Antifreeze Protein Produced Endogenously in Winter Rye Leaves

Abstract

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.Overwintering plants survive freezing temperatures by forming ice within their tissues. In these plants, it has generally been believed that ice formation occurs spontaneously in large vessels in plant tissues and then spreads throughout the plant at a rate determined by the environmental temperature (14). However, it has been clearly demonstrated in other frost-tolerant organisms such as insects, reptiles, and mollusks that the process of ice formation is controlled by specific proteins. These organisms all produce proteins that function as ice nucleators to initiate extracellular ice formation (6,15,19). Moreover, freezing-tolerant insects also produce antifreeze proteins that modify the ice crystals that form outside the cells (6).We hypothesized that frost-tolerant plants may also produce proteins that control extracellular ice formation. We chose to conduct our experiments using winter rye (Secale cereale L.), an overwintering, herbaceous monocot that can survive temperatures below -200C (11). It has been shown by scanning EM that cold-acclimated winter rye leaves survive freezing temperatures by forming ice only in intercellular spaces (17). In the experiments reported here, we examined cold-acclimated rye leaves for the capacity to modify ice in a manner similar to that observed for antifreeze proteins purified from arctic and antarctic fish (5) and frost-tolerant insects (6).To prove that antifreeze proteins exist in plants, we must demonstrate that plant extracts possess two characteristics shown to be unique to previously purified antifreeze proteins. The first characteristic of antifreeze proteins is a distinctive, concentration-dependent effect on ice crystal morphology (5). In pure water, ice normally grows parallel to the basal plane (a axes) of the crystal lattice, with little growth perpendicular to the basal plane (the c axis), so that the ice crystals appear flat and round. Low concentrations (nM) of antifreeze proteins preferentially inhibit the growth of ice along the a axes so that the hexagonal prism faces of the crystal become evident. At higher concentrations (Mm) of antifreeze protein, the crystals grow predominantly along the c axis to form hexagonal bipyramids and needle-like crystals (5). The second unique characteristic of antifreeze proteins is a measurable degree of thermal hysteresis. Thermal hysteresis, the difference between freezing and melting temperatures, is determined by observing the effect of temperature on the g...

Immunolocalization of Antifreeze Proteins in Winter Rye Leaves, Crowns, and Roots by Tissue Printing

“…Winter rye has been shown to accumulate proteins in the leaf apoplast during cold acclimation (Griffith et al, 1992;Marentes et al, 1993;Hon et al, 1994). These apoplastic proteins, which have the ability to modify ice crystal growth, were identified as GLPs, CLPs, and TLPs using amino-terminal amino acid sequence comparisons, immuno-cross-reactions, and enzyme activity assays (Hon et al, 1995).…”

“…During cold acclimation, winter rye leaves produce intrinsic ice nucleators that have been shown to initiate the formation of extracellular ice during freezing under controlled conditions (Brush et al, 1994). Moreover, winter rye has been shown to accumulate AFPs, which have the ability to modify the growth of ice (Griffith et al, 1992;Marentes et al, 1993). AFP secretion in winter rye is associated This work was supported by grants from the Natural Science and Engineering Research Council of Canada to M.G., the Canadian Space Agency to D.S.C.Y., the Academy of Finland to K.P.-M., and the fellowships from the Jenny and Antti Wihuri Foundation and the Turku University Foundation to M.A.…”

“…' I 1 O, 1996 know where these proteins accumulate in CA tissue. Although winter rye AFPs have been extracted from the leaf apoplast (Griffith et al, 1992), their presence in other cellular compartments or their tissue specificity has not been determined. In this paper, we report, to our knowledge for the first time, the localization of AFPs in plant tissues.…”

Antifreeze Proteins in Winter Rye Are Similar to Pathogenesis-Related Proteins

“…When extracellular ice begins to form, its further growth and propagation through the plant is restricted by a number of factors, including cell-wall modifications (Wisniewski et al, 1991(Wisniewski et al, , 1993Chalker-Scott, 1992), arabinoxylans (Olien, 1965;Kindel et al, 1989), and AFPs (Griffith et al, 1992;Urrutia et al, 1992). AFPs adsorb onto ice This work was supported by research grants from the Natural Science and Engineering Research Council of Canada (M.G.…”

Antifreeze proteins in naturally cold acclimated leaves of Drimys angustifolia, Senecio icoglossus, and Eucalyptus ssp.

“…Since Griffith et al (1992) described, in a pioneering way, the presence of AFPs in winter rye leaves (Secale cereale), which were later studied by Hon et al (1994), Antikainen and Griffith (1997) and Lim et al (2013), the presence of AFPs with different characteristics have been detected in wheat (Triticum aestivum) (ANTIKAINEN; GRIFFITH, 1997;ZHANG et al, 2007), carrots (Daucus carota) (SMALLWOOD et al, 1999;WANG et al, 2002;ZHANG et al, 2004;GOMEZ GALINDO et al, 2005;DING et al, 2014), ryegrass (Lolium perenne) (SIDEBOTTOM et al, 2000;PUDNEY et al, 2003;MIDDLETON et al, 2012), Solanum tuberosum (URRUTIA et al, 1992), Solanum dulcamara (URRUTIA et al, 1992;DUMAN, 2002), Forsythia suspensa (SIMPSON et al, 2005), Picea abis and Picea pungens (JARZABEK et al, 2009), among many others.…”