogenetic tree constructed using the neighbor-joining method based on the multiple sequences alignment. Accessions numbers of published sequences in the GenBank are as follows: Amaranthus cruentus (AcLEA, KX852451), Beta vulgaris LEA_Dc3 (XP_010691209.1), Vitis amurensis LEA5 (ADY17817.1), Brassica napus LEA76, Vitis vinifera (XP_002285360.1), Arabidopsis thaliana LEA7 (AT1G00010), Arabidopsis thaliana LEA 1X08_A (AT1G01470), Arabidopsis lyrata subsp. lyrata (ARALDYDRAFT_47395), Cicer arietinum LEA3 (XP_004506901.1), Medicago truncatula LEA3 (XP_013454682.1), Lupinus angustifolius LEA3 (XP_019454903.1), Arachis hypogaea LEA3 (ADQ91835.1), Oryza sativa Indica LEA3 (CAA92106.1), Oryza sativa Japonica LEA3 (ABS44867.1), Triticum aestivum (AHZ35571.1), PVLEA4-25 and PvLEA18 from Phaseolus vulgaris (AAC49862.1 and AAC49859.1, respectively), Zea mays (NP_001150813.1), Catharanthus roseus (AAY84145.1), Solanum tuberosum LEA 2-like (XP_006364193.1), Solanum lycopersicum (NP_001238798.1), Capsicum annuum DC3 (XP_016562822.1), Nicotiana sylvestris Dc3-like (XP_009770536.1), Nicotiana tabacum Dc3-like (XP_016459037.1).
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Figure 1: (A) MUSCLE multiple sequence alignment of AcLEA isolated from immature seeds of Amaranthus cruentus. The red boxes show the category and position of the conserved motifs. (B) Phylogenetic tree constructed using the neighbor-joining method based on the multiple sequences alignment. Accessions numbers of published sequences in the GenBank are as follows: Amaranthus cruentus (AcLEA, KX852451), Beta vulgaris LEA_Dc3 (XP_010691209.1), Vitis amurensis LEA5 (ADY17817.1), Brassica napus LEA76, Vitis vinifera (XP_002285360.1), Arabidopsis thaliana LEA7 (AT1G00010), Arabidopsis thaliana LEA 1X08_A (AT1G01470), Arabidopsis lyrata subsp. lyrata (ARALDYDRAFT_47395), Cicer arietinum LEA3 (XP_004506901.1), Medicago truncatula LEA3 (XP_013454682.1), Lupinus angustifolius LEA3 (XP_019454903.1), Arachis hypogaea LEA3 (ADQ91835.1), Oryza sativa Indica LEA3 (CAA92106.1), Oryza sativa Japonica LEA3 (ABS44867.1), Triticum aestivum (AHZ35571.1), PVLEA4-25 and PvLEA18 from Phaseolus vulgaris (AAC49862.1 and AAC49859.1, respectively), Zea mays (NP_001150813.1), Catharanthus roseus (AAY84145.1), Solanum tuberosum LEA 2-like (XP_006364193.1), Solanum lycopersicum (NP_001238798.1), Capsicum annuum DC3 (XP_016562822.1), Nicotiana sylvestris Dc3-like (XP_009770536.1), Nicotiana tabacum Dc3-like (XP_016459037.1).
Mentions: AcLEA cDNA contains an ORF of 516 bp that codifies for a 172 amino acids protein with a molecular mass calculated of 18.34 kDa and a theoretical pI of 8.58, values that corresponded to experimental data previously reported (Maldonado-Cervantes et al., 2014). The sequence (Supplementary Figure S2A) was deposited in the GenBank with access code KX852451. In order to identify AcLEA similar proteins and consensus sequences, a search was performed using protein BLAST algorithm and multiple alignment was carried out with the sequences of the highest similarity matches (Figure 1A). A search of related sequences in the LEAPdb database (Hunault and Jaspard, 2010) confirmed that all these sequences are grouped in the LEA_4 Pfam (PF02987). According to the classification proposed by Battaglia et al. (2008), in this family are included LEA proteins from Group 3, such as the cotton protein D-7 (Dure, 1993). Group 3 LEA proteins are characterized by a repetitive motif of 11 amino acids TAQAAKDKTSE (motif 3) in the middle of the sequence that is preceded or followed by ATEAAKQKASE (motif 5); in the N-terminal region is usually conserved the SYKAGETKGRKT (motif 4); meanwhile GGVLQQTGEQV (motif 1) and AADAVKHTLGM (motif 2) are frequently observed in the C-terminal. In many proteins motifs 3 and 5 are present more than once. Motifs 1 to 5 were detected in AcLEA wherein the motif arrangement is M4-M5-M3-M1-M2 with only one complete motif of each type (Figure 1A). On the other hand, the motifs arrangement for LEA group 6 is in the order M3-M1-M2-M4 (Rivera-Najera 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.