Genes encoding chitinase-antifreeze proteins are regulated by cold and expressed by all cell types in winter rye shoots.
ABSTRACT One group of antifreeze proteins (AFPs) is composed of two chitinases that accumulate in the apoplast of winter rye leaves during cold acclimation. In this study, the 28- and 35-kDa chitinase-AFPs were localized in nonacclimated and cold-acclimated rye leaves by immunoelectron microscopy with an antiserum produced against the purified winter rye 35-kDa chitinase-AFP. In cold-acclimated winter rye leaves, labelled chitinase-AFPs were abundant in the walls of epidermal, parenchymal sheath and mesophyll cells and xylem vessels, while less label was present in walls of vascular parenchyma cells. In contrast, chitinase labelling was essentially absent in the nonacclimated cells except in xylem vessels. As shown by RNA blotting, the transcripts of chitinase-AFPs accumulated to a high level in rye leaves during cold acclimation, to a lesser extent in crowns and were not detectable in roots. mRNA transcripts of the 28-kDa chitinase-AFP were localized in rye leaves by in situ hybridization. The chitinase-AFP transcripts were found in the same cell types as the protein itself. We conclude that all metabolically active cell types in cold-acclimated winter rye leaves and crowns are able to synthesize chitinase-AFPs and secrete them into adjacent cell walls, where they may interact with ice to delay its propagation through the plant and modify its growth.
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ABSTRACT: Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.Plant physiology 05/1999; 119(4):1361-70. · 6.56 Impact Factor
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ABSTRACT: The induction of pollen embryogenesis in Capsicum annuum L. has been studied at the cellular level using various in situ approaches with several molecular probes for DNA, RNA and proteins. The late vacuolated microspore and the young bicellular pollen grain are stages of gametophytic development in which embryogenesis can be induced. Our results show that the late vacuolated microspore stage is most responsive to embryogenesis induction. The proliferating cell nuclear antigen (PCNA) has been immunolocalized at the electron microscopy level, in order to map replication sites in relation to the fine structure of chromatin. It shows different patterns of labelling at both developmental stages studied, revealing that the late vacuolated microspore is in a period of replication. Other in situ studies have been performed to characterize the state of nuclear activity at the specific developmental stages in which the embryogenic induction can occur. The modern in situ terminal-deoxy-nucleotidyl transferase (TdT) reaction for DNA, the immunolocalization of various nuclear antigens (as snRNPs, fibrillarin, RNA) and the ultrastructural in situ hybridization using 18S and 25S ribosomal probes provided valuable data bout the specific features displayed by the functional nuclear compartments of the microspore, and the young vegetative and generative cells. They are related not only to the state of gene activity but also with probably the ability to switch to the sporophytic pathway at specific developmental times of their gametophytic program.European Journal of Cell Biology 05/1996; 69(4):373-86. · 3.21 Impact Factor
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ABSTRACT: During cold acclimation, winter rye (Secale cereale L.) plants secrete antifreeze proteins that are similar to pathogenesis-related (PR) proteins. In this experiment, the secretion of PR proteins was induced at warm temperatures by infection with pink snow mold (Microdochium nivale), a pathogen of overwintering cereals. A comparison of cold-induced and pathogen-induced proteins showed that PR proteins accumulated in the leaf apoplast to a greater level in response to cold. The PR proteins induced by cold and by snow mold were similar when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting. Both groups of PR proteins contained glucanase-like, chitinase-like, and thaumatin-like proteins, and both groups exhibited similar levels of glucanase and chitinase activities. However, only the PR proteins induced by cold exhibited antifreeze activity. Our findings suggest that the cold-induced PR proteins may be isoforms that function as antifreeze proteins to modify the growth of ice during freezing while also providing resistance to the growth of low-temperature pathogens in advance of infection. Both functions of the cold-induced PR proteins may improve the survival of overwintering cereals.Plant physiology 11/1999; 121(2):665-74. · 6.56 Impact Factor