Dehydrin accumulation and extreme low-temperature tolerance in Siberian spruce (Picea obovata).

To investigate the role of dehydrins (DHNs) in extreme low-temperature (LT) tolerance, we sampled needle tissue of Siberian spruce (Picea obovata Ledeb.) from trees growing in an arboretum in Trondheim, Norway from August 2006 to April 2007 and tracked changes in LT tolerance via relative electrolyte leakage. We used western blotting to estimate relative amounts of proteins binding a DHN K-segment antibody, measured relative amounts of nine transcripts for small (<25 kDa) DHNs by quantitative reverse transcription-polymerase chain reaction (PCR) using primers developed for DHN transcripts in a closely related species, Picea abies (L.) Karsten, and isolated and sequenced PCR products for five P. obovata DHNs. Three protein bands of 53, 35 and 33 kDa were detected on western blots of SDS-PAGE-separated protein extracts. The 53-kDa DHN was already present late in the growing season, but accumulated during acclimation, and levels decreased rapidly during deacclimation. The 33- and 35-kDa proteins, identified as Picg5 class DHNs by mass spectrometry, first appeared in detectable amounts late in the acclimation process and remained at detectable levels throughout the period of maximum LT tolerance. Levels of the 53-kDa DHN correlated with two LT tolerance parameters, while results for the 33- and 35-kDa proteins were equivocal due to limited sample size and variation in LT tolerance during the mid-winter period. Three additional bands of 30, 28 and 26 kDa were detected in extracts from needles collected in November 2010 using an immunity-purified antibody. Immunoblotting of two-dimensional gel electrophoresis gels loaded with proteins extracted from October and November samples corroborated the results obtained by SDS-PAGE western blots. One large spot in the 53 kDa range and two trains of spots in the same size range as the 33 and 35 kDa DHNs were detected using the K-segment antibody. Eight of the nine DHN transcripts closely tracked LT tolerance parameters, whereas the ninth DHN transcripts followed a reverse pattern, decreasing during winter and increasing again during deacclimation. Multiple regression models using principal components of the transcripts to predict two different LT tolerance parameters suggest separate but overlapping functions for different DHNs in establishing and maintaining extreme LT tolerance.

[1]  Jianquan Liu,et al.  A new phylogeny for the genus Picea from plastid, mitochondrial, and nuclear sequences. , 2013, Molecular phylogenetics and evolution.

[2]  G. Gröbner,et al.  Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein[W] , 2011, Plant Cell.

[3]  Anton S. Shiriaev,et al.  Pair-wise multicomparison and OPLS analyses of cold-acclimation phases in Siberian spruce , 2011, Metabolomics.

[4]  A. Barker.  Plant Cold Hardiness: From the Laboratory to the Field , 2010 .

[5]  W. Schröder,et al.  Proteomics of extreme freezing tolerance in Siberian spruce (Picea obovata). , 2010, Journal of proteomics.

[6]  T. Close,et al.  The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes1[W][OA] , 2009, Plant Physiology.

[7]  P. Schaberg,et al.  Dynamics of low-temperature acclimation in temperate and boreal conifer foliage in a mild winter climate. , 2008, Tree physiology.

[8]  J. Partanen,et al.  Dehydrins expression related to timing of bud burst in Norway spruce , 2008, Planta.

[9]  P. Schaberg,et al.  Cold in the common garden: comparative low-temperature tolerance of boreal and temperate conifer foliage , 2007, Trees.

[10]  T. Rorat,et al.  Plant dehydrins — Tissue location, structure and function , 2006, Cellular & Molecular Biology Letters.

[11]  Wei Wang,et al.  A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis , 2006, Electrophoresis.

[12]  M. Sutinen,et al.  Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation. , 2006, Tree physiology.

[13]  O. Junttila,et al.  Analysis of gene expression during bud burst initiation in Norway spruce via ESTs from subtracted cDNA libraries , 2006, Tree Genetics & Genomes.

[14]  S. Kasapis.  Glass Transitions in Frozen Foods and Biomaterials , 2005 .

[15]  L. Hoffmann,et al.  Biochemical and physiological mechanisms related to cold acclimation and enhanced freezing tolerance in poplar plantlets , 2005 .

[16]  J. Buitink,et al.  Glass formation in plant anhydrobiotes: survival in the dry state. , 2004, Cryobiology.

[17]  Jun-jun Liu,et al.  Characterization of Picg5 novel proteins associated with seasonal cold acclimation of white spruce (Picea glauca) , 2004, Trees.

[18]  R. Arora,et al.  Dehydrin variability among rhododendron species: a 25-kDa dehydrin is conserved and associated with cold acclimation across diverse species. , 2004, The New phytologist.

[19]  P. Heino,et al.  Photoperiod and temperature differentially regulate the expression of two dehydrin genes during overwintering of birch (Betula pubescens Ehrh.). , 2004, Journal of experimental botany.

[20]  Ch. R. Allagulova,et al.  The Plant Dehydrins: Structure and Putative Functions , 2003, Biochemistry (Moscow).

[21]  J. Cushman,et al.  Conformation of a Group 2 Late Embryogenesis Abundant Protein from Soybean. Evidence of Poly (l-Proline)-type II Structure1 , 2003, Plant Physiology.

[22]  T. Close,et al.  The binding of Maize DHN1 to Lipid Vesicles. Gain of Structure and Lipid Specificity1 , 2003, Plant Physiology.

[23]  S. Kontunen-Soppela,et al.  Seasonal fluctuation of dehydrins is related to osmotic status in Scots pine needles , 2001, Trees.

[24]  J. Svensson,et al.  Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography. , 2000, Protein expression and purification.

[25]  M. A. Hemminga,et al.  High critical temperature above T(g) may contribute to the stability of biological systems. , 2000, Biophysical journal.

[26]  Y. P. Lei,et al.  Effects of vitrified and nonvitrified sugars on phosphatidylcholine fluid-to-gel phase transitions. , 2000, Biophysical journal.

[27]  P. Rinne,et al.  Dehydrins in cold-acclimated apices of birch (Betula pubescens Ehrh.): production, localization and potential role in rescuing enzyme function during dehydration , 1999, Planta.

[28]  G. Bryant,et al.  Freezing, drying, and/or vitrification of membrane- solute-water systems. , 1999, Cryobiology.

[29]  P. Rinne,et al.  Onset of freezing tolerance in birch (Betula pubescens Ehrh.) involves LEA proteins and osmoregulation and is impaired in an ABA‐deficient genotype , 1998 .

[30]  B. Fowler,et al.  Accumulation of an Acidic Dehydrin in the Vicinity of the Plasma Membrane during Cold Acclimation of Wheat , 1998, Plant Cell.

[31]  T. Close,et al.  Temporal accumulation and ultrastructural localization of dehydrins in Zea mays , 1997 .

[32]  P. Rinne,et al.  Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens. Involvement of ABA and dehydrins , 1997 .

[33]  T. Close.  Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins , 1996 .

[34]  H. Goff,et al.  Glass transitions in aqueous carbohydrate solutions and their relevance to frozen food stability , 1996 .

[35]  T. Close,et al.  Seasonal patterns of dehydrins and 70-kDa heat-shock proteins in bark tissues of eight species of woody plants , 1996 .

[36]  T. Close,et al.  Cold-Specific Induction of a Dehydrin Gene Family Member in Barley , 1995, Plant physiology.

[37]  F. Bergmann,et al.  Introgressive hybridization and phylogenetic relationships between Norway, Picea abies (L.) Karst., and Siberian, P. obovata Ledeb., spruce species studied by isozyme loci , 1995, Heredity.

[38]  M. Wisniewski,et al.  Cold Acclimation in Genetically Related (Sibling) Deciduous and Evergreen Peach (Prunus persica [L.] Batsch) (II. A 60-Kilodalton Bark Protein in Cold-Acclimated Tissues of Peach Is Heat Stable and Related to the Dehydrin Family of Proteins) , 1994, Plant physiology.

[39]  T. Close,et al.  A view of plant dehydrins using antibodies specific to the carboxy terminal peptide , 1993, Plant Molecular Biology.

[40]  M. Wisniewski,et al.  Cold Acclimation in Genetically Related (Sibling) Deciduous and Evergreen Peach (Prunus persica [L.] Batsch): I. Seasonal Changes in Cold Hardiness and Polypeptides of Bark and Xylem Tissues. , 1992, Plant physiology.

[41]  P. Quinn,et al.  The phase behavior of lipids in photosynthetic membranes , 1987, Journal of bioenergetics and biomembranes.

[42]  W. Larcher,et al.  Frost Survival of Plants: Responses and Adaptation to Freezing Stress , 1987 .

[43]  A. Hirsh.  Vitrification in plants as a natural form of cryoprotection. , 1986, Cryobiology.

[44]  E. Beck,et al.  Frost Resistance in Spruce (Picea abies (L.) Karst.): V. Influence of Photoperiod and Temperature on the Membrane Lipids of the Needles , 1982 .

[45]  M. Senser.  Frost Resistance in Spruce [Picea abies(L.) Karst]: III. Seasonal Changes in the Phospho- and Galactolipids of Spruce Needles , 1982 .

[46]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[47]  A. Sakai.  Survival of the Twig of Woody Plants at −196° C. , 1960, Nature.

[48]  P. Schaberg,et al.  Going to extremes: low temperature tolerance and acclimation in temperate and boreal conifers , 2009 .

[49]  V. Lumbreras,et al.  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 , 2007 .

[50]  M. Uemura,et al.  Responses of the plasma membrane to low temperatures , 2006 .

[51]  T. Artlip,et al.  Seasonal expression of a dehydrin gene in sibling deciduous and evergreen genotypes of peach (Prunus persica [L.] Batsch) , 2004, Plant Molecular Biology.

[52]  S. Colombo,et al.  Conifer Cold Hardiness , 2001, Tree Physiology.

[53]  M. Tesche.  BuchbesprechungA. Sakai, W. Larcher, Frost Survival of Plants. Responses and Adaptation to Freezing Stress., Springer-Verlag, Berlin-Heidelberg-New York-LondonParis-Tokyo (1987), Series Ecological Studies 62. 321 S . , 200 Abb., zahlr. Tab. , Preis : DM 198. , 1988 .

[54]  Professor Dr. Walter Larcher,et al.  Frost Survival of Plants , 1987, Ecological Studies.

[55]  P. Wareing.  Tree Physiology , 1957, Nature.