Characterization of a group 1 late embryogenesis abundant protein in encysted embryos of the brine shrimpArtemia franciscana

Abstract

Late embryogenesis abundant (LEA) proteins are hydrophilic molecules that are believed to function in desiccation and low-temperature tolerance in some plants and plant propagules, certain prokaryotes, and several animal species. The brine shrimp Artemia franciscana can produce encysted embryos (cysts) that enter diapause and are resistant to severe desiccation. This ability is based on biochemical adaptations, one of which appears to be the accumulation of the LEA protein that is the focus of this study. The studies described herein characterize a 21 kDa protein in encysted Artemia embryos as a group 1 LEA protein. The amino acid sequence of this protein and its gene have been determined and entered into the NCBI database (no. EF656614). The LEA protein consists of 182 amino acids and it is extremely hydrophilic, with glycine (23%), glutamine (17%), and glutamic acid (12.6%) being the most abundant amino acids. This protein also consists of 8 tandem repeats of a 20 amino acid sequence, which is characteristic of group 1 LEA proteins from non-animal species. The LEA protein and its gene are expressed only in encysted embryos and not in larvae or adults. Evidence is presented to show that the LEA protein functions in the prevention of drying-induced protein aggregation, which supports its functional role in desiccation tolerance. This report describes, for the first time, the purification and characterization of a group 1 LEA protein from an animal species.

Improved tolerance to salt and water stress in Drosophila melanogaster cells conferred by late embryogenesis abundant protein

“…Two unique nucleotide sequences (AfLEA-1.1 and AfLEA-1.3) were amplified from diapause embryos of Artemia franciscana with primers based on a previously-published sequence for a group 1 LEA gene (Sharon et al, 2009). The sequences encode for polypeptides of 180 and 197 amino acids, respectively, and both share approximately 86% sequence homology to the previously published protein sequence (Gene Bank: ABR67402).…”

“…The resulting cDNA was amplified with primers based on a previously published nucleotide sequence (Sharon et al, 2009) µg/ml blasticidine (Invitrogen, Grand Island, NY) was added, and for co-transfections with pCoPURO puromycin was used at 5 µg/ml. Resistant cell lines were obtained after 2-4 weeks of selection, and successful transfection was confirmed by Western blotting in cases of pIB/V-5-HIS-DEST constructs or by microscopy in case of GFP-labeled proteins using pAWG/pCoPURO.…”

“…The brine shrimp A. franciscana has served as an important model for animal desiccation tolerance, and multiple LEA proteins that belong to group 1 (PF00477), 3 (PF02987), and 4 (PF04927) are found in developmental stages that survive severe desiccation (Hand et al, 2007;Sharon et al, 2009;Wu et al, 2011). Common features of group 1 and 3 LEA proteins are the overrepresented occurrence of hydrophilic amino acids (i.e., glycine content above 6%), and the presence of identical repeating motifs, while group 4 proteins are conserved in their 70-80 amino acid long N-terminal portion (Battaglia et al, 2008;Wise, 2003).…”

The expression pattern of dormancy-associated genes in multiple life-history stages in the rotifer Brachionus plicatilis

“…LEA proteins are associated with desiccation tolerance throughout the life cycle of major plant taxa and especially in seeds. They have been found also in non-plant species (nematodes, bdelloid rotifers, Artemia cysts, springtail insects) and almost all non-plant LEA proteins belong to Group 3 (Tunnacliffe & Wise, 2007;Clark et al, 2007), although a Group 1 LEA protein was recently reported in Artemia cysts (Sharon et al, 2009). The exact functions of LEAs are not completely known but they were shown to play a role in protein stabilization, prevent protein aggregation (Chakrabortee et al, 2007) and participate in the intracellular glassy state formation during desiccation in orthodox seeds (Berjak, 2006).…”

Stress tolerance during diapause and quiescence of the brine shrimp, Artemia

“…The need for a termination cue differentiates diapause from quiescence, a hypometabolic, stress-tolerant state requiring only return to favorable growth parameters for termination (Drinkwater and Clegg 1991). Stress tolerance during diapause and quiescence in Artemia has been examined from several perspectives including the synthesis, composition, and structure of the cyst shell (Liu et al 2009;Dai et al 2011a;, the role of trehalose (Clegg 1965;Clegg and Jackson 1992;Yang et al 2013), the composition and activity of late embryogenesis abundant (LEA) proteins (Sharon et al 2009;Wu et al 2011;Toxopeus et al 2014), and the function of molecular chaperones such as small heat shock proteins (sHsps) and artemin (King and MacRae 2012;King et al 2013King et al , 2014.…”