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Plant Cene Register
Nucleotide Sequence of a Gene Encoding a 58.5-Kilodalton Barley Dehydrin That Lacks a Serine Tract' Timothy J. Close*, Nicole C. Meyer, and Judy Radik Department of Botany and Plant Sciences, University of California, Riverside, California 92521-01 24
(T.J.C., N.C.M.); and Commonwealth Scientific and Industrial Research Organization Division of Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia (J.R.) Dehydrins, also known as the late embryogenesis abundant (LEA) D-11 family of proteins (Close et al., 1993, and refs. therein), generally accumulate in plants in response to water deficit, embryo desiccation, cold temperature, reduction in externa1 osmotic potential, or application of ABA. Over 40 examples of deduced amino acid sequences of higher plant dehydrins have been published to date, and there is immunological evidence of related proteins in organisms as distant from higher plants as cyanobacteria (Close and Lammers, 1993). Known members of this family of proteins are characterized by the presence of highly conserved repeating motifs including the Lys-rich 15amino acid consensus EKKGIMDKIKEKLPG, which is always located near the carboxy terminus (the lone exception being cotton LEA D-11). A related but slightly less conserved consensus sequence is usually repeated at least once and up to 10 additional times in published examples of plant dehydrins. Dehydrins can be divided into two main classes, those containing approximately nine consecutive Ser residues and those without this Ser tract. The Ser residues in this tract can be phosphorylated, and it has been proposed that phosphorylation of Ser's is related to the binding of nuclear localization signal peptides (Goday et al., 1994). Dehydrins have a nucleocytoplasmic location (Asghar et al., 1994; Goday et al., 1994). The majority of dehydrins identified to date fall into the class that contains the Ser tract, and the minority fall into the latter class, without the Ser tract. Those in the former class usually contain two or three Lys-rich consensus blocks. Dehydrins in the latter class usually contain six or more consensus blocks. There are exceptions to these generalizations, and each class can be subdivided further based on intervening amino acid sequences located between the consensus blocks. Examples of dehydrins that lack a Ser tract include spinach CAP85 (61.5 kD with 11 repeats) (Neven et al., 1993), wheat WCS120 (39.0 kD with 6 repeats) (Houde et al., 1992)' and wheat COR39 (39.1 kD
Table I. Characteristics of Himalaya barley dhn5 sequence Organism: Hordeum vulgare L. cv Himalaya. Source of Clones: Sau3A partially digested, size-fractionated barley DNA ligated to BamHIISall digested EMBL4 DNA. Approximately 15-kb barley DNA insert in clone HV5. dhn5 located on approximately 6-kb EcoRl subfragment of HV5. Method of Identification: Primary library plaques formed on host Escherichia coli NW2, probed by hybridization with radiolabeled mixture of Himalaya barley dhnl, dhn2, dhn3, and dhn4 cDNA inserts. Nucleotide Sequencing Techniques: EcoRl 6-kb subfragment containing dhn5 subcloned to pTZ plasmid vector as clone HV5-54. Further subclones and seria1 deletion clones were constructed. Both strands of complete coding region were sequenced by dideoxy chain termination using deazaguanine dGTP. Subclone HV5-54 contained approximately 0.6 kb 5' flanking DNA, 1.73 kb open reading frame, and 3.6 kb 3' flanking DNA. We determined 2432 bp including the 5' flanking region and entire open reading frame. Features of Gene Sequence: One continuous open reading frame with no introns. Features of Predicted Amino Acid Sequence: Open reading frame of 575 amino acids (1725 nucleotides); predicted molecular mass of 58,548 D. Nine Lys-rich consensus blocks, no Ser tract. Antibodies: Anti-dehydrin consensus peptide antibodies (Close et al., 1993) detect DHN5 polypeptide produced by in vitro translation and a polypeptide of similar apparent molecular mass in water deficit-stressed seedlinas.
with 6 repeats) (Guo et al., 1992), each of which is induced by low temperature. Four Himalaya barley (Hordeum vulgare L.) dehydrin cDNA clones (dhnl, dhn2, dhn3, and dhn4, originally named dhn8, dhn9, dhnl7, and dhn28, respectively) were described severa1 years ago (Close et al., 1989). The genes dhnl and dhn2 are located on barley chromosome 7 (5H) (Close and Chandler, 1990) and are tightly linked both to each other and to the major winter hardiness determinant of Dicktoo barley (Pan et al., 1994). The genes dhn3 and dhn4 are
This work was supported in part by U.S. Department of Agriculture, National Research Initiative Competitive Grants Program grant No. 91-37100-6615. * Corresponding author; e-mail
[email protected]; fax 1-909-787-4437.
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tightly linked to each other and map to barley chromosome 6 (6H) (Close and Chandler, 1990; Pan et al., 1994). Dehydrins encoded by these four genes a11 contain the Ser tract. Another dehydrin gene, dhn6 (T.J. Close, DNA sequence unpublished), is located on barley chromosome 4 (4H) (Pan et al., 1994). Here w e report the nucleotide sequence of a Himalaya barley dehydrin genomic clone, dhn5, that encodes a 58.5-kD protein containing nine Lys-rich consensus blocks and no Ser tract (Table I). This arrangement of motifs is similar to the known examples of cold-induced dehydrins from spinach and wheat noted above. However, the dhn5 gene is located on barley chromosome 6 (6H) near dhn3 and dhn4, which is not the location of the major winter hardiness determinant in barley (Pan et al., 1994). Based on the sequence similarity of DHN5 to previously identified coldinduced dehydrins and the barley genetic mapping data, it appears possible that DHN5 may be a component of general cold acclimation, but not the major determinant of winter hardiness in Dicktoo barley, which may involve other unlinked dehydrin genes that are present on barley chromosome 7 (5H). Physiological experiments on the effect of low temperature on barley dehydrin expression and genetic tests on the role of dehydrin genes in winter hardiness are underway. Received July 11, 1994; accepted July 18, 1994. Copyright Clearance Center: 0032-0889/95/107/0289/02. The GenBank accession number for the sequence reported in this article is M95810.
Plant Physiol. Vol. 107, 1995 LITERATURE ClTED
Asghar R, Fenton RD, DeMason DA, Close TJ (1994)Nuclear and cytoplasmic localization of maize embryo and akurone dehydrin. Protoplasma 177 87-94 Close TJ, Chandler PM (1990) Cereal dehydrins: sr,rology,gene mapping and potential functional roles. Aust J Plant Physiol 1 7 333-344 Close TJ, Fenton RD, Moonan F (1993)A view of plmt dehydrins using antibodies specific to the carboxy terminal peptide. Plant Mo1 Biol 23: 279-286 Close TJ, Kortt AA, Chandler PM (1989) A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Mo1 Biol 13: 95-108 Close TJ, Lammers PJ (1993) An osmotic stress protein of cyanobacteria is immunologically related to plant dehydrins. Plant Physiol 101: 773-779 Goday A, Jensen AB, Culianez-Macia FA, Mar Alb(sM, Figueras M, Serratosa J, Torrent M, Pages M (1994) The Inaize abscisic acid-responsive protein Rabl7 is located in the nucleus and interacts with nuclear localization signals. Plant Cell 6: 351-360 Guo W, Ward RW, Thomashow MF (1992) Characterization of a cold-regulated wheat gene related to Arubidopsis: Cor47. Plant Physiol 100: 915-922 Houde M, Danyluk J, Laliberte J-F, Rassart E, Dhindsa RS, Sarhan F (2992) Cloning, characterization,and expression of a cDNA encoding a 50-kilodalton protein specifically induced by cold acclimation in wheat. Plant Physiol 99: 1381-1387 Neven LG, Haskell DW, Hofig A, Li Q-B, Guy C1. (1993) Characterization of a spinach gene responsive to lovr temperature and water stress. Plant Mo1 Biol 21: 291-305 Pan A, Hayes PM, Chen F, Chen THH, Blake T, Wi,ight S, Karsai I, Bedo 2 (1994) Genetic analysis of the componcints of winterhardiness in barley (Hordeum vulgure L.). Theor Appl Genet (in press)
