Functional characterization of DHN-5, a dehydrin showing a differential phosphorylation pattern in two Tunisian durum wheat (Triticum durum Desf.) varieties with marked differences in salt and drought tolerance
Introduction
Progenitors of Triticum sp. (aestivum and durum) cultivated wheat, are native to the Mediterranean region and are a basis of human nutrition in many countries. Wheat production in this region is limited mainly by the availability of water resources. Plants have developed different strategies to face water deficit and over the past few years much attention has been focused on the identification of genes and proteins induced in response to environmental stress [1], [2]. Developing embryos have been largely used as an experimental model to study desiccation tolerance, due to their ability to survive extreme water loss during the final maturation stage of development. Some of the polypeptides accumulating in the mature embryos are directly involved in the dehydration tolerance and in many cases similar polypeptides have been shown to accumulate both in dry embryos and in vegetative tissues of plants subjected to stresses such as dehydration, cold, high salinity and abscisic acid (ABA) treatments [3], [4]. ABA plays an active role in the control of physiological and molecular processes leading to a desiccation tolerance in seeds as well as in vegetative tissues [5]. Effects of endogenous ABA concentrations on gene expression have been widely discussed [6], [7], [8]. Among the proteins that accumulate in the mature embryo and in response to water stress and ABA treatments in vegetative tissues, the RAB (responsive to ABA) proteins, also known as dehydrins (DHN) and late-embryogenesis-abundant (LEA) proteins, constitute an important class and are thought to be involved in protein repair. Since they have chaperone like properties, LEA proteins may help other proteins to recover their native conformation after denaturation or mis-folding during water stress [9]. LEA proteins are ubiquitous among photosynthetic organisms including cyanobacteria, monocotyledon and dicotyledon plants [10]. These soluble proteins are induced by dehydration stress and range in size from 9 kDa for rice Wsi724 [11] to 200 kDa for wheat Wcs200 [12]. Dhn loci tend to be multigenic and occur in clusters on more than one chromosome. In wheat and wheat progenitors, Dhn genes are located on chromosome 6D of Triticum tauschii [13], chromosomes 4A, 5A and 6A of Triticum monococcum [14].
Based on their common amino acid sequence domains, LEA proteins were classified into three major groups [15], [16]. The group 2 LEA proteins contain the lysine rich repeat K-segment (EKKGIMDKIKEKLPG) and have been proposed to form an amphipathic helix [17]. This structure allows both hydrophilic and hydrophobic interactions to stabilize proteins in water stress environments [18]. A cluster of eight serine residues, the S segment, is present in many but not all members of the group 2. The S segment can undergo phosphorylation by the casein kinase 2, CK2 as was shown in RAB 17 of maize [19], [20] and ERD14 of Arabidopsis [21]. Goday et al. [22] reported that phosphorylation is important for RAB17 capacity to bind to nuclear localization signal (NLS) peptides in vitro. Another conserved domain present in many dehydrins, the Y-segment with the consensus sequence DEYGNP, shares some similarities to the nucleotide binding site of chaperones of plants and bacteria [10]. The known physical properties of dehydrins suggest roles as stabilizers of nuclear or cytoplasmic macromolecules under low water or high salt conditions, thereby preserving structural integrity [10], [23], [24]. Transgenic plants overexpressing the barley Lea gene HVA1 or the maize Rab17 show enhanced tolerance to water and salt stresses [25], [26], supporting the hypothesis that dehydrins may play an important role in the protection of plants under water, or salt stress conditions.
In this study, we report the molecular cloning of a wheat dehydrin cDNA (Dhn-5), the product of which was recognized by an antibody raised against the maize LEA protein RAB17 [27]. Immunoblot analyses showed that DHN-5 is differentially phosphorylated in two Tunisian durum wheat (Triticum durum Desf.) varieties with marked differences in salt and drought tolerance [28], [29]. We propose the use of this dehydrin as a marker of drought and salt tolerance between the different natural wheat varieties.
Section snippets
Plant material and treatment
Two Tunisian cultivars of durum wheat (T. durum Desf.) with marked differences in salt and drought tolerance were obtained from INRAT, Laboratoire de Physiologie Végétale (Tunis, Tunisia). The tolerant (R) variety is named Oum Rabiaa and the sensitive (S) one is named Mahmoudi. Plants were grown in a greenhouse (16/8 h daily light period, 25 °C temperature (night/day) and 60–70% relative humidity). Seeds were germinated both in Petri dishes and in pots in compost soil (leafmould:
Isolation and characterization of the wheat Dhn-5 cDNA
During our search for wheat dehydrins belonging to the group 2 LEA proteins, we focused on the one highly homologous to the maize RAB17, a key component in plant response to water and osmotic stresses. The full-length cDNA encoding this dehydrin called Dhn-5 was cloned and sequenced as described in Section 2. Sequence analysis of the Dhn-5 cDNA (GenBank accession no. AY619566) revealed an ORF of 684 bp with a 3′-UTR of 344 bp and a 5′-UTR of 96 bp. The analysis of the deduced amino acid sequence
Discussion
Plant dehydrins belonging to the large RAB/DHN/LEA protein family are known to play a protective role during the dehydration process. Based on homology to maize (RAB17) and barley dehydrins (DHN-4 and DHN-5), we have isolated and characterized a novel durum wheat dehydrin called DHN-5. The deduced amino acid sequence of DHN-5 showed conserved tracts of amino acid sequence with the maize RAB17 protein. Both proteins are rich in glycine and contain a central region of high homology of 30 amino
Acknowledgements
We thank Mónica Pons for confocal microscopy support. This work was supported jointly by grants from the Ministry of Scientific Research, Technology and Development of Competencies, Tunisia and the Agence Espagnole de cooperation Internationale (AECI) Officina Técnica de Cooperación, Spain.
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