Accumulation of Dehydrin Transcripts and Proteins in Response to Abiotic

Antarctic Science 16 (2): 175–184 (2004) © Antarctic Science Ltd Printed in the UK DOI: 10.1017/S0954102004001920 Accumulation of dehydrin transcripts and proteins in response to abiotic stresses in Deschampsia antarctica NÉLIDA OLAVE-CONCHA1, SIMÓN RUIZ-LARA2, XIMENA MUÑOZ1, LEÓN A. BRAVO1 and LUIS J. CORCUERA1* 1Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Chile 2Instituto de Biología Vegetal y Biotecnología. Universidad de Talca, Chile *[email protected] Abstract: Deschampsia antarctica Desv. is one of two vascular plants from the Maritime Antarctic. It is usually exposed to cold, salt, and desiccating winds. We hypothesize that D. antarctica has genes that encode dehydrin proteins and their expression is regulated by low temperature, salt or osmotic stress. To test this hypothesis a fragment of a dehydrin gene from D. antarctica was identified and used as a probe to study dehydrin expression under low temperature, salt, and osmotic stress, and exogenous ABA (abscisic acid) treatments. An anti-dehydrin antibody was also used to study dehydrin protein accumulation under the same treatments. Southern analysis of genomic DNA treated with different endonucleases showed more than four bands recognized by the probe, suggesting that D. antarctica has several dehydrin genes. Northern analysis showed two putative dehydrin transcripts of 1.0 kb accumulated only under exogenous ABA and 1.6 kb under osmotic and salt treatments, suggesting that D. antarctica would have ABA-dependent and - independent pathways for regulation of dehydrin expression. Western analysis showed seven dehydrin proteins (58, 57, 55, 53, 48, 30 and 27 kDa) under the different stress treatments. Cold-accumulated dehydrin proteins were immunolocalized, showing that they are associated with vascular and epidermal tissue, which are preferential ice nucleation zones. Received 30 September 2003, accepted 26 January 2004 Key words: ABA, Antarctica, dehydration, environmental stress, grass, Poaceae, South Shetland Islands Introduction a DEYGNP motif and a lysine rich block Plants have to deal with various environmental stresses (EKKGIMDKIKEKLPG). This block is also called K- during their life cycle. A common element in response to segment and is present in one or more copies and string of many environmental stresses is cellular dehydration. serine residues, which is also a common feature of many Insufficient soil moisture, low relative humidity, low and dehydrins (Close & Lammers 1983, Dure et al. 1989, Close elevated temperatures are among the conditions that can 1997). Other features, such as glycine-rich repeats, can also affect tissue water potential in plants and cause water be present in the dehydrin sequence (reviewed in Close deficiency symptoms (Shinozaki & Yamaguchi-Shinozaki 1996, 1997). Dehydrins are hydrophilic, thermostable and 1996, Bray 1997). Plants have evolved physiological have a high amount of charged amino acids (Close 1997). It responses to defend themselves against environmental has been hypothesized that dehydrins function by stress. Among these responses is the expression of stabilizing large-scale hydrophobic interactions, such as dehydrins, which are also known as late-embryogenesis- membrane structures or hydrophobic patches of proteins abundant protein Group II (LEA) (Ingram & Bartels 1996). (Tytler et al. 1993, Koag et al. 2003). Highly conserved They have been studied in several plants, yet we still have polar regions of dehydrins have been suggested to form an incomplete knowledge of their fundamental role in the hydrogen bonds with polar regions of macromolecules, cell. They are evolutionarily conserved among acting essentially as a surfactant, to prevent coagulation photosynthetic organisms including angiosperms, under conditions of cellular dehydration or low temperature gymnosperms, ferns, mosses, liverworts, algae and (Campbell & Close 1997). Immunoelectron microscopy cyanobacteria, as well as in some non-photosynthetic analyses of the dehydrin Wcor410 from wheat have organisms such as yeast and nematodes (Close & Lammers revealed that these proteins accumulate in the vicinity of the 1993, Mtwisha et al. 1998, Browne et al. 2002). Many plasma membranes of the cells where freeze-induced dehydrins have been identified in plants in response to low dehydration is likely to be more detrimental (Danyluk et al. temperature, salt, drought and exogenous ABA (abscisic 1998). These results support the hypothesis of a stabilizing acid) (Close & Lammers 1993, Labhilili et al. 1995, Close function for dehydrins. Most dehydrins have been found in 1996, Pelah et al. 1997, Nylander et al. 2001). Dehydrins the cytoplasm and nucleus (Svensson et al. 2002). Most are characterized by conserved amino acid motifs, including dehydrin studies have been done in crop plants (Barley, 175 176 N. OLAVE-CONCHA et al. Wheat, Peach) or Arabidopsis thaliana. Few studies have for 24 h, except for low temperature in which long term been made in wild species from dry or cold regions (Sauter treatments were included (5 h, 10 h, 2 d, 4 d, 10 d, 14 d, 18 d et al. 1999, Ndima et al. 2001). and 21 d). Two samples of 0.1 g fresh leaf tissue were taken Deschampsia antarctica Desv. (Poacea) is one of two at 24 h for Western analysis. All the assays were performed vascular plants that have naturally colonized the maritime in duplicate with the same light intensity and photoperiod as Antarctic (Edwards & Smith 1988, Casaretto et al. 1994). described above. The native Antarctic vegetation must have various mechanisms that allow the maintenance of metabolism at RNA blot analysis and probe low temperature during the Antarctic summer (growing season) and survival during winter (Bravo et al. 2001). Total RNA was isolated from control and treated plants Nonetheless, no special features that allow only them to using a phenol-guanidine hydrochloride procedure. RNA survive in the Antarctic have been identified so far (Smith was separated onto a 1.2% agarose gel containing 2003). Deschampsia antarctica does not have unusual formaldehyde. Gels were stained with ethidium bromide to contents of total polar lipids or a high degree of unsaturation visualize rRNA bands for verification of equal loading, then of fatty acids compared with other Poaceae (Zúñiga et al. photographed and transferred onto a nylon membrane by 1994). High amounts of sucrose and fructans are found in capillary blotting with 20X SSC (1X SSC, 0.15 M NaCl, this plant mainly towards the end of summer under field 0.015 M sodium citrate), fixed with UV light (UVP) and by conditions (Zúñiga et al. 1996). This plant also has high baking at 80°C. Blots were prehybridized overnight at 45°C sucrose phosphate synthase activity (Zúñiga-Feest et al. in 5X SSC, 0.1% (w/v) SDS, 50% (w/v) Formamide, 5X 2003). This is consistent with the findings that D. antarctica Denhart, 100 µg ml-1 salmon sperm DNA. Blots were has relatively high net photosynthetic rates on cool days (Xiong et al. 1999). At 0ºC, this plant maintains about 30% of the photosynthetic rate found at the optimum temperature a. (Edwards & Smith 1988). Since D. antarctica is usually exposed to cold, salt, and desiccant winds in the field, we hypothesize that D. antarctica has genes that encode dehydrin proteins and that their accumulation is likely to be affected by low temperature, salt or osmotic stress. These conditions are common during the growing season. This paper reports the characterization of a partial sequence of a D. antarctica dehydrin gene and the effect of low temperature, salt, osmotic stress and ABA treatments on dehydrin mRNA level and protein accumulation. Materials and methods Plant material, growth conditions and stress treatments Deschampsia antarctica was collected in the Coppermine b. Peninsula on Robert Island, South Shetland Islands (62°22'S, 59°43'W). A description of the environmental conditions of the habitat has been published elsewhere (Zúñiga et al. 1996, Alberdi et al. 2002). Plants were reproduced vegetatively in pots with a rich organic soil:peat mixture (3:1) and maintained at 15°C in a growth chamber, with light intensity of 150 µmol m-2 s-1 and a photoperiod of 21/3 L/D (Long Day). Plants were watered daily and Fig. 1. Nucleotide and deduced amino acid sequence of a genomic supplemented with Phostrogen, using 0.12 g l-1 once every fragment of a dehydrin gene obtained by PCR. a. Nucleotide two weeks. There were four basic treatments: low sequence of 454 nucleotide, from nucleotide 135 to 213 temperature, salinity, osmotic stress and ABA. Low correspond to intron (underlined). b. Amino acid deduced temperature treatment was performed at 4 ± 2ºC day/night. sequence from the genomic fragment. Amino acid repeats NaCl (salt stress), polyethylene glycol 15000–20000 typical for proteins belonging to the DHN/LEA/RAB proteins (osmotic stress) and ABA, were added separately to the are underlined: dark line = the φ-segments, dotted line = the K- growth medium to reach final concentrations of 500 mM, segments, bold = S-segment, ≈ corresponds to intron. (NCBI, 24% (w/w) and 100 µM, respectively. All treatments were accession: AY266052). DEHYDRINS IN DESCHAMPSIA ANTARCTICA 177 hybridized with a dehydrin specific probe consisting of a repeated twice. 454 bp PCR–amplified genomic fragment from D. antarctica. This probe was obtained by using the DNA blot analysis following degenerate primers: 5`- GAYGARTAYGGIAAYCC-3´ that corresponds to Deschampsia antarctica genomic DNA was extracted from DEYGNP

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