Hyperosmotic conditions and low temperatures cause cellular dehydration, i.e. removal of water from the cytoplasm into the extracellular space, resulting in the reduction of cytosolic volumes and the alteration of cellular mechanisms. Dehydrins (DHNs) are a group of heat-stable plant proteins believed to play a protective role during cellular dehydration [1
]. They accumulate during dehydrative stress caused by or associated with low or freezing temperatures, drought, salinity, embryo desiccation and abscissic acid synthesis. Dehydrins are very rich in glycine residues, while cysteine and tryptophane are lacking or under-represented [3
]. They are characterized by highly conserved 15-mer lysin rich sequences, called K-segments, which may be present one or several times, one or more Y-segments (DEYGNP) and/or S-segments (serine cluster) [2
]. The K-segment can form a putative amphipathic α-helix structure, with the potential for both hydrophilic and hydrophobic interaction [4
]. Due to this property, dehydrins potentially have a chaperone-like function in stabilizing partially denatured proteins or membranes, coating them with a cohesive water layer and preventing their coagulation during desiccation [3
]. Rinne et al [5
] demonstrated that dehydrins could help hydrolytic enzymes maintain their activity even in desiccating environmental conditions, such as freezing. This result confirms the general belief that dehydrins help the cell to survive desiccation, probably creating local pools of water that are required for survival and re-growth.
Dehydrins were initially found in flowering plants, but immunological studies and screenings of cDNA and genome libraries revealed that dehydrins are widely distributed in the plant kingdom [6
]. In fact, they were found in the brown algae Fucus spiralis
, F. vesciculosus
, and F. evanescens
], in the lichen Selaginella lepidophylla
] as well as in the cyanobacterium Anabaena sp
]. Dehydrin-homolog sequences are also present in Escherichia coli
] and Chlamidia trachomatis
], and even in Drosophila melanogaster
]. To our knowledge, dehydrins have never been reported in fungi, even if some fungal proteins are classified as l
bundant' (LEA) or LEA-like proteins. Dehydrins belong in fact to this larger protein family. The LEA protein classification proposed by Dure [4
] and Bray [11
] was recently revised by Wise [12
] on the basis of the Kyte and Doolitle hydrophobicity metric, predicted secondary structures, expression patterns and sequence features. Dehydrins are now classified in Class IIa and Class IIb of LEA proteins, corresponding to the previous D11 family or Group 2.
LEA proteins belonging to different classes do not share any evident sequence similarity, even if Garay-Arroyo et al. [13
] found that they are characterized by high hydrophilicity and high percentage of glycines, leading to their denomination as "hydrophilins". They are synthesised in the later stages of plant embryogenesis, when seeds are maturing and their water content is decreasing and, in vegetative tissues, in response to water stress [14
]. Their precise function is still unknown, but it has been suggested that they are involved in protecting cellular or molecular structures from the damaging effects of water loss by sequestration of ions, replacement of hydrogen bonding function of water or renaturation of unfolded proteins [11
]. Although primarily found in plants, a number of putative LEA genes have been found in non-plant species, including bacteria [16
], nematodes [18
] and fungi. The first study on a LEA-like protein in fungi was carried out by Mtwisha et al. [14
] who suggested that HSP12
from Saccharomyces cerevisiae
should be considered as a LEA-like protein on the basis of its expression pattern and amino acid composition. Also GRE1
from S. cerevisiae
] and CON6
from Neurospora crassa
] can be ascribed to the family of LEA proteins, because they exhibit a high content of hydrophilic amino acids and their corresponding transcripts accumulate respectively in response to hyperosmosis and desiccation. Moreover, 12 fungal proteins are already classified as 'LEA 4' (named as LEA Class III proteins by Wise [12
]) under the Pfam domain family PF02987 on the basis of the presence of at least one IPR004238 (InterPro ID) domain.
In the framework of an expressed sequence tag project aimed at identifying key regulators and master genes controlling the fruiting body formation in the white truffle Tuber borchii
], we found that the EST called M6G10 was the most up-regulated gene of the reproductive stage compared to the vegetative stage. Truffles are ectomycorrhizal fungi, producing ascocarps which are highly appreciated and commercialised for their organoleptic properties [22
]. Since truffle fruiting bodies cannot yet be obtained under controlled conditions, most studies on truffle primary and secondary metabolism are based on vegetative mycelium cultivated in axenic conditions.
In this study, bioinformatics tools and expression studies were used to support the hypothesis that M6G10 can be considered not only as a LEA protein coding gene, but as the first DHN-like coding gene isolated in fungi. In addition, homologs of this gene, all still defined as "coding for hypothetical proteins" in public databases, were found in other fungal ascomycetous and plant genomes. On the basis of some physiochemical similarities to known plant dehydrins, the identification of a new conserved signature pattern and the expression profile in osmotic and cold stress, we support the classification of these "hypothetical proteins" as dehydrins belonging to Class II LEA proteins.