Summary
Apoplastic antifreeze proteins (AFPs) accumulate in winter rye (Secale cereale L. cv. Musketeer) leaves during cold acclimation. Two of the rye AFPs with molecular masses of 32 and 35 kDa are similar in their amino acid sequences and epitopes to β-1, 3-endoglucanase. Localization of these AFPs, which we refer to as glucanase-like proteins (GLPs), was carried out with antiserum raised against the 32 kDa AFP. Specimens from leaves and roots of non-acclimated (NA) plants and cold acclimated (CA) plants were prepared by freeze-substitution for high resolution immunoelectron microscopy. In CA leaves, high levels of GLPs were observed in cell walls of mesophyll cells adjacent to intercellular spaces and in secondary thickenings of xylem vessels. Taken together with the absence of GLPs in vacuoles, these results confirm the apoplastic accumulation of AFPs in CA winter rye. Within the cells of CA leaves, GLPs were localized in cisternae of the rough endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which indicates that GLPs are secreted via an exocytic bulk-flow pathway. The occurrence of high levels of GLPs in CA leaves, their low presence in NA leaves and the lack of GLPs in roots all suggest that there is a correlation between increased accumulation of GLPs and increased freezing tolerance of these plant materials. Furthermore, the localization of GLPs in the immediate vicinity of pathways for free water within the tissues supports the view that these proteins have an important role in the crystallization and/or recrystallization of water when the leaves of CA winter rye are exposed to freezing temperatures.
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Abbreviations
- AFP:
-
antifreeze protein
- BSA:
-
bovine serum albumin
- CA:
-
cold acclimated
- GAR:
-
goat antirabbit antiserum conjugated with colloidal gold
- GLP:
-
glucanase-like protein
- NA:
-
non-acclimated
- PBS:
-
phosphate buffered saline
- PR:
-
pathogenesis related
References
Abeles FB, Forrence LE (1970) Temporal and hormonal control of β-1,3-glucanase inPhaseolus vulgaris L. Plant Physiol 45: 395–400
Antikainen M, Griffith M, Zhang J, Hon W-C, Yang DSC, Pihakas-ki-Maunsbach K (1996) Immunolocalization of antifreeze proteins in winter rye leaves, crowns, and roots by tissue printing. Plant Physiol (in press)
Benhamou N, Grenier J, Asselin A, Legrand M (1989) Immunogold localization of β-1,3-glucanases in two plants infected by vascular wilt fungi. Plant Cell 1: 1209–1221
Bol JF, Linhorst HJM, Cornelissen BJC (1990) Plant pathogenesis-related proteins induced by virus infection. Annu Rev Phytopathol 28: 113–138
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 341–374
Cabello F, Jorrín JV, Tena M (1994) Chitinase and β-1,3-glucanase activities in chickpea (Cicer arietinum). Induction of different isoenzymes in response to wounding and ethephon. Physiol Plant 92: 654–660
Carlemalm E, Garavito RM, Villiger W (1982) Resin development for electron microscopy and an analysis of embedding at low temperature. J Microsc 126: 123–143
Cassab GI, Varner JE (1988) Cell wall proteins. Annu Rev Plant Physiol Plant Mol Biol 39: 321–353
Chrispeels MJ (1991) Sorting of proteins in the secretory system. Annu Rev Plant Physiol Plant Mol Biol 42: 21–53
Denecke J, Botterman J, Deblaere R (1990) Protein secretion in plant cells can occur via a default pathway. Plant Cell 2: 51–59
DeVries AL (1986) Antifreeze glycopeptides and peptides: interactions with ice and water. Methods Enzymol 127: 293–303
Griffith M, Antikainen M (1996) Extracellular ice formation in freezing-tolerant plants. Adv Low Temp Biol 3 (in press)
—, Brown GN (1982) Cell wall deposits in winter ryeSecale cereale L. “Puma” during cold acclimation. Bot Gaz 143: 486–490
—, Mclntyre HCH (1990) The effect of photoperiod and temperature on growth and frost resistance of winter rye root systems. Physiol Plant 79: 519–525
—, Huner NPA, Espelie KE, Kolattukudy PE (1985) Lipid polymers accumulate in the epidermis and mestome sheath cell walls during low temperature development of winter rye leaves. Protoplasma 125: 53–64
—, Ala P, Yang DSC, Hon W-C, Moffatt BA (1992) Antifreeze protein produced endogenously in winter rye leaves. Plant Physiol 100: 593–596
Griffiths G (1993) Fine structure immunocytochemistry. Springer, Berlin Heidelberg New York Tokyo
Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Calif Agric Exp Stat Circ 347: 1–32
Hon W-C, Griffith M, Chong P, Yang DSC (1994) Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves. Plant Physiol 104: 971–980
—, Griffith M, Mlynarz A, Kwok YC, Yang DSC (1995) Antifreeze proteins in winter rye are similar to pathogenesis-related proteins. Plant Physiol 109: 879–889
Huner NPA, Palta JP, Li PH, Carter JV (1981) Anatomical changes in leaves of Puma rye in response to growth at cold-hardening temperatures. Bot Gaz 142: 55–62
Kauffmann S, Legrand M, Geoffrey P, Fritig B (1987) Biological function of “pathogenesis-related” proteins: four PR proteins of tobacco have 1,3-β-glucanase activity. EMBO J 6: 3209–3212
Keefe D, Hinz U, Meins F Jr (1990) The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves. Planta 182: 43–51
Kurosaki F, Tokitoh Y, Morita M, Nishi A (1989) Localization and secretion of β-1,3-glucanase of cultures carrot cells. Plant Sci 65: 39–43
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Marentes E, Griffith M, Mlynarz A, Brush RA (1993) Proteins accumulate in the apoplast of winter rye leaves during cold acclimation. Physiol Plant 87: 499–507
Mauch F, Staehelin LA (1989) Functional implications of the subcellular localization of ethylene-induced chitinase and β-1,3-glucanase in bean leaves. Plant Cell 1: 447–457
—, Meehl JB, Staehelin LA (1992) Ethylene-induced chitinase and β-1,3-glucanase accumulate specifically in the lower epidermis and along vascular strands of bean leaves. Planta 186: 367–375
Maunsbach AB (1994) Embedding of cells and tissues for ultrastructural and immunocytochemical analysis In: Cells JE (ed) Cell biology, a laboratory handbook, vol 2. Academic Press, San Diego, pp 117–125
Newman GR, Hobot JA (1993) Resin microscopy and on-section immunocytochemistry. Springer, Berlin Heidelberg New York Tokyo
Pearce RS (1988) Extracellular ice and cell shape in frost-stressed cereal leaves: a low-temperature scanning-electron-microseopy study. Planta 175: 313–324
—, Asworth EN (1992) Cell shape and localization of ice in leaves of overwintering wheat during frost stress in the field. Planta 188: 324–331
Peterson CA, Griffith M, Huner NPA (1985) Permeability of the suberized mestome sheath in winter rye. Plant Physiol 77: 157–161
Pihakaski-Maunsbach K, Puhakainen T (1995) Effect of cold exposure on cortical microtubules of rye (Secale cereale) as observed by immunocytochemistry. Physiol Plant 93: 563–571
Raikhel NV, Lee H-I, Broekaert WF (1993) Structure and function of chitin-binding proteins. Annu Rev Plant Physiol Plant Mol Biol 44: 591–615
Raymond JA, DeVries AL (1977) Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci USA 74: 2589–2593
Rohringer R, Ebrahim-Nesbat F, Wolf G (1983) Proteins in intercellular washing fluids from leaves of barley (Hordeum vulgare L.). J Exp Bot 34: 1589–1605
Sock J, Rohringer R, Kang Z (1990) Extracellular β-1,3-glucanases in stem rust-affected and abiotically stressed wheat leaves. Plant Physiol 94: 1376–1389
Sticher L, Hinz U, Meyer AD, Meins F Jr (1992) Intracellular transport and processing of a tobacco vacuolar β-1,3-glucanase. Planta 188: 559–565
Van Den Bulcke M, Bauw G, Castresana C, Van Montagu M, Vandekerckhove J (1989) Characterization of vacuolar and extracellular β(1,3)-glucanases of tobacco: evidence for a strictly compartmentalized plant defence system. Proc Natl Acad Sci USA 86: 2673–2677
Wubben JP, Joosten MHAJ, Van Kan JAL, DeWit PJGM (1992) Subcellular localization of plant chitinases and 1,3-β-glucanases inCladosporium fulvum (syn.Fulvia fulva)-infected tomato leaves. Physiol Mol Plant Pathol 41: 23–32
—, Eijkelboom CA, De Wit PJGM (1993) Accumulation of pathogenesisrelated proteins in the epidermis of tomato leaves infected byCladosporium fulvum. Neth J Plant Pathol 99 Suppl 3: 231–239
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Pihakaski-Maunsbach, K., Griffith, M., Antikainen, M. et al. Immunogold localization of glucanase-like antifreeze protein in cold acclimated winter rye. Protoplasma 191, 115–125 (1996). https://doi.org/10.1007/BF01281809
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DOI: https://doi.org/10.1007/BF01281809