A carrot leucine-rich-repeat protein that inhibits ice recrystallization

Science

Volumn 282, Issue 5386, 1998, Pages 115-117

  Worrall, Dawn ^a   Elias, Luisa ^a   Ashford, David ^a   Smallwood, Maggie ^a   Sidebottom, Chris ^b   Lillford, Peter ^b   Telford, Julia ^b   Holt, Chris ^b   Bowles, Dianna ^a  

^a UNIVERSITY OF YORK   (United Kingdom)

^b UNILEVER RESEARCH   (United Kingdom)

Author keywords

[No Author keywords available]

Indexed keywords

ANTIFREEZE PROTEIN; INHIBITOR PROTEIN; LEUCINE;

AMINO ACID SEQUENCE; ARTICLE; CARROT; COLD TOLERANCE; CRYSTALLIZATION; FISH; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN EXPRESSION; SEQUENCE ANALYSIS;

AMINO ACID SEQUENCE; ANTIFREEZE PROTEINS; CLONING, MOLECULAR; CRYSTALLIZATION; DAUCUS CAROTA; DNA, COMPLEMENTARY; GLYCOPROTEINS; GLYCOSYLATION; ICE; ISOELECTRIC POINT; LEUCINE; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; MOLECULAR WEIGHT; PLANT PROTEINS; PLANT ROOTS; PLANTS, GENETICALLY MODIFIED; PLANTS, TOXIC; TOBACCO;

EID: 0032475833     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5386.115     Document Type: Article

References (41)

1. J. G. Duman, D. W. Wu, T. M. Olsen, M. Urrutia, D. Tursman, Adv. Low Temp. Biol. 2, 131 (1993); P. L. Davies and C. L. Hew, FASEB J. 4, 2460 (1990).
2. J. G. Duman, D. W. Wu, T. M. Olsen, M. Urrutia, D. Tursman, Adv. Low Temp. Biol. 2, 131 (1993); P. L. Davies and C. L. Hew, FASEB J. 4, 2460 (1990).
3. P. L. Davies and B. D. Sykes, Curr. Opin. Struct. Biol. 7, 828 (1997).
4. Y. Yeh and R. E. Feeney, Chem. Rev. 96, 601 (1996).
5. M. Griffith and K. V. Ewart, Biotechnol. Adv. 13, 375 (1995); J. G. Duman and T. M. Olsen, Cryobiology 30, 322 (1993); M. E. Urrutia, J. G. Duman, C. A. Knight, Biochim. Biophys. Acta 1121, 199 (1992).
6. M. Griffith and K. V. Ewart, Biotechnol. Adv. 13, 375 (1995); J. G. Duman and T. M. Olsen, Cryobiology 30, 322 (1993); M. E. Urrutia, J. G. Duman, C. A. Knight, Biochim. Biophys. Acta 1121, 199 (1992).
7. M. Griffith and K. V. Ewart, Biotechnol. Adv. 13, 375 (1995); J. G. Duman and T. M. Olsen, Cryobiology 30, 322 (1993); M. E. Urrutia, J. G. Duman, C. A. Knight, Biochim. Biophys. Acta 1121, 199 (1992).
8. W.-C. Hon, M. Griffith, A. Mlynarz, Y. C. Kwok, D. S. C. Yang, Plant Physiol. 109, 879 (1995).
9. J. G. Duman, Biochim. Biophys. Acta 1206, 129 (1994).
10. T. Huang and J. G. Duman, Cryobiology 32, 577 (1995).
11. M. Smallwood et al., unpublished results. Details of carrot AFP purification, antibody preparation, and the modified version of the Splat assay [C. A. Knight, J. Hallett, A. L. DeVries, ibid. 2, 55 (1988)] for RI activity will be published elsewhere.
12. M. Smallwood et al., unpublished results. Details of carrot AFP purification, antibody preparation, and the modified version of the Splat assay [C. A. Knight, J. Hallett, A. L. DeVries, ibid. 2, 55 (1988)] for RI activity will be published elsewhere.
13. TH was assayed as described by A. L. DeVries [Methods Enzymol. 127, 293 (1986)]. Purified AFP was adjusted to a protein concentration of 1 mg/ml; homogenate of leaves from transgenic tobacco line 35-9-CA was assayed at a total protein concentration of 14.2 mg/ml, and that from line 35-9-CB was assayed at a total protein concentration of 7.5 mg/ml.
14. Monoclonal antibody YZ1/2.23 [M. T. McManus et al., Planta 175, 56 (1988)] was used for immunodetection of N-glycans. Enzymic deglycosylation: Purified carrot AFP (2.5 mg/ml) was incubated with α-mannosidase (50 U/ml), β-hexosaminidase (10 U/ml), and β-galactosidase (1 U/ml) from jack bean; β-mannosidase (4 U/ml) from Helix pomatia (all from Oxford Glycosciences, Abingdon, UK); β-xylosidase (0.1 U/ml) from tobacco (F. Khan and D. Ashford, unpublished data); and α-fucosidase (2 U/ml) from bovine kidney (Boehringer-Mannheim) in 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.02% sodium azide and bovine serum albumin (1.1 mg/ml) for 16 hours at 37°C together with appropriate controls.
15. Monoclonal antibody YZ1/2.23 [M. T. McManus et al., Planta 175, 56 (1988)] was used for immunodetection of N-glycans. Enzymic deglycosylation: Purified carrot AFP (2.5 mg/ml) was incubated with α-mannosidase (50 U/ml), β-hexosaminidase (10 U/ml), and β-galactosidase (1 U/ml) from jack bean; β-mannosidase (4 U/ml) from Helix pomatia (all from Oxford Glycosciences, Abingdon, UK); β-xylosidase (0.1 U/ml) from tobacco (F. Khan and D. Ashford, unpublished data); and α-fucosidase (2 U/ml) from bovine kidney (Boehringer-Mannheim) in 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.02% sodium azide and bovine serum albumin (1.1 mg/ml) for 16 hours at 37°C together with appropriate controls.
16. A. L. DeVries, Science 172, 1152 (1971).
17. Internal peptide sequences were obtained according to J. Rosenfeld, J Capdevielle, J. C. Guillemot, and P. Ferrara [Anal. Biochem. 203, 173 (1992)]. The peptide sequences are as follows: CP1, PNLFGKFN; CP2, QFFDXTAYL; CP208, IPEESALK; CP209, LTXLDLSFNK; CP3, LRLSSTSLSGPVPLFFPQL; and CP4, LL(G/R)(V/L) IPKQLSTLPNL (26).
18. 2 and primer concentrations of 1 μM. The ∼800-base pair amplified fragment was cloned and used to screen a cold-acclimated carrot tap root cDNA library (ZAP cDNA kit; Stratagene) to obtain the full-length AFP clone. Seventeen hybridizing plaques were identified, seven were partially sequenced, and two were sequenced to completion, both of which contained identical coding regions representing the carrot AFP.
19. J. Draper, R. Scott, J. Hamil, in Plant Genetic Transformation and Gene Expression, J. Draper, R. Scott, P. Armitage, R. Walden, Eds. (Blackwell Scientific, Oxford, 1988), pp. 69-160.
20. K. D. Kenward, M. Altschuler, D. Hildebrand, P. L. Davies, Plant Mol. Biol. 23, 377 (1993); R. Hightower, C. Baden, E. Penzes, P. Lund, P. Dunsmuir, ibid. 17, 1013 (1991).
21. K. D. Kenward, M. Altschuler, D. Hildebrand, P. L. Davies, Plant Mol. Biol. 23, 377 (1993); R. Hightower, C. Baden, E. Penzes, P. Lund, P. Dunsmuir, ibid. 17, 1013 (1991).
22. Leaves were either homogenized in buffer A [50 mM tris-HCl, 10 mM EDTA, and 20 mM ascorbic acid (pH 7.5)] containing 30% (w/w) sucrose or vacuum infiltrated with buffer A and ICF prepared as described [R. Rohringer, F. Ebrahim-Nesbat, G. Wolf, J. Exp. Bot. 34, 1589 (1983)]. Where necessary, protein was concentrated by precipitation with 5× volume ice-cold acetone. Protein concentration was determined by the Bio-Rad assay according to the manufacturer's instructions.
23. A. Collmer and N. T. Keen, Annu. Rev. Phytopathol. 24, 383 (1986).
24. Ten microliters of PG enzyme [A. niger PG (1 U/ml); Sigma] was added to 40 μl of 300 mM sodium acetate buffer with purified carrot AFP (±0.2 mg/ml). After 15 min of preincubation at 37°C, 100 μl of polygalacturonan substrate [0.5% (w/v) deesterified citrus polygalacturonic acid] was added and incubated at 37°C for 20 min. Reducing end groups released by PG were assayed [K. C. Gross, Hortic. Sci. 17, 933 (1982)]. PG activity was eluted from a cell wall fraction of ripe tomato fruit [L. S. Zheng, R. C. Heupel, D. Dellapenna, Plant Cell 4, 1147 (1992)] and assayed as above. When testing for inhibition of the tomato enzyme, AFP was also preincubated with substrate present at limiting concentrations (>0.05% polygalacturonan) before assay.
25. Ten microliters of PG enzyme [A. niger PG (1 U/ml); Sigma] was added to 40 μl of 300 mM sodium acetate buffer with purified carrot AFP (±0.2 mg/ml). After 15 min of preincubation at 37°C, 100 μl of polygalacturonan substrate [0.5% (w/v) deesterified citrus polygalacturonic acid] was added and incubated at 37°C for 20 min. Reducing end groups released by PG were assayed [K. C. Gross, Hortic. Sci. 17, 933 (1982)]. PG activity was eluted from a cell wall fraction of ripe tomato fruit [L. S. Zheng, R. C. Heupel, D. Dellapenna, Plant Cell 4, 1147 (1992)] and assayed as above. When testing for inhibition of the tomato enzyme, AFP was also preincubated with substrate present at limiting concentrations (>0.05% polygalacturonan) before assay.
26. Sequence analyses were done with Wisconsin Package Version 9.1 (Genetics Computer Group, Madison, WI, 1997).
27. J. M. Logsdon and W. F. Doolittle, Proc. Natl. Acad. Sci. U.S.A. 94, 3485 (1997).
28. C. B. Rajeshekar and A. Lafta, Plant Physiol. 111, 605 (1996); B. L. Porwal, Y. K. Mathur, S. P. Goswami, P. L. Maliwal, Indian J. Agron. 34, 293 (1989); M. Wisniewski, E. N. Ashworth, K. Schaffer, Protoplasma 141, 160 (1987).
29. C. B. Rajeshekar and A. Lafta, Plant Physiol. 111, 605 (1996); B. L. Porwal, Y. K. Mathur, S. P. Goswami, P. L. Maliwal, Indian J. Agron. 34, 293 (1989); M. Wisniewski, E. N. Ashworth, K. Schaffer, Protoplasma 141, 160 (1987).
30. C. B. Rajeshekar and A. Lafta, Plant Physiol. 111, 605 (1996); B. L. Porwal, Y. K. Mathur, S. P. Goswami, P. L. Maliwal, Indian J. Agron. 34, 293 (1989); M. Wisniewski, E. N. Ashworth, K. Schaffer, Protoplasma 141, 160 (1987).
31. B. Kobe and J. Deisenhofer, Curr. Opin. Struct. Biol. 5, 409 (1995); Trends Biochem. Sci. 19, 415 (1994).
32. B. Kobe and J. Deisenhofer, Curr. Opin. Struct. Biol. 5, 409 (1995); Trends Biochem. Sci. 19, 415 (1994).
33. _, Nature 366, 751 (1993).
34. Z. C. Jia, C. I. Deluca, H. M. Chao, P. L. Davies, ibid. 384, 285 (1996); F. D. Sönnichsen, B. D. Sykes, H. Chao, P. L. Davies, Science 259, 1154 (1993).
35. Z. C. Jia, C. I. Deluca, H. M. Chao, P. L. Davies, ibid. 384, 285 (1996); F. D. Sönnichsen, B. D. Sykes, H. Chao, P. L. Davies, Science 259, 1154 (1993).
36. J. A. Bertrand et al., EMBO J. 15, 2678 (1996); F. D. Sonnichsen, B. D. Sykes, P. L. Davies, Protein Sci. 4, 460 (1995).
37. J. A. Bertrand et al., EMBO J. 15, 2678 (1996); F. D. Sonnichsen, B. D. Sykes, P. L. Davies, Protein Sci. 4, 460 (1995).
38. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; X, any amino acid; and Y, Tyr.
39. P. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975).
40. H. U. Stotz, J. J. A. Contos, A. L. T. Powell, A. B. Bennett, J. M. Labavitch, Plant Mol. Biol. 25, 607 (1994).
41. D. Worrall, L. Elias, and M. Smallwood were supported by a grant from Unilever Central Research Fund. L. Doucet and A. Sutcliffe are thanked for technical support. S. Sparrow and P. Crosby are thanked for help with manuscript preparation.