Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth

doi:10.1016/j.bbrc.2006.01.151

Biochemical and Biophysical Research Communications

Volume 342, Issue 2, 7 April 2006, Pages 406-413

https://doi.org/10.1016/j.bbrc.2006.01.151 Get rights and content

Abstract

Late embryogenesis abundant (LEA) proteins constitute a set of proteins widespread in the plant kingdom that show common physicochemical properties such as high hydrophilicity and high content of small amino acid residues such as glycine, alanine, and serine. Typically, these proteins accumulate in response to water deficit conditions imposed by the environment or during plant normal development. In this work, we show that the over-expression in Escherichia coli of proteins of the LEA 2 and the LEA 4 families from Arabidopsis thaliana leads to inhibition of bacterial growth and that this effect is dependent on discrete regions of the proteins. Our data indicate that their antimicrobial effect is achieved through their interaction with intracellular targets. The relevance of the cationic nature and the predicted structural organization of particular protein domains in this detrimental effect on the bacteria growth process is discussed.

Section snippets

Materials and methods

Cloning of the open-reading frames of selected LEA genes from A. thaliana. The coding regions for the different A. thaliana LEA proteins were obtained by polymerase chain reactions (PCR) using gene-specific primers (Supplemental Table) and Vent polymerase (New England BioLabs). Primers were designed according to the sequences in the Arabidopsis genome database [24]. The initial methionine was created by introduction of an NcoI site (CCATGG) in the sense primers. For group 4 LEA D113, this

Over-expression of LEA proteins in E. coli

The open reading frames of 4 different LEA proteins were over-expressed in bacteria under the control of the trp/lac promoter. The extracts of bacterial cultures expressing the recombinant proteins showed new bands, which are not present in those extracts obtained from bacteria carrying an empty vector (Fig. 1A). In most cases, the new bands corresponded to abundant polypeptides, which are maintained soluble after a boiling treatment in a low-salt buffer (Fig. 1B). This characteristic has been

Discussion

The high correlation established between water deficit conditions, imposed by the environment and/or by normal development, and the accumulation of LEA proteins in plants has led to a particular interest on this set of proteins. As for many other proteins, to better characterize their properties, it has been very helpful to over-express them in heterologous systems such as E. coli to obtain large quantities that allow the generation of additional tools such as antibodies, structural studies,

Acknowledgments

We thank G. del Rio for a critical review of the manuscript, the Oligonucleotide Synthesis and DNA Sequencing Facilities of the Instituto de Biotecnología (IBt) for providing us with the oligonucleotides and DNA sequences used in this work, B. Welin for the kind donation of plasmid pCOR47R, and the IBt Informatic Facility for their technical support. We are also grateful to Dr. L. Possani for allowing us to use the protein sequencing facilities of his laboratory. This work was partially

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negro Jamapa grown under water deficit for 48 h, as described previously [39] (LEA4-51-62, AtEM1 and PvLEA4B-7 PCR products were inserted into pJET1.2 plasmid (Thermo Scientific). The integrity of the cloned inserts was confirmed by DNA sequencing and cDNAs were transferred into pTrc99A expression vector as described before [28]. Oligonucleotides synthesis and DNA sequencing services were provided by Oligonucleotide Synthesis and DNA Sequencing Facilities of the Instituto de Biotecnología-UNAM.
Late embryogenic abundant proteins (LEA) are a group of proteins that accumulate during the desiccation phase of the seed and in response to water deficit in the plant. Most LEA proteins are highly hydrophilic and have physicochemical characteristics similar to those of intrinsically disordered proteins (IDPs). Although the function of LEA proteins is not fully understood, there is evidence indicating that these proteins have an important role in reducing the effects caused by water limitation. The analysis of the biochemical and physicochemical characteristics of LEA proteins is crucial to determine their function, for which it is necessary to obtain large amounts of pure protein. Within this current work, we have improved our previous TCA purification method used for basic recombinant LEA proteins to obtain acidic IDPs, the method reported here is fast and simple and is based on the enrichment of the protein of interest by boiling of the bacterial extract followed by a precipitation with different concentrations of TCA and salt. This protocol was applied to acidic and basic IDPs, represented by eight recombinant LEAs, resulting in milligram quantities of highly enriched proteins, which keep their in vitro functionality.
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Dehydrins, a family of hydrophilic and intrinsically disordered proteins, are a subgroup of late embryogenesis abundant proteins that perform different protective roles in plants. Although the transition from a disordered to an ordered state has been associated with dehydrin function or interactions with specific partner molecules, the question of how the primary and secondary dehydrin protein structure is related to specific functions or target molecule preferences remains unresolved. This work addresses the in silico sequencing analysis and in vitro functional characterization of two dehydrin isoforms, VviDHN2 and VviDHN4, from Vitis vinifera. Conformational changes suggest potential interactions with a broad range of molecules and could point to more than one function. The in silico analysis showed differences in conserved segments, specific amino acid binding sequences, heterogeneity of structural properties and predicted sites accessible for various post-translational modifications between the sequence of both dehydrins. Moreover, in vitro functional analysis revealed that although they both showed slight antifungal activity, only VviDHN4 acts as a molecular shield that protects proteins from freezing and dehydration. VviDHN4 also demonstrated high potential as a chaperone and reactive oxygen species scavenger, in addition to presenting antifreeze activity, all of which confirms its multifunctional nature. Our findings highlight the significant role of Y-segments and the differential and specific amino acid composition of less conserved segments that are rich in polar/charged residues between S- and K-segments, coupled with post-translational modifications, in modulating and switching dehydrin biological function.
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The relatively rich of Lys and Arg residues in proteins are consistent with the nature of many cationic proteins that possess strong antimicrobial activity [49]. The derivative polypeptide of the LEA4-D113 protein with the highest number of cationic residues was responsible for the growth inhibitory effect [41]. The analysis of OsLEA1a cationic nature showed that its total Lys and Arg content is 22.6%, indicating OsLEA1a polypeptide contained high content of Lys and Arg residues and showing a potential a-helix structure, however, the overexpression of the TrxA-OsLEA1a fusion protein or native OsLEA1a did not interfere with the bacterial normal growth process under normal conditions.
OsLEA1a is a late embryogenesis abundant (LEA) protein gene from Oryza sativa L, which contains an open reading frame of 282-bp that encodes a putative polypeptide of 93 amino acids. OsLEA1a protein contains abundant of Lys, Ala, Glu, Asp, Gly, Arg and Leu, but depleted in Cys, His, Phe, Trp and Tyr residues; and is strongly hydrophilic. OsLEA1a includes six helical domains and a β-sheet domain. Real-time PCR analysis showed that OsLEA1a was expressed in roots, leaves and panicles of rice, with no or only a few transcripts in stem tissues, and remained at a relatively higher level in leaves during the tillering period, the heading period, the filling period and the full ripe period. To make sense of OsLEA1a functions, TrxA-OsLEA1a fusion protein expression vector and OsLEA1a protein expression vector were transformed into Escherichia coli DL21 (DE3), respectively. The accumulation of the TrxA-OsLEA1a fusion protein or OsLEA1a protein interfered with the resistance of E. coli to high salinity, metal ions, hyperosmotic, oxidation, heat and freeze-thaw stresses. The purified TrxA-OsLEA1a fusion protein reduced stabilization of LDH and increased damage of diverse abiotic stresses to LDH. The findings suggested that the OsLEA1a may confor antibacterial activity under abiotic stresses.
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Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.

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Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth

Abstract

Section snippets

Materials and methods

Over-expression of LEA proteins in E. coli

Discussion

Acknowledgments

Cryobiology

J. Biol. Chem.

Trends Biochem. Sci

J. Biol. Chem.

Trends Plant Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Chromatogr. B

J. Biol. Chem.

Anal. Biochem.

Prot. Expr. Purif.

J. Mol. Biol.

Peptides

Trends Biotechnol.

J. Lipid Res.

Common amino acid sequence domains among the LEA proteins of higher plants

Plant Mol. Biol.

Isolation and characterization of hardening-induced proteins in Chlorella vulgaris C-27: identification of late embryogenesis abundant proteins

Plant Cell Physiol.

The molecular basis of dehydration tolerance in plants

Annu. Rev. Plant Mol. Biol.

Tr288, a rehydrin with a dehydrin twist

Plant Mol. Biol.