Environmental and Experimental Botany 106 (2014) 60–69

Contents lists available at ScienceDirect Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

The use of proteins for frost protection in sensitive crop

John G. Duman a,∗, Michael J. Wisniewski b a Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA b United States Department of Agriculture, Agricultural Research Service, Kearneysville, WV 25430, USA article info abstract

Article history: Antifreeze proteins (AFPs), also known as ice binding proteins (IBPs), have evolved as an important adap- Received 14 November 2013 tation in numerous organisms exposed to subzero temperatures. AFPs have only been identified Received in revised form in freeze tolerant species (those able to survive extracellular ). Consequently, plant AFPs have 17 December 2013 very low specific activities as they have not evolved to completely prevent ice formation in the plant. Accepted 3 January 2014 In contrast, fish and most insect AFPs function to prevent freezing in species that have evolved freeze Available online 13 January 2014 avoidance mechanisms. Therefore, the activity of these AFPs, especially those of insects (as they are gen- erally exposed to considerably lower temperatures than fish), is much greater. The ability of AFPs to Keywords: Antifreeze proteins non-colligatively lower the freezing point of water (thermal hysteresis) has led to the idea that frost- Plant cold-tolerance sensitive crop plants could avoid damage resulting from common minor frost events in late spring and Transgenic plants early autumn by expressing high activity AFPs that permit them to remain unfrozen to temperatures of ◦ Plant frost injury approximately −5 C. Over the past 20 years, the efficacy of this concept has been tested in a variety of Prevention of plant frost injury studies that produced transgenic plants (including Arabidopsis thaliana, and several crop plants) express- ing various AFPs. Initially, fish AFPs were employed in these studies but as insect AFPs, with higher levels of antifreeze activity, were discovered these have become the AFPs of choice in plant transformation studies. Some studies have produced transgenic plants that have exhibited improved cold tolerance of 1–3 ◦C compared to the wild-type. None of the studies with transgenic plants, however, have yet attained a sufficient level of protection. Progress to this point indicates that more significant results are achievable. If so, the billions of dollars lost annually to frost damage of sensitive crops could be avoided. Geographic ranges and growing seasons could also be expanded. This review provides an overview of the studies of transgenic plants producing AFPs, and makes suggestions for future advancements in this field of study. © 2014 Elsevier B.V. All rights reserved.

1. Introduction apples, cherries, etc.) were lost in the Northeast and Midwest in the spring of 2012. In 2013, Chile experienced a late spring frost Despite overall increases in mean daily temperatures, it is that resulted in a 22% reduction in exportable fruit representing expected that there will be an increase in the number of devas- a loss of 800 million dollars. While efforts were made to pro- tating spring frosts due to the erratic weather patterns associated vide frost protection during all these events, most were futile. with global climate change (Gu et al., 2008). In April, 2007 the Partially to blame for this failure is our continuing lack of knowl- midwest, central and southern plains, and southeast portions of edge about what makes plants freeze at a particular temperature the U.S. experienced a record breaking freezing event that cause and a lack of effective, economical, and environmentally-friendly unprecedented damage to many economically important crops methods of frost protection (Wisniewski et al., 2008). Lower- in excess of 2 billion dollars (Gu et al., 2008). Since that time, ing ice nucleation temperatures and inhibiting the propagation several spring frost events have caused significant losses to the of ice from the outside to the interior of plants is an approach fruit industry. For example, 50–90% of fruit crops (grapes, peaches, to frost protection whose potential has been noted by several authors (Ball et al., 2002; Hacker and Neuner, 2007; Lindow, 1995; Wisniewski et al., 2003, 2008, 2009). The use of highly active antifreeze proteins (AFPs), and possibly antifreeze glycol- Abbreviations: AFP, antifreeze protein; DAFP, antifreeze protein from Den- ipids (AFGLs) (Walters et al., 2009, 2011), represents a logical droides canadensis; IBP, ice binding protein; kDa, kiloDalton; NMR, nuclear approach to limiting frost damage by inhibiting inoculative freezing magnetic resonance; CAT, chloramphenical acetyl transferase; LT50, temperature and enhancing in freeze-sensitive, annual plants and lethal to 50% of the individuals. ∗ newly emerging plant parts on perennial plants. Transgenic plants Corresponding author. Tel.: +1 574 631 9499. E-mail address: [email protected] (J.G. Duman). expressing AFPs could also potentially extend the growing seasons

0098-8472/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.envexpbot.2014.01.001 J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69 61 and expand the geographic ranges of several crops and horticultural factor (e.g. CBF) controlling a cold regulon (defined as a suite of plants. cold-inudcible genes) or overexpression of a specific “cryoprotec- Plant phenology, such as the timing of budbreak and the onset tive” gene. The former approach, however, requires the use of a of flowering and vegetative growth, is strongly controlled by cli- cold-inducible promoter to avoid the adverse effects of overex- mate and as such has become a strong bioindicator of ongoing pression of the transcription factor on growth (Wisniewski et al., climate change (Gordo and Sanz, 2010). An analysis of 29 years 2011). While true cold acclimation requires numerous adaptations, (1971–2000) of phenological data in Europe has indicated that 78% it is still possible that less encompassing short-term approaches of all leafing, flowering and fruiting records have advanced and that can be identified that are capable of protecting sensitive plants, or the average advance of spring has been 2.5 days decade−1 (Menzel plant parts such as blossoms, from spring frosts, where tempera- et al., 2006). A report on Mediterranean ecosystems, which includes tures reach only a few degrees below freezing. The use of antifreeze 29 perennial plant species monitored from 1943 to 2003, stated proteins obtained from freeze avoiding such as insects, are that spring phenological events are changing more than autumn potential candidates. events as the former events are more sensitive to climate condi- Antifreeze proteins (AFPs) are an important component of the tions and are thus undergoing the greatest alterations (Gordo and repertoire of adaptations to subzero temperatures in many orga- Sanz, 2010). Khanduri et al. (2008) in a study of 650 temperate, nisms, including numerous plants. AFPs are thought to function by globally-distributed plant species has reported that spring-related adsorbing onto the surface of ice , blocking the addition of phenological events have advanced 1.9 days decade−1 and autumn- water molecules to growth sites and thereby lowering the temper- related events an average of 1.4 days decade−1. Therefore, the ature at which the will grow (Jia and Davies, 2002; Knight impact of the predicted increase in episodes of spring frosts (Gu et al., 1991; Raymond and DeVries, 1977; Raymond et al., 1989). et al., 2008) will be exacerbated in many species due to the early Recent evidence has demonstrated that some AFPs can also affect onset of spring growth. Additionally, Ball and Hill (2009), in a review water structure at some distance from the actual surface of the of several studies, indicated that elevated atmospheric CO2 concen- AFP and this may also be important in the antifreeze capabilities of trations can have a negative impact on plant cold acclimation and these proteins (Ebbinghaus et al., 2010, 2012; Meister et al., 2013). as a result enhance vulnerability to frost damage. Consequently, in By conventional definition, the freezing and melting points of an spite of anticipated global warming the need for protecting vul- aqueous sample are identical. The temperature at which a small nerable plants from freeze damage will continue and become even crystal will melt completely if the temperature is raised slightly is more critical. the melting point and the temperature at which the crystal will The process by which plants actively undergo changes in gene begin to grow as the temperature is lowered slightly is the freez- expression and biochemistry resulting in an enhanced ability to ing point. However, this is not the case if antifreeze proteins are withstand freezing temperatures and desiccation stress is referred present. AFPs have only a small effect on the normal melting point to as cold acclimation (Weiser, 1970; Wisniewski et al., 2003). of water, raising it slightly (Celik et al., 2010; Knight and DeVries, Mechanisms associated with cold hardiness are generally divided 1989). AFPs, however, decrease the temperature at which an ice into two categories: freeze avoidance and freeze tolerance (Sakai crystal grows, defined as the hysteretic freezing point, by an aver- and Larcher, 1987). Plants that are cold acclimated and can with- age of 1–2 ◦C below the melting point in fishes and 2–5 ◦C in insects, stand exposure to subzero temperatures are principally freeze although the difference can be as much as 13 ◦C in the Alaskan bee- tolerant or exhibit combined mechanisms of freeze tolerance in tle Cucujus clavipes in winter when the insect is dehydrated and the some tissues and freeze avoidance (deep supercooling) in other AFPs concentrated (Duman et al., 2010). This difference between tissues (Kasuga et al., 2007, 2008). In contrast, many animals com- the melting and hysteretic freezing points is termed thermal hys- pletely avoid freezing in winter, and antifreeze proteins are often teresis (TH) and is characteristic of AFPs (DeVries, 1971, 1986). The involved in this ability. For example, freeze-avoiding larvae of the usual technique for determining the presence of AFPs in a sam- Alaskan Cucujus clavipes routinely supercool to −40 ◦Cin ple is to assay for this unique thermal hysteresis activity (DeVries, winter, and when exposed to especially cold temperatures further 1986). The magnitude of the measured thermal hysteresis is often, adapt to avoid freezing to as low as −100 ◦C while vitrifying at depending on the AFP, inversely correlated with the size of the crys- temperatures near −70 ◦C by means of cryoprotective dehydration, tal(s) present in the sample being tested (Husby and Zachariassen, antifreeze proteins and glycolipids, and high molar concentrations 1980; Nicodemus et al., 2006). Consequently, the level of freez- of glycerol (Sformo et al., 2010, 2011; Walters et al., 2009), as well ing protection provided by AFPs in insects to combat inoculative as numerous other mechanisms (Carrasco et al., 2011, 2012). freezing across the cuticle is generally greater than the measured Cold acclimation is a multigenic, quantitative trait that involves thermal hysteresis, because the size of the cuticular water pores biochemical and structural changes that have a dramatic effect on through which external ice might propagate are much smaller than the physiology of a plant (Levitt, 1980; Weiser, 1970). There is no the size of the crystals used in the thermal hysteresis measurements consensus on the number and identity of genes causally related to (Duman et al., 2010). AFPs can also inhibit ice nucleators, thereby cold acclimation. Various reports have estimated that from <100 lowering the nucleation temperature and promoting supercooling to 1000 genes are up-regulated and a similar number are down- (Duman, 2001, 2002). Therefore, AFPs can promote freeze avoid- regulated (Bassett and Wisniewski, 2009; Fowler and Thomashow, ance by inhibition of (1) inoculative freezing across the body surface 2002). Cold acclimation is an inducible process requiring both low and (2) ice nucleators (Duman et al., 2010). temperature (<10 ◦C) and moderate to high light (generally above Although AFPs were first found in Antarctic fish (DeVries, 1971), 400 ␮mol m−2 s−1) to achieve maximum hardiness. The specific thermal hysteresis has been identified in numerous diverse orga- conditions required to achieve maximum cold hardiness are species nisms including many insects (Duman, 1977, 1979a, 2001; Duman specific. It is a dynamic process that may require days or weeks. et al., 2010), collembola (Graham and Davies, 2005; Lin et al., 2007; Because of the complex changes in physiology, metabolism, Zettel, 1984), spiders (Duman, 1979b; Zachariassen and Husby, structure, and water content associated with cold acclimation, 1982), mites (Block and Duman, 1989), nematodes (Wharton et al., plants that are actively growing, flowering, or breaking dormancy 2005), plants (Duman, 1994; Duman and Olsen, 1993; Griffith and typically have little to no frost tolerance (Sakai and Larcher, 1987; Yaish, 2004; Griffith et al., 1992; Huang and Duman, 2002; Simpson Wisniewski et al., 2003) and thus are very susceptible to frost et al., 2005; Smallwood et al., 1999; Urrutia et al., 1992; Worrall damage. Therefore, current approaches to improving frost toler- et al., 1998), and fungi and (Duman and Olsen, 1993; ance typically involve the overexpression of a specific transcription Hoshino et al., 2003; Sun et al., 1995). AFPs have been purified from 62 J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69 many of these species (for reviews see DeVries, 2005; Duman et al., during recent relatively warm winters (Nickell et al., 2013), but 2010; Graether and Sykes, 2004; Griffith and Yaish, 2004). Based supercooled to a mean of −25 ◦C exhibiting a thermal hystere- on their extremely varied structures in different organisms, it is sis of 3–5 ◦C during colder winters (Duman, 1982). During these clear that AFPs have evolved independently numerous times, even very cold winters it was not unusual to identify individual larvae within a given taxon such as fish (Cheng and Chen, 1999; Cheng having 6–9 ◦C of thermal hysteresis in their hemolymph. The AFPs and DeVries, 2002; DeVries, 2005). Also, the specific thermal hys- responsible for the thermal hysteresis in D. canadensis (Andorfer teresis activities of different AFPs vary considerably. For example, and Duman, 2000; Duman et al., 1998; Nickell et al., 2013) are while highly active insect AFPs produce several degrees of thermal similar to those described in other such as Tenebrio molitor hysteresis, in plants the activity is limited to only a few tenths of (Graham et al., 1997; Liou et al., 1999), and Cucujus clavipes (Duman a degree, or less. It is important to note, however, that AFPs have et al., 2004a,b, 2010). As previously mentioned, C. clavipes from only been identified in freeze tolerant plants, where the absolute Alaska typically supercool to −40 ◦C, and below if they are exposed prevention of freezing is not the goal. At times the only indica- to extreme low temperatures (Bennett et al., 2005; Sformo et al., tion of AFP presence in a plant is the recrystallization inhibition of 2010, 2011). Structurally, these beetle AFPs are families of 7–16 kDa ice and/or unusual ice crystal structure, both of which require only proteins consisting of 12-13-mer repeating units in which certain minimal AFP activity. In spite of this low specific activity, plant AFPs amino acids are highly conserved. NMR and X-ray studies (Graether are functionally significant in freeze tolerant species because of and Sykes, 2004; Graether et al., 1999, 2000; Leinala et al., 2002) their recrystallization inhibition, and perhaps other abilities such as revealed the 3-dimensional structure of the T. molitor AFP to be a their impact on crystal structure (Griffith and Yaish, 2004; Griffith flattened cylinder formed by a right-handed ␤-helix. Every sixth et al., 2005; Knight and Duman, 1986). Recrystallization refers to residue of these insect AFPs is a cysteine that forms a disulfide the process that normally occurs after the initial formation of ice. Ice bridge across the interior of the cylinder to provide stability (Li et al., crystals tend to be small when they first form but over time increase 1998a,b). residues adjacent to each cysteine are spaced in size due to the migration of water molecules from the high sur- to permit their hydroxyl groups to hydrogen bond to oxygen atoms face free energy of small (high radius of curvature) crystals onto in the ice lattice. Alternatively, they bind water molecules in an larger crystals. This process is referred to as recrystallization and ice-like fashion and these then bind to the ice. This T-C-T sequence it can result in physical damage to cells. AFPs, even at low concen- forms the ␤-helical structure that presents a flat ice-binding sur- trations, prevent recrystallization, thus promoting freeze tolerance face. Beetle AFPs bind to both the prism and basal planes of ice (Tursman and Duman, 1995). Since all of the known AFP-producing (Graether et al., 2000; Pertaya et al., 2008), and this may account for plants are freeze tolerant, their function in plants is not to prevent their unusually high TH activity. Additionally, Meister et al. (2013) freezing entirely, as is the case with AFPs of freeze avoiding ani- reported that D. canadensis AFPs (DAFPs) can affect water struc- mals. Because of the variability in specific activity and function, the ture up to a distance of 28 A˚ from their surface and that this plays terms ice-binding proteins (IBPs) or ice-structuring proteins (ISPs) are a role in their TH activity. Certain DAFPs exhibit a self-enhancing sometimes used to include true AFPs, as well as those proteins from synergistic effect on the thermal hysteresis activity of other DAFPs freeze tolerant species with minimal thermal hysteresis activity (Wang and Duman, 2005). The >30 AFPs comprising the family of and those with only the ability to inhibit recrystallization or affect DAFPs in D. canadensis are expressed in a tissue specific fashion the shape of ice crystals (Wharton et al., 2009). (Duman et al., 2002; Nickell et al., 2013), and just four are rou- In contrast to the low specific thermal hysteresis activity of tinely present in the hemolymph: DAFPs-1, 2, 4, and 6. DAFP-1 and plant AFPs, those found in freeze avoiding animals, especially cer- -2 enhance one another, as do DAFPs-2 and DAFP-4, and DAFPs-4 tain insects, produce thermal hysteresis of the magnitude that and -6. In addition, a thaumatin-like protein found in D. canadensis, could protect sensitive plants from damage resulting from typi- that lacks thermal hysteresis activity itself, also enhances the activ- cal spring frosts. The thermal hysteresis measured in fish serum is ities of DAFPs-1 and -2 (Wang and Duman, 2006). This synergistic approximately 1.0–2.0 ◦C(DeVries, 2005). Maximum thermal hys- effect has the potential to be of considerable importance in gen- teresis produced by fish AFPs is ∼2.5 ◦C, or a bit higher. While erating enhanced freeze avoidance in transgenic plants expressing this is not exorbitant, it is sufficient to protect fish since the tem- the proper combination of DAFPs. perature of seawater does not generally drop below −1.9 ◦C. In addition to the lower specific activity of fish AFPs relative to those 2. Transgenic AFP plants of insects, there are other problems associated with the use of cer- tain fish AFPs for plant protection. The antifreeze glycoproteins A number of studies have produced transgenic plants express- (AFGPs) present in the prevalent and largely endemic Antarctic fish ing AFPs from other plants, fish, and insects. Most of these did not of the group Nototheniiformes, as well as some northern species produce lowered freezing temperatures in the transgenic plants. such as cods, have relatively simple protein sequences, largely In some systems, AFP expression in transgenic plants produced consisting of repeating tri- of -threonine-alanine plants whose extracts exhibited low levels of thermal hysteresis (DeVries, 1971). Post-translational glycosylation of the threonine (0.37 ◦C/mg apoplastic protein by nanoliter osmometer, Holmberg residues, however, seems to rule-out the use of the genes cod- et al., 2001), recrystallization inhibition in vitro (Worrall et al., ing these proteins for use in transgenic plants since the activity 1998), or reduced electrolyte leakage in leaves after freezing (Wallis of the AFGPs is dependent on the disaccharides (galactosyl-N- et al., 1997; Wang et al., 2008; Zhu et al., 2010). Only the expres- acetylgalactoseamine). On the other hand, a potentially positive sion of AFPs derived from beetle larvae (Huang et al., 2002; Lin et al., aspect of using certain fish AFPs is that, in addition to lowering the 2011) unambiguously resulted in increased levels of supercooling freezing temperature, type-I (winter flounder, etc.) and III (zooar- in the transformants, however, the freezing temperatures of the cids, etc.) AFPs and the AFGLs can provide membrane protection plants were often not reported in other studies. at hypothermic temperatures (Kamijima et al., 2013; Tomczak and Crowe, 2002). Terrestrial insects in cold climates generally experience much 2.1. Transformation with fish AFPs lower temperatures than fish. Therefore, they have evolved more active AFPs. For example, larvae of the beetle Dendroides canaden- Winter flounder, Pseudopleuronectes americanus, fish type sis from northern Indiana supercooled to approximately −20 ◦C and I, AFP was infiltrated into the extracellular spaces of leaves had a mean hemolymph thermal hysteresis of approximately 3 ◦C of canola (Brassica napus), potato (Solanum tuberosum) and J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69 63

Arabidopsis thalianna using vacuum infiltration. This resulted in a AFP in this study, however, may be the difference in codon usage 1.8 ◦C reduction in the average freezing temperature, as measured between fish and plants. by differential thermal analysis, of the leaves relative to control The winter flounder AFP, with codon usage frequencies con- leaves infiltrated with cytochrome C or water (Cutler et al., 1989). sistent with those of higher plants, was also used to successfully The level of protection relative to controls varied with the plant transform potato using Agrobacterium mediated transformation type (1.8 ◦CinB. napus; 4.0 ◦CinA. thalianna. The AFP-infiltrated (Wallis et al., 1997). Inclusion of a native signal directed the leaves also exhibited a decrease in the rate of ice crystal forma- expressed protein to the apoplast. Electrolyte leakage tests (LT50) tion and the amount of freezable water at various temperatures (as (Sukumarin and Weiser, 1972) were used to determine the efficacy measured by NMR). These results provided the impetus for several of the expressed AFP. Leaves from wild-type Russet Burbank potato later studies whose goal was the production of transgenic plants and transformed lines expressing varying levels of AFP (as deter- producing fish, especially winter flounder, AFPs. mined by Western blots) were tested for damage after being held The first successful reported expression of an AFP in for 30 min at temperatures between −2 and −4 ◦C. Freezing was plant material was in corn protoplasts (Georges et al., 1990). A induced by the application of external ice. The wild-type plants ◦ plasmid that included a translational fusion of a gene encoding exhibited an LT50 of −2.5 C while transformed lines expressing the winter flounder type-I fish AFP and the N-terminal of chloram- AFP exhibited a range of lower LT50 values depending on the level phenical acetyltransferase (CAT), along with a signal peptide, with of AFP production. The line expressing the most AFP exhibited an ◦ ◦ codons based on plant preferences, and the cauliflower mosaic virus LT50 of −3.5 C, indicating that the AFP provided approximately 1 C 35S promoter, was introduced into the protoplasts by electropo- of protection relative to the wild-type. The described results were ration. The incorporation and expression of the AFP-cat transcript obtained when leaves were frozen for 30 min periods. Tests were was confirmed in protein extracts of the protoplasts by measuring also conducted using longer freezing times ranging from 1 to 4 h at CAT activity and examining Western blots probed with anti-AFP −2.5 ◦C. The line with the most AFP expression exhibited approxi- and anti-CAT antibodies. In spite of inclusion of a signal peptide mately 22% leakage after a one hour freeze and leakage increased sequence in the afp-cat gene, only a small percentage of the fusion with increased time of freezing, but 50% electrolyte leakage was protein was properly secreted into the medium while approxi- not obtained until leaves had been frozen for four hours. The study mately 75% of the protein remained in the protoplasts, presumably did not determine if the decreased electrolyte leakage in the AFP because the signal peptide was not properly cleaved by proteases. producing transformants resulted from decreased ice nucleation, a The level of cold tolerance of the resulting protoplasts was not decreased rate of ice crystal growth, or an inhibition of recrystal- reported. lization. In other tests, in which leaves were held at temperatures of Hightower et al. (1991) produced transformed tomato and −2to−4 ◦C without external ice nucleation, the AFP producing lines tobacco plants expressing winter flounder AFPs. The authors syn- had less electrolyte leakage, suggesting that decreased ice nuclea- thesized an afa3 gene encoding a native AFP that contains three tion played at least some role in these results. For example, at −4 ◦C units of the alanine rich 11 amino acid repeat from winter flounder, the highest producing AFP line exhibited only 2.3% leakage, while as well as the gene for a fusion protein (Spa-Afa5) encoding a win- the wild-type had 34.4%. In addition to this freeze protection, the ter flounder based AFP with five repeats of the 11 amino acid repeat transgenic plants were also reported to exhibit increased tolerance linked to a truncated Stapholococcus protein A. The constructs to hypothermic above-freezing temperatures. At 4–6 ◦C, transgenic were transformed into E. coli and then introduced into Agrobac- lines exhibited growth of new tissue and robust leaf color. Perhaps terium tumefaciens. Plants were transformed by co-cultivation with these features could be attributed to the hypothermic protection of wounded leaf discs. The purpose of the study was to determine if membranes provided by winter flounder type-I fish AFP (Kamijima the recrystallization inhibition properties of the native AFP and/or et al., 2013; Tomczak and Crowe, 2002). the enhanced recrystallization inhibition activity of the fusion Another study used modified winter flounder AFP genes, plus protein (Mueller et al., 1991) could provide some level of freeze pro- and minus a signal peptide, to transform spring wheat (Khanna tection that might improve the post-harvest freeze/thaw qualities and Daggard, 2006). Results were similar to those reported by of frozen fruits and vegetables. Both afa3 and the spa-afa5 mRNAs Wallis et al. (1997) for transgenic potato. A synthetic gene encod- were detected in tomato and tobacco. Only the fusion protein, how- ing a larger form of the winter flounder AFP was used and this ever, was identified in Western blots, and only in tissue from the was suggested to be responsible for the increased thermal stability transgenic tomato. Recrystallization inhibition activity was mea- of the expressed protein relative to an earlier study by Kenward sured in extracts from the tomato tissue where the fusion protein et al. (1993). In the study reported by Khanna and Daggard (2006), was detected. The authors did not determine whether this activity substantial amounts of the AFP accumulated in both the cyto- was sufficient to provide either frost protection or improve super- plasm and the apoplast when plants were grown at 14–18 ◦C. The cooling so as to prevent freezing in the transgenic plants, but, this is apoplast targeted AFP represented 1.61% of the total soluble pro- unlikely to have been the case because the low level of recrystalliza- tein and 90% of the AFP was apoplastic. This is important as ice tion inhibition reported indicates very limited antifreeze protein is typically initiated in the apoplast. Recrystallization inhibition activity. was observed in whole plant extracts and in apoplastic fluid, indi- Kenward et al. (1993) also attempted expression of winter cating the presence and accumulation of active AFP. Electrolyte flounder AFP in tobacco using the cauliflower mosaic virus 19S pro- leakage tests indicated that transgenic lines expressing AFP had moter and cDNA encoding for the pre-pro-AFP. Although mRNA improved freeze tolerance relative to wild-type plants. This was was detected, the protein was not, unless the transgenic plants especially true in the line demonstrating the highest level of AFP were held at 4 ◦C for 24–48 h, after which the pro-AFP was identi- (measured by Western blot), with 90% of the AFP being secreted fied. The authors suggested that the proper alpha-helical secondary to the apoplast. While wild-type plants exhibited >50% electrolyte structure of the flounder AFP was not maintained at high temper- leakage at −5 ◦C, the highest AFP producing line exhibited only 27% atures, perhaps resulting in the breakdown of the protein. If true, leakage at −5 ◦C, 40% at −6 ◦C, and approximately 55% at −7 ◦C. this would indicate that this AFP may be not be useful for protec- Consequently, once again the best AFP producing lines provided tion of frost sensitive plants because higher daytime temperatures about 1 ◦C of frost protection relative to the wild-type. The reduc- prior to a frost may negate any beneficial effect that may have been tion in freezing injury in the transgenic lines could not be absolutely achieved by the presence of the properly folded AFP. Another fac- attributed to inhibition of freezing (avoidance) or to less freezing tor that may account for the low expression of the winter flounder damage (tolerance). 64 J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69

Type II fish AFP (found in sea raven, smelt and herring) con- typically migrate on gels as if they are larger than they actually tains five disulfide bridges, so once they are properly folded they are, MALDI-TOFF mass spectrometry was used to better identify are more stable than type I fish AFPs. Kenward et al. (1999) the mass of the proteins in the bands. The larger band matched using Agrobacterium mediated transformation of tobacco leaf disks the mature DAFP, indicating that the smaller band was a partial introduced a gene encoding the mature sea raven type-II fish pro- breakdown product, perhaps resulting from proteases in the plants tein, driven by the cauliflower mosaic virus 35S promoter. Lines homogenates. Immunoblots of leaf apoplastic extracts showed that with and without the tobacco mosic virus signal peptide were pro- only plants expressing DAFP-1 with a signal peptide secreted the duced in order to target the protein to either the apoplast or the mature DAFP into the apoplast. A thermal hysteresis of 0.8 ◦C was cytosol. The mature form of Type II fish AFP representing up to 2% observed in the highest activity line, demonstatrating that the of apoplastic protein was present in the transgenic tobacco lines. signal peptide was recognized and cleaved in the plants. The break- No accumulation was observed in the cytoplasm. Active AFP was down product present in the homogenates was not present in the identified by the presence of bipyramidal ice crystals typical of leaf apoplast, so proteolysis was not responsible for the lower than crystal growth in the presence of this AFP, but thermal hystere- expected apoplastic activity. The lines expressing DAFP-1 lacking sis activity was very low. Field trials to test the frost resistance of the signal peptide did not secrete DAFP into the apoplast but ther- the transformed plants relative to wild-type plants were conducted mal hysteresis activity was present in homogenates, although it at the Agriculture Canada Research Station in Delhi, Canada dur- was not as high as in lines expressing DAFP with the signal peptide. ing the autumn. The transformed plants did not exhibit increased To determine if there was a difference between the lines in frost resistance, however, the authors did suggest that the trans- the temperature at which plants froze, Huang et al. (2002) used genic AFP-producing plants could be be grown for the purpose of high-resolution infrared thermography (Wisniewski et al., 1997) harvesting type-II fish protein. to observe the freezing process and determine the temperature at which freezing was initiated (Fig. 1). Although plants were frozen 2.2. Transformation with insect AFPs using four different protocols, transgenic lines expressing DAFP-1 with the signal peptide (DAFP in the apoplast) always froze at a The studies that expressed fish, mainly winter flounder, AFPs in statistically lower temperature than wild-type plants, while those transgenic plants resulted in the production of plants that were, with DAFP-1 confined to the cytoplasm did not. The four different at best, approximately 1 ◦C more freezing tolerant than wild-type freezing protocols were as follows. (1) Whole plants with exposed plants. While this was a significant accomplishment, a greater roots were placed in groups such that adhering soil was contiguous level of protection is required to provide adequate levels of frost and freezing was initiated in the soil to insure that all plants were tolerance in plants. The sequencing and descriptions of insect equally exposed to inoculation at the same temperature. Plants AFPs with significantly greater specific thermal hysteresis activ- were cooled at 5 ◦Ch−1. The mean freezing temperature of trans- ity (Andorfer and Duman, 2000; Duman et al., 1998; Graham et al., genic plants expressing DAFP-1 with the signal peptide (DAFP in 1997; Graether et al., 1999) offered an opportunity to improve on the apoplast) was −4.6 ◦C, which was significantly lower (p ≤ 0.05) this level of frost protection by making the transgenic plants frost than that of the wild-type which froze at an average of −3.3 ◦C. avoiding. The freezing temperature of transgenics lacking the signal peptide, Holmberg et al. (2001) generated the first transgenic plants and therefore lacking DAFP in the apoplast, was −3.9 ◦C which was expressing an insect AFP. A synthetic gene, based on the AFP present not statistically different from the wild-type. In this protocol, plant in the spruce budworm, Choristoneura fumiferana,(Tyshenko et al., freezing was initiated by inoculation from ice in the soil and spread 1997) was transformed into tobacco with a plant signal peptide and first to the lower stem and then through the rest of the plant. (2) driven by the cauliflower mosaic virus 35S promoter. Success of Plants were cut at the base, the cut stems were sealed with vac- the transformation was measured by the presence of appropriately uum grease to prevent leakage of xylem fluids, the surface of the sized transcripts using RT-PCR, recrystallization inhibition activity plants were sprayed with an ice nucleating active strain (Cit7) of in crude leaf homogenates and apoplastic extracts of the plants, bacteria, and cooled at a rate of 5 ◦Ch−1. Once and most importantly, thermal hysteresis (0.37 ◦C) in the apoplas- again transgenics with DAFP in the apoplast froze at a lower tem- ◦ tic fluid. The freezing response or LT50 of the transgenic tobacco perature (mean temperature of −5.1 C), which was significantly was not reported. lower (p ≤ 0.05) than the wild-type which froze at −4.2 ◦C. In these Huang et al. (2002) successfully produced transgenic Arabidop- plants, freezing began in the leaves or stems, probably initiated by sis thalianna (ecotype WS-2) expressing DAFP-1 from Dendroides the surface bacteria. (3) Plants were treated as in 2, except that canadensis. This AFP was chosen because it is the most abun- they were sprayed with water that did not contain ice nucleat- dant DAFP in the D. canadensis hemolymph and has high specific ing bacteria. The results were similar to those of 2 above, except antifreeze (thermal hysteresis) activity. D. canadensis AFPs rou- that all freezing temperatures were slightly lower. Under all three tinely produce a hemolymph thermal hysteresis of 5–6 ◦C(Li et al., of the above freezing protocols, freezing of the plants began at 1998a,b), and inhibit ice nucleators (Duman, 2002) and inocula- fairly high sub-zero temperatures and was initiated by extrinsic tive freezing initiated by external ice (Olsen et al., 1998). An earlier nucleating agents present on the surface of the plants. The pres- study also indicated that DAFPs could inhibit the freeze damage ence of apoplastic DAFP-1, even at fairly modest levels of activity, in isolated cells resulting from external ice formation, probably produced a small, but significant, decrease in the temperature at by preventing cytoplasmic freezing and recrystallization inhibition which freezing was initiated. (4) The fourth cooling protocol pro- (Tursman and Duman, 1995). Genes encoding the native DAFP- duced freezing that began at much lower temperatures. Stems were 1, with and without the insect signal peptide, were cloned into severed at the base, mounted in vacuum grease, and cooled at expression vectors containing the cauliflower mosaic virus 35S 5 ◦Ch−1. These plants were not sprayed with water or Cit7 bacte- RNA promoter and terminator. Arabidopsis was transformed via ria. Once again, the transgenics with apoplastic DAFP-1 froze at a Agrobacterium-mediated transformation. Transformed plants were significantly lower (p ≤ 0.05) temperature (−16.6 ◦C) than the wild- initially selected for growth on kanamycin, and those with the high- type (−13.3 ◦C), an average of 3.3 ◦C colder. The transgenics with est levels of dafp-1 were identified using Northern blots and chosen cytoplasmic, but not apoplastic, DAFP-1 did not freeze at a signif- for further analysis. Western blots demonstrated expression of icantly lower temperature than the wild-type. Since freezing is a DAFP-1, ± signal peptide, in T4 and later generation plants, although stochastic event, the lower freezing temperatures observed using two bands were present on the electrophoresis gels. Because DAFPs the last protocol, were reflective of the small plant size and the J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69 65

Fig. 1. Freezing of Arabidopsis plants expressing the DAFP-1 antifreeze protein (AFP) with (line 340) and without (line 270) a signal peptide targeting the protein for secretion to the appoplast. The gene coding the DAFP-1 protein was obtained from the beetle, Dendroides canadensis. Individual plants were removed from the soil, mounted on cardboard with silicon grease, and cooled at approximately 5 ◦C/h. Plants were not sprayed with water and so supercooled to very low temperatures before freezing. Freezing was monitored using high resolution infrared thermography and is visualized as an exothermic event that raises the temperature of the plant above ambient air temperature. Top left—Wild-type and transformed Arabidopsis plants prior to conducting the freezing test. Top right—Freezing of a wild type plant at −9.4 ◦C. Average freezing temperature of wild type plants was −13.3 ◦C. Bottom left—Freezing of a 270-23 transgenic plant (DAFP-1 without a signal peptide) at −11.6 ◦C. Average freezing temperature of transgenic 270-23 plants was −13.8 ◦C. No significant difference was observed between wild-type and 270 transgenic plants. Bottom right—Freezing of two 340-29 plants (DAFP-1 with a signal peptide) at −17.9 ◦C. Average freezing temperature of 340-29 plants was −16.6 ◦C which was significantly different (p ≤ 0.05) than the wild-type and 270-23 plants. The high level of supercooling indicates that the DAFP-1 may have inhibited ice nucleators present in the apoplast.

absence of high populations of extrinsic nucleators, and the fact Although the results of the experiments demonstrating that that the plants were dry (no surface moisture) thus the plants transgenic Arabidopsis expressing apoplastic DAFP-1 froze at signif- were able to supercool. Although the purpose of the transforma- icantly lower temperatures than wild-type plants is encouraging, tion experiments was to determine if the transgenic plants froze at a degree of freeze protection greater than 1–3 ◦C will be neces- lower temperatures than wild-type plants, once plants did freeze, sary to have a major impact on the ability of plants to survive a regardless of the freezing conditions, no difference in survivorship significant frost event. The small amount of freeze avoidance was observed. All plants that froze were dead seven days after (1–3 ◦C) achieved was attributed primarily to the low level of ther- thawing. mal hysteresis activity accomplished, only 0.8 ◦C at best in the 66 J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69 apolast, present in the transgenic plants. After these experiments plants were identified by RT-PCR of leaf homogenates, and plants were completed, however, it was discovered that certain DAFPs, with the highest transcript levels were selected for further analysis. and certain other proteins, are able to interact with one another The expressed AFP was localized to the cell walls of the transgen- to synergistically increase thermal hysteresis activity (Wang ics using immuno-gold labeling, and Western blots demonstrated and Duman, 2005, 2006). This suggested that transgenic plants the presence of the AFP in apoplastic fluid. Thermal hysteresis or expressing a combination of DAFPs with synergistic interactions, recrystallization inhibition tests to determine if the expressed pro- might exhibit greater thermal hysteresis, and therefore greater tein was active were not conducted, although it can be assumed that freeze protection, even if the total level of DAFP expression was at least some level of activity was present. The cold tolerance of the not raised. transgenics relative to wild-type was tested by visual inspection Therefore, Lin et al. (2011) introduced two synergistic DAFP of plants and measurements of ion leakage and malondialdehyde cDNAs (dafp-1 and dafp-4) into the genome of Arabidopsis (a measure of lipid peroxidation) release following exposure of the using Agrobacterium-mediated floral dip transformation. To gen- plants to −1 ◦C for variable periods of time up to 72 h. After 2 and erate transgenic Arabidopsis expressing both DAFP-1 and -4 3 days of exposure to −1 ◦C, wild-type plants were reported to be co-transformation of multiple binary vectors containing different more negatively affected than were the transgenics, as assessed by genes was performed. Insect DAFP signal peptides were used to tar- the presence of more wilted leaves in the wild-type plants. After get the DAFPs to the apoplast and transgenic lines expressing either one day of recovery at 25–28 ◦C the transgenic plants appeared to DAFP-1 or DAFP-4, and both DAFP-1 and -4 were generated. Lines be fully recovered while the wild-type plants still appeared to be with the greatest amount of thermal hysteresis were selected for stressed as indicated by wilted leaves. This was evident even after further study. Southern blots indicated that these lines all had mul- five days of recovery. Ion leakage and malondialdehyde levels of tiple copies of the , and RT-PCR confirmed that the genes both wild-type and transgenics were low after one day at −1 ◦C, but were being expressed. Western blots of total soluble protein from the levels in wild-type plants rose significantly relative to the trans- the lines demonstrated the presence of DAFPs, however, the level genics after 2–3 days at −1 ◦C. Therefore, the presence of the beetle of expression of DAFP-4 was low. The thermal hysteresis measured AFP provided some level of protection to the tobacco at this rela- − in the protein extracts (50 mg ml 1 total protein) was consistent tively mild, but lengthy, subzero temperature exposure. Whether with the Western blots as the two lines expressing both DAFP-1 + 4 this was due to providing freeze avoidance or increasing freezing ◦ had the greatest activity (1.39 and 1.20 C). The DAFP-1 line had the tolerance was not determined. ◦ next highest activity (0.75 C) and the DAFP-4 line did not exhibit Zhu et al. (2010) synthesized a spruce bud worm (C. fumifer- thermal hysteresis but did produce hexagonal ice crystals indicat- ana) AFP and expressed it in Arabidopsis thaliana using a plant ing a very low level of DAFP activity. The measurment of thermal codon bias and a tobacco PR-S signal peptide. The expression vec- hysteresis in apoplastic fluids provided similar results: tor contained the synthetic AFP gene and a double cauliflower ◦ ◦ 1.15 C in the DAFP-1 + 4 lines and 0.75 C in the DAFP-1 line. mosaic virus 35S promotor. Three transformed lines exhibiting the The DAFP-4 line had no activity, not even an effect on ice crytstal highest levels of afp transcript were selected based on RT-PCR of structure (i.e., hexagonal crystals). total RNA from leaves. The levels of AFP expressed and the AFP Lin et al. (2011) used the following protocol to determine the activities (thermal hysteresis or recrystallization inhibition) were temperature at which freezing was initiated in the plants. Stems not determined or reported, so the claim of overexpression of the were cut at the base, sealed with vacuum grease, placed in Petri AFP was unsubstantiated. After three weeks growth at 23 ◦C under dishes in an environmental chamber, cooled, and a high-resolution long photoperiod, wild-type and transformed plants were trans- infrared camera was used to detect freezing events. The mean freez- ferred to 4 ◦C for two days (at either long or short photoperiods) ing temperature of the transgenic lines expressing both DAFPs-1 and then subjected to −20 ◦C for 30 min. Plants were then held ◦ and -4 was significantly lower than in wild-type plants (2.9 C at 4 ◦C overnight, then transferred back to a 23 ◦C growth cham- lower), and lines producing only DAFP-1 or DAFP-4. The freezing ber. Visual inspection of the plants several days later indicated temperature of the lines producing only DAFP-1 was also signifi- that while most of the wild-type plants had died, a higher level ◦ cantly lower than in the wild-type (1.8 C lower), but was not in of survival was evident in the transgenic lines, although actual those lines producing only DAFP-4. Tests with individual leaves numbers were not reported. The line with the highest level of afp excised from these plants showed similar trends. Although these transcript also had the best survival and the line with the low- results were again encouraging, a greater level of freeze avoid- est level of transcript exhibited much poorer survival. Electrolyte ance was expected in the lines producing both DAFP-1 and -4 due leakage and malondialdehyde content in all of the plants increased to expected synergy between the two DAFPs. A greater level of after cold treatment, but the levels were significantly greater in thermal hystereis in the apoplastic fluid of these lines was also wild-type plants than in the transformed plants. The damage in the expected. The lower than expected level of synergy and concomi- transformed lines was inveresly correlated with afp transcript lev- tant thermal hysterisis activity was suspected to be the result of low els in the three transformant lines. These results again indicated levels of expression of DAFP-4 in the transgenic lines that expressed that expression of an insect AFP in transgenic plants ameliorated DAFP-4 alone or in conjuction with DAP-1. This premise was con- the injury resulting from exposure of plants to freezing temper- firmed by RT-qPCR. In fact, expression of dafp-1 was 116-fold and atures. It should be noted, however, that the freezing protocol 53-fold greater than dafp-4 in the two DAFP-1 + 4 expressing lines, used was very atypical for freezing stress studies. Whether the and when the thermal hysteresis of pure DAFP-1 and DAFP-4 was plants had actually frozen during the protocol was not substanti- tested at ratios of 53/1 or 116/1, no synergy in thermal hysteresis ated and such a short exposure (30 min) would not have guaranteed activity was observed. Consequently, the freeze protection in the freezing unless ice nucleation was induced at a warm, sub-zero transgenic plants was consistent with the levels of thermal hystere- temperature. Therefore, whether the reduction in injury in the sis measured in the apoplastic fluid, as was the case in the earlier transgenic plants was due to freeze avoidance (promotion of super- study (Huang et al., 2002). cooling), a reduction in the amount of physical injury to cells by the Wang et al. (2008) expressed an AFP from another beetle, growth of large ice crystals being inhibited by recrystallization, or Microdera punctipennis, from the Xinjiang desert region of China an increase in freezing tolerance (protection of cell membranes, in tobacco using the same approach to vector construction and especially the plasmalemma) could not be determined. The impact transformation as previously described. This AFP is similar to the of growing the plants under short vs. long photoperiods was not AFPs in D. canadensis and Tenebrio molitor beetles. Transformed reported. J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69 67

2.3. Transformation with plant AFPs (IBPs) environmental conditions that are present during natural frosts are often different and more complex than those used in environmen- Given the previously mentioned low specific activity of known tal chambers, field tests are eventually necessary to provide reliable plant AFPs, perhaps better described as ice binding proteins (IBPs), proof of the ability of the plants to avoid or tolerate freezing. (6) it seems unlikely that expression of these proteins in frost-sensitive While use of Arabidopsis or other model plants for “proof of concept” crop plants would result in increased freeze avoidance, and there- studies is often the most efficient approach, it is necessary to even- fore frost protection. Also, because freeze tolerance is a complex tually extend the study to the actual crop plant for which protection process involving a host of biochemical changes and adaptations, is desired. Initiation of freezing, whether resulting from inoculative it is improbable that expression of IBPs in frost-sensitive plants freezing from the surface or from internal ice nucleators, varies would render them freeze tolerant. It is possible, however, that between species. Additionally, since ice nucleation is a stochastic plant IBPs could exhibit ice nucleation inhibition activity. If so, event, the size or mass of the plant will have an impact on the prob- this could be of value in situations where inhibition of intrinsic ability of a nucleation event occurring. Most crop plants are much ice nucleators is critically important. Most plant IBPs appear to be larger than Arabidopsis, and therefore they may freeze at a higher present in the apoplast, where ice forms in freeze tolerant plants, temperature. Also, the developmental stage of the plant when frost and consequently these IBPs apparently do not inhibit ice nuclea- protection is required may also play a significant role since frost tion. Wisniewski et al. (1999), however, reported a with tolerance and/or avoidance can vary on a seasonal and develop- thermal hysteresis activity, in the cytoplasm of acclimated peach mental basis. In spite of these and other considerations, such as the bark and xylem cells, including xylem ray parenchyma cells, which resistance of the public to genetically engineered crop plants, it is are known for their ability to supercool to very low temperatures. likely that the development of transgenic plants expressing AFPs A leucine rich carrot AFP (IBP) gene, structurally similar to the will eventually result in crops and horticulturally important plants polygalacturonase inhibitor protein (Worral et al., 1998) was used that survive exposure to frosts of −5or6◦C, and perhaps lower to produce transformed Arabidopsis that accumulated the IBP at temperatures. Such developments will have tremendous positive low temperatures (Meyer et al. (1999). AFP activity was defined as results for growers by reducing or eliminating losses to damag- the ability of extracts from transformed plants to affect ice crystal ing frosts, and expanding both the geographical range and growing shape. These transgenic plants would not be expected to exhibit periods for crops. a lower freezing temperature (i.e., increased freezing avoidance) since the specific thermal hysteresis activity of plant IBPs (including the one from carrot) are low. The plants were not tested for cold References tolerance. Andorfer, C.A., Duman, J.G., 2000. Isolation and characterization of cDNA clones endcoding antifreeze proteins of the Pyrochroid beetle Dendroides canadensis.J. 3. Conclusions and future directions Insect Physiol. 46, 365–372. Ball, M.C., Hill, M.J., 2009. Elevated atmospheric CO2 concentrations enhance vul- nerability to frost damage in a warming world. In: Gusta, L., Wisniewski, M., While the studies outlined in this review provide evidence that Tanino, K. (Eds.), Plant Cold Hardiness: From the Laboratory to the Field. CAB the expression of AFPs can be used to produce more frost resistant International, Cambridge, MA, USA, pp. 183–189. Ball, M.C., Wolfe, J., Canny, M., Hofmann, A.B., Nicotra, J., Hughes, D., 2002. Space and crop plants, there is obviously considerable need for improvement. time dependence of temperature and freezing in evergreen leaves. Funct. Plant Transgenic plants with the ability to avoid freezing at temperatures Biol. 29, 1259–1272. below −5or−6 ◦C would be of great value, providing protection Bassett, C.L., Wisniewski, M., 2009. Global expression of cold-responsive genes in fruit trees. In: Gusta, L., Wisniewski, M., Tanino, K. (Eds.), Plant Cold Hardiness: from freeze damage during the minor freezes that occur in early From the Laboratory to the Field. CABI, Oxford, England. pp. 72–79. spring and early autumn. Based on the results achieved in trans- Bennett, V.A., Sformo, T., Walters, K., Toien, O., Jeannet, K., Hochstrasser, R., Pan, Q., genics thus far, producing this level of freezing avoidance and/or Serianni, A.S., Barnes, B.M., Duman, J.G., 2005. Comparative overwintering physi- freezing tolerance by the ectopic expression of AFPS (especially ology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause. J. Exp. Biol. 208, insect AFPS) seems plausible. A few points should be kept in mind, 4467–4477. however, in pursuing this line of study. (1) The higher specific ther- Block, W., Duman, J.G., 1989. The presence of thermal hysteresis producing mal hysteresis activity AFPs, probably those of insects, should be antifreeze proteins in the Antarctic mite, Alaskozetes antarcticus. J. Exp. Zool. 250, 229–231. employed. (2) A high level of expression of active proteins, tar- Carrasco, M.A., Buechler, S., Arnold, R., Sformo, T., Barnes, B.M., Duman, J.G., 2011. geted to the apoplastic fluid, is critical, as is the use of AFPs with Elucidating the biochemical overwintering adaptations of larvae of the Alaskan the ability to act synergistically in increasing thermal hysteresis. beetle Cucujus clavipes puniceus, a non-model organism, via high throughput proteomics. J. Proteome Res. 10, 4634–4646. Proper folding of the proteins is essential to their activity. Therefore, Carrasco, M.A., Buechler, S., Arnold, R., Sformo, T., Barnes, B.M., Duman, J.G., 2012. afp transcript levels, protein concentration, and thermal hysteresis Investigating the deep supercooling ability of an Alaskan beetle, Cucujus clavipes activity should always be determined and reported so that if opti- puniceus, via high throughput proteomics. J. Proteomics 75, 1220–1234. Celik, Y., Graham, L.A., Mok, Y.-F., Bar, M., Davies, P.L., Braslavsky, I., 2010. Super- mal results are not achieved in the transgenic plants the potential heating of ice crystals in antifreeze protein solutions. Proc. Natl. Acad. Sci. USA problem can be identified and addressed in subsequent work. (3) 107, 5423–5428. When expression of the AFPs throughout the whole plant is not Cheng, C.-H.C., Chen, L., 1999. Evolution of an antifreeze glycoprotein. Nature 401, 443–444. essential, the efficacy of site-specific promoters should be explored. Cheng, C.-C.H., DeVries, A.L., 2002. Origins and evolution of fish antifreeze pro- For example, restricting AFP expression to flowers using a flower teins. In: wart, K.V., Hew, C.L. (Eds.), Fish Antifreeze Proteins. New Jersey, World specific promoter could provide protection to sensitive flowers of Scientific Publishing, pp. 83–107. fruit trees in spring. (4) The actual temperature at which freezing Cutler, A.J., Saleem, M., Kendall, E., Gusta, L.V., Georges, F., Fletcher, G.L., 1989. Winter flounder antifreeze protein improves the cold hardiness of plant tissues. J. Plant is initiated in the plants being studied should be monitored, along Physiol. 135, 351–354. with ice propagation, by infrared video thermography if posssible. DeVries, A.L., 1971. Glycoproteins as biological antifreeze agents in Antarctic fishes. This will enable the identification not only of the freezing tem- Science 172, 1152–1155. DeVries, A.L., 1986. Antifreeze glycopeptides and peptides: interactions with ice and perature but also the determination of whether or not decreased water. Methods Enzymol. 127, 293–303. freeze injury in transgenic plants is the result of the formation of DeVries, A.L., 2005. Ice, antifreeze proteins and antifreeze genes in polar fishes. In: less ice, variation in the site of formation, rate of ice propagation, Barnes, B.M., Carey, H.V. (Eds.), Life in the Cold, University of Alaska Press. Alaska, pp. 307–315. etc. (5) Care should be taken to use well described, realistic, and Duman, J.G., 1977. The role of macromolecular antifreeze in the darkling beetle, multiple freezing regimes to test the resistance of plants. Since the Meracantha contracta. J. Comp. Physiol. B 115, 279–286. 68 J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69

Duman, J.G., 1979a. Thermal hysteresis factors in overwintering insects. J. Insect Husby, J.A., Zachariassen, K.E., 1980. Antifreeze agents in the body fluid of winter Physiol. 25, 805–810. active insects and spiders. Experientia 36, 963–964. Duman, J.G., 1979b. Subzero temperature tolerance in spiders: the role of thermal Jia, Z., Davies, P.L., 2002. Antifreeze proteins: an unusual receptor-ligand interaction. hysteresis factors. J. Comp. Physiol. B 131, 347–352. Trends Biochem. Sci. 27, 101–106. Duman, J.G., 1982. Insect and ice nucleating agents. Cryobiology 19, Khanduri, V.P., Sharma, C.M., Singh, S.P., 2008. The effects of climate change on plant 613–627. phenology. Environmentalist 28, 143–147. Duman, J.G., 1994. Purification and characterization of a thermal hysteresis protein Kamijima, T., Sakashita, M., Miura, A., Nishimiya, Y., Tsuda, S., 2013. Antifreeze pro- from a plant, the bittersweet nightshade Solanum dulcamara. Biochim. Biophys. tein prolongs the life-time of insulinoma cells during hypothermic preservation. Acta 1206, 129–135. PLoS ONE 8 (9), e73643, http://dx.doi.org/10.1371/journal.pone.0073643 Duman, J.G., 2001. Antifreeze and ice nucleator proteins in terrestrial arthropods. Kasuga, J., Mizuno, K., Arakawa, K., Fujikawa, S., 2007. Anti-ice nucleation activity Ann. Rev. Physiol. 63, 327–357. in xylem extracts from trees that contain deep supercooling xylem parenchyma Duman, J.G., 2002. The inhibition of ice nucleators by insect antifreeze proteins is cells. Cryobiology 55, 305–314. enhanced by glycerol and citrate. J. Comp. Physiol. B 172, 163–168. Kasuga, J., Hashidoko, Y., Nishioka, A., Yoshiba, M., Arakawa, K., Fujikawa, S., 2008. Duman, J.G., Olsen, T.M., 1993. Thermal hysteresis protein activity in bacteria, fungi, Deep supercooling xylem parenchymas cells of katsura tree (Cercidiphyllum and phylogenetically diverse plants. Cryobiology 30, 322–328. japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activ- Duman, J.G., Li, N., Verleye, D., Goetz, F.W., Wu, D.W., Andorfer, C.A., Benjamin, T., ity. Plant Cell Environ. 31, 1335–1348. Parmelee, D.C., 1998. Molecular characterization and sequencing of antifreeze Kenward, K.D., Altschuler, M., Hildebrand, D., Davies, P.L., 1993. Accumulation of proteins from larvae of the beetle Dendroides canadensis. J. Comp. Physiol. B 168, type I fish antifreeze protein in transgenic tobacco is cold-specific. Plant Mol. 225–232. Biol. 23, 377–385. Duman, J.G., Verleye, D., Li, N., 2002. Site-specific forms of antifreeze protein in the Kenward, K.D., Brandle, J., McPherson, J., Davies, P.L., 1999. Type II fish antifreeze beetle Dendroides canadensis. J. Comp. Physiol. B 172, 547–552. protein accumulation in transgenic tobacco does not confer frost resistance. Duman, J.G., Bennett, V.A., Li, N., Wang, L., Huang, L., Sformo, T., Barnes, B.M., 2004, Transgen. Res. 8, 105–117. Antifreeze proteins in terrestrial arthropods. In: Barnes, B.M. Carey, H.V. (Eds.), Khanna, H.K., Daggard, G.E., 2006. Targeted expression of redesigned and codon Life in the Cold. University of Alaska Press, pp. 527–542. optimized synthetic gene leads to recrystallization inhibition and reduced elec- Duman, J.G., Bennett, V., Sformo, T., Hochstrasser, R., Barnes, B.M., 2004b. Antifreeze trolyte leakage in spring wheat at sub-zero temperatures. Plant Cell. Rep. 25, proteins in Alaskan insects and spiders. J. Insect Physiol. 50, 259–266. 1336–1346. Duman, J.G., Walters, K.R., Sformo, T., Carrasco, M.A., Nickell, P., Lin, X., Barnes, B.M., Knight, C.A., Duman, J.G., 1986. Inhibition of recrystallization of ice by insect 2010. Antifreze and ice nucleator proteins. In: Denlinger, D.L., Lee, R.E. (Eds.), thermal hysteresis proteins: a possible cryoprotective role. Cryobiology 23, Low Temperature Biology of Insects. Cambridge University Press, Cambridge, 256–262. pp. 59–90. Knight, C.A., DeVries, A.L., 1989. Melting inhibition and superheating of ice by an Ebbinghaus, S., Meister, K., Born, B., DeVries, A.L., Gruebele, M., Havenith, M., antifreeze glycopeptide. Science 245, 505–507. 2010. Antifreeze glycoprotein activity correlates with long-range protein-water Knight, C.A., Cheng, C.C., DeVries, A.L., 1991. of alpha-helical antifreeze dynamics. J. Am. Chem. Soc. 132, 12210–12212. peptides on specific ice crystal surface planes. Biophys. J. 59, 409–418. Ebbinghaus, S., Meister, K., Prigozhin, M.B., DeVries, A.L., Havenith, M., Dzubiella, Leinala, E.K., Davies, P.L., Doucet, D., Tyshenko, M.G., Walker, V.K., Jia, Z., 2002. A J., Gruebele, M., 2012. Functional importance of short-range binding and beta-helical antifreeze protein isoform with increased activity: structural and long-range solvent interactions in helical antifreeze peptides. Biophys. J. 103, functional insights. J. Biol. Chem. 277, 33349–33352. L20–L22. Levitt, J., 1980. Responses of Plants to Environmental Stresses. Chilling, Freezing and Fowler, S., Thomashow, M.F., 2002. Arabidopsis transcriptome profiling indicates Environmental Stresses, Vol. 1., 2nd ed. New York, Academic Press. that multiple regulatory pathways are activated during cold acclimation in addi- Li, N., Chibber, B.A.K., Castellino, F.J., Duman, J.G., 1998a. Mapping of disulfide tion to the CBF cold responsive pathway. Plant Cell 14, 1675–1690. bridges in antifreeze proteins from overwintering larvae of the beetle Dendroides Georges, F., Saleem, M., Cutler, A., 1990. Design and cloning of a synthetic gene for the canadensis. Biochem. 37, 6343–6350. flounder antifreeze protein and its expression in plant cells. Gene 91, 159–165. Li, N., Andorfer, C.A., Duman, J.G., 1998b. Enhancement of insect antifreeze protein Gordo, O., Sanz, J.J., 2010. Impact of climate change on plant phenology in Mediter- activity by solutes of low molecular mass. J. Exper. Biol. 201, 2243–2251. ranean ecosystems. Global Change Biol. 16, 1082–1106. Lin, F.-H., Graham, L.A., Campbell, R.L., Davies, P.L., 2007. Structural modeling of Graether, S.P., Sykes, B.D., 2004. Cold survival of freeze intolerant insects: the struc- snow flea antifreeze protein. Biophys. J. 92, 1717–1723. ture and function of beta-helical antifreeze proteins. Euro. J. Biochem. 271, Lin, X., Wisniewski, M.E., Duman, J.G., 2011. Expression of two self-enhancing 3285–3296. antifreeze proteins from the beetle Dendroides canadensis in Arabidopsis thaliana. Graether, S.P., Ye, Q.L., Davies, P.L., Sykes, D.B., 1999. and preliminary Plant Mol. Biol. Rep. 29, 802–813. x-ray crystallographic analysis of spruce budworm antifreeze protein. J. Struct. Lindow, S.E., 1995. Control of epiphytic ice-nucleation-active bacteria for manage- Biol. 126, 72–75. ment of frost injury. In: Lee, Jr. R.E., Warrant, G.J., Gusta, L.V. (Eds), Biological Ice Graether, S.P., Kuiper, M.J., Gagne, S.M., Walker, V.K., Jia, Z., Sykes, B.D., Davies, P.L., Nucleation and its Applications. APS Press, St. Paul, Minn, pp. 239–256. 2000. Beta-helix structure of a hyperactive antifreeze protein from an insect. Liou, Y.-C., Thibault, P., Walker, V.K., Davies, P.L., Graham, L.A., 1999. A complex Nature 406, 325–328. family of highly heterogeneous and internally repetitive hyperactive antifreeze Graham, L.A., Liou, Y.-C., Walker, V.K., Davies, P.L., 1997. Hyperactive antifreeze proteins from the beetle Tenebrio molitor. Biochem. 38, 11415–11424. proteins from beetles. Nature 188, 727–728. Meister, K., Ebbinghaus, Y., Xu, Y., Duman, J.G., DeVries, A.L., Gruebele, D.M., Graham, L.A., Davies, P.L., 2005. Glycine-rich antifreeze proteins from snow fleas. Havenith, M., 2013. Long-range protein-water dynamics in hyperactive insect Science 310, 461. antifreeze proteins. Proc. Natl. Acad. Sci., U.S.A. 110, 1617–1622. Griffith, M., Yaish, M.W., 2004. Antifreeze proteins in overwintering plants: a tale of Menzel, A., Sparks, T.H., Estrella, N., Koch, E., Aasa, A., Ahas, R., Alm-Kübler, K., Bissolli, two activities. Trends Plant Sci. 9, 399–405. P., Braslavská, O., Briede, A., Chmielewski, F.M., Crepinsek, Z., Curnel, Y., Dahl, Griffith, M., Ala, P., Yang, D.S.C., Hon, W.-C., Moffit, B.A., 1992. Antifreeze A., Defila, C., Donnelly, A., Filella, Y., Jatczak, K., Måge, F., Mestre, A., Nordli, Ø., protein produced endogenously in winter rye leaves. Plant Physiol. 100, Penuelas,˜ J., Pirinen, P., Remisová,ˇ V., Scheifinger, H., Striz, M., Susnik, A., van 593–596. Vliet, A.J.H., Wielgolaski, F.-E., Zach, S., Zus, A., 2006. European phonological Griffith, M., Lumb, C., Wiseman, S.B., Wisniewski, M., Johnson, R.W., Marangoni, A.G., response to climate change matches the warming pattern. Global Change Biol. 2005. Antifreeze proteins modify the freezing process in planta. Plant Physiol. 12, 1969–1976. 138, 330–340. Meyer, K., Keil, M., Naldrett, M.J., 1999. A leucine-rich repeat protein of carrot that Gu, L., Hanson, P.J., Post, W.M., Kaiser, D.P., Yang, B., Nemani, R., Pallardy, S.G., Meyers, exhibits antifreeze activity. FEBS Lett. 447, 171–178. T., 2008. The 2007 Eastern US spring freeze: Increased cold damage in a warming Mueller, G.M., McKnown, R.L., Corotto, L.V., Hague, C., Warren, G.J., 1991. Inhibition world? BioScience 58, 253–262. of recrystallization in ice by chimeric proteins containing antifreeze domains. J. Hacker, J., Neuner, G., 2007. Ice propagation in plants visualized at the tissue level Biol. Chem. 266, 7339–7344. by infrared differential thermal analysis (IDTA). Tree Physiology 27, 1661–1670. Nickell, P.K., Sass, S., Verleye, D., Blumenthall, E.M., Duman, J.G., 2013. Antifreeze pro- Hightower, R., Baden, C., Penzes, E., Lund, P., Dunsmuir, P., 1991. Expression of teins in the primary urine of larvae of the beetle Dendroides canadensis (Latreille). antifreeze proteins in transgenic plants. Plant Mol. Biol. 17, 1013–1021. J. Exp. Biol. 216, 1697–1703. Holmberg, N., Farres, J., Bailey, J.E., Kallio, P.T., 2001. Targeted expression of a Nicodemus, J., O’Tousa, J.E., Duman, J.G., 2006. Expression of a beetle, Dendroides synthetic codon optimized gene, encoding the spruce budworm antifreeze pro- canadensis, antifreeze protein in Drosophila melanogaster. J. Insect Physiol. 52, tein, leads to accumulation of antifreeze activity in the apoplasts of transgenic 888–896. tobacco. Gene 275, 115–124. Olsen, T.M., Sass, S., Li, N., Duman, J.G., 1998. Factors contributing to seasonal Hoshino, T., Kiriaki, M., Ohgiya, S., Fujiwara, M., Kondo, H., Nishimiya, Y., Yumoto, increases in inoculative freezing resistance in overwintering fire-colored beetle I., Tsuda, S., 2003. Antifreeze proteins from snow mold fungi. Can. J. Bot. 81, larvae Dendroides canadensis (Pyrochroidae). J. Exp. Biol. 201, 1585–1594. 1175–1181. Pertaya, N., Marshall, C.B., Celik, Y., Davies, P.L., Braslavsky, I., 2008. Direct visual- Huang, T., Duman, J.G., 2002. Cloning and characterization of a thermal hysteresis ization of spruce budworm antifreeze protein interacting with ice: basal plane (antifreeze) protein with DNA-binding activity from winter bittersweet night- affinity confers hyperactivity. Biophys. J. 95, 333–341. shade, Solanum dulcamara. Plant Mol. Biol. 48, 339–350. Raymond, J.A., DeVries, A.L., 1977. Adsorption inhibition as a mechanism of freezing Huang, T., Nicodemus, J., Zarka, D.G., Thomashow, M.F., Wisniewski, M., Duman, resistance in polar fishes. Proc. Natl. Acad. Sci. U.S.A. 74, 2589–2593. J.G., 2002. Expression of an insect (Dendroides canadensis) antifreeze protein in Raymond, J.A., Wilson, P.W., DeVries, A.L., 1989. Inhibition of growth on nonbasal Arabidopsis thaliana results in a decrease in plant freezing temperature. Plant planes in ice by fish antifreeze. Proc. Natl. Acad. Sci. U.S.A. 86, 881–885. Mol. Biol. 50, 333–344. Sakai, A., Larcher, W., 1987. Frost Survival of Plants. Berlin, Springer. J.G. Duman, M.J. Wisniewski / Environmental and Experimental Botany 106 (2014) 60–69 69

Sformo, T., Walters, K., Jeannet, K., McIntyre, J, Wowk, B., Fahy, G., Barnes, Wang, Y., Qiu, L., Dai, C., Wang, J., Luo, J., Zhang, F., Ma, J., 2008. Expression B.M., Duman, J.G., 2010. Deep supercooling, vitrification, and limited survival of insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 to -100 ◦C in larvae of the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: confers the cold tolerance to transgenic tobacco. Plant Cell Reporter 27, Cucujuidae). J. Exp. Biol. 213, 502–509. 1349–1358. Sformo, T., McIntyre, J., Walters, K.R., Barnes, B.M., Duman, J.G., 2011. Probabil- Weiser, C.J., 1970. Cold resistance and injury in woody plants: knowledge of hardy ity of freezing in the freeze avoiding beetle larvae Cucujus clavipes puniceus plant adaptations to freezing stress may help us to reduce winter damage. Sci- (Coleoptera, Cucujidae) from Interior Alaska. J. Insect Physiol. 57, 1170–1177. ence 169, 1269–1278. Simpson, D.J., Smallwood, M., Twigg, S., Doucet, C.J., Ross, J., Bowles, D.J., 2005. Purifi- Wharton, D.A., Barrett, J., Goodall, G., Marshall, C.J., Ramlov, H., 2005. Ice-active cation and characterisation of an antifreeze protein from Forsythia suspensa (L.). proteins from the Antarctic nematode Panagrolaimus davidi. Cryobiology 51, Cryobiology 51, 230–234. 198–207. Smallwood, M., Worrall, D., Byass, L., Elias, L., Ashford, D., Doucet, C.J., Holt, C., Wharton, D.A., Pow, B., Kristensen, M., Ramlov, H.R., Marshall, C.J., 2009. Ice-active Telford, J., Lillford, P., Bowles, D.J., 1999. Isolation and characterization of a novel proteins and cryoprotectants from the New Zealand alpine cockroach Celato- antifreeze protein from carrot (Daucus carota). Biochem. J. 340, 385–391. blatta quinquemaculata. J. Insect Physiol. 55, 27–31. Sukumarin, M.P., Weiser, C.J., 1972. Freezing injury in potato leaves. Plant Physiol. Wisniewski, M., Lindow, S.E., Ashworth, E.N., 1997. Observations of ice nucleation 50, 564–567. and propagation in plants using infrared video thermography. Plant Physiol. 113, Sun, X., Griffith, M., Pasternak, J.J., Glick, B.R., 1995. Low temperature growth, freez- 327–334. ing survival, and production of antifreeze protein by the plant growth promoting Wisniewski, M., Webb, R., Balsamo, R., Close, T.J., Yu, X.M., Griffith, M., rhizobacterium PseudomonasputidaGR12-2. Can. J. Microbiol. 41, 776–784. 1999. Purification, immunolocalization, cryoprotective and antifreeze activ- Tomczak, M.M., Crowe, J.H., 2002. The interaction of antifreeze proteins with model ity of PCA60: a dehydrin from peach (Prunus persica). Physiol. Plants 105, membranes and cells. In: Ewart, K.V., Hew, C.L. (Eds.), Fish Antifreeze Proteins. 600–608. World Scientific, London, pp. 187–212. Wisniewski, M., Bassett, C.L., Gusta, L., 2003. An overview of cold hardiness in woody Tursman, D., Duman, J.G., 1995. Cryoprotective effects of thermal hysteresis protein plants: seeing the forest through the trees. Hort. Sci. 38, 952–959. on survivorship of frozen gut cells from the freeze tolerant centipede Lithobius Wisniewski, M., Glenn, M., Gusta, L., Fuller, M.P., 2008. Using infrared thermography forficatus. J. Exp. Zool. 272, 249–257. to study freezing in plants. Hort. Sci. 43, 1648–1651. Tyshenko, M.G., Doucet, D., Davies, P.L., Walker, V.K., 1997. The antifreeze potential Wisniewski, M., Gusta, L.V., Fuller, M.P., Karlson, D., 2009. Ice nucleation, propaga- of spruce budworm thermal hysteresis protein. Nat. Biotech. 15, 887–890. tion, and deep supercooling: the lost tribes of freezing studies. In: Gusta, L.V., Urrutia, M.E., Duman, J.G., Knight, C.A., 1992. Plant thermal hysteresis proteins. Wisniewski, M., Tanino, K. (Eds.), Plant Cold Hardiness: From the Laboratory to Biochim. Biophys. Acta 1121, 199–206. the Field. CABI Cambridge. pp. 1–11. Wallis, J.G., Wang, H., Guerra, D.J., 1997. Expression of a synthetic antifreeze protein Wisniewski, M., Norelli, J., Bassett, C., Artlip, T., Macarisin, D., 2011. Ecotopic expres- in potato reduces electrolyte release at freezing temperatures. Plant Mol. Biol. sion of a novel peach (Prunus persica) CBF transcription factor in apple (Malus x 35, 323–330. domestica) results in short-day induced dormancy and increased cold hardiness. Walters, K.R., Serianni, A.S., Sformo, T., Barnes, B.M., Duman, J.G., 2009. A thermal Planta 233, 971–983. hysteresis-producing xylomannan antifreeze in a freeze tolerant Alaskan beetle. Worrall, D., Elias, L., Ashford, D., Smallwood, M., Sidebottom, C., Lillford, P., Telford, Proc. Nat. Acad. Sci., U.S.A. 106, 20210–20215. J., Holt, C., Bowles, D., 1998. A carrot leucine-rich-repeat protein that inhibits ice Walters, K.R., Serianni, A.S., Voituron, Y., Sformo, T., Barnes, B.M., Duman, J.G., 2011. A recrystallization. Science 282, 115–117. thermal hysteresis-producing xylomannan glycolipid antifreeze associated with Zachariassen, K.E., Husby, J.A., 1982. Antifreeze effects of thermal hysteresis agents cold-tolerance is found in diverse taxa. J. Comp. Physiol. B 181, 631–640. protect highly supercooled insects. Nature 298, 865–867. Wang, L., Duman, J.G., 2005. Antifreeze proteins of the beetle Dendroides canadensis Zettel, J., 1984. Cold hardiness strategies and thermal hysteresis in Collembola. Revue enhance one another’s activities. Biochem. 44, 10305–10312. d’Ecol. Biol. du Sol 21, 189–203. Wang, L., Duman, J.G., 2006. A thaumatin-like protein from larvae of the beetle Den- Zhu, B., Xiong, A.S., Peng, R.H., Xu, J., Jin, X.F., Meng, X.R., Yao, Q.H., 2010. Over- droides canadensis enhances the activity of antifreeze proteins. Biochem. 45, expression of ThpI from Choristoneura fumiferana enhances tolerance to cold in 1278–1284. Arabidopsis. Mol. Biol. Rep. 37, 961–966.