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Environmental and Experimental Botany 106 (2014) 60–69

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

he use of antifreeze proteins for frost protection in sensitive croplants

ohn G. Dumana,∗, Michael J. Wisniewskib

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USAUnited States Department of Agriculture, Agricultural Research Service, Kearneysville, WV 25430, USA

r t i c l e i n f o

rticle history:eceived 14 November 2013eceived in revised form7 December 2013ccepted 3 January 2014vailable online 13 January 2014

eywords:ntifreeze proteinslant cold-toleranceransgenic plantslant frost injuryrevention of plant frost injury

a b s t r a c t

Antifreeze proteins (AFPs), also known as ice binding proteins (IBPs), have evolved as an important adap-tation in numerous organisms exposed to subzero temperatures. Plant AFPs have only been identifiedin freeze tolerant species (those able to survive extracellular freezing). Consequently, plant AFPs havevery low specific activities as they have not evolved to completely prevent ice formation in the plant.In contrast, fish and most insect AFPs function to prevent freezing in species that have evolved freezeavoidance 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 tonon-colligatively lower the freezing point of water (thermal hysteresis) has led to the idea that frost-sensitive crop plants could avoid damage resulting from common minor frost events in late spring andearly autumn by expressing high activity AFPs that permit them to remain unfrozen to temperatures ofapproximately −5 ◦C. Over the past 20 years, the efficacy of this concept has been tested in a variety ofstudies 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 levelsof 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 of1–3 ◦C compared to the wild-type. None of the studies with transgenic plants, however, have yet attaineda 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. Geographicranges and growing seasons could also be expanded. This review provides an overview of the studies of

ing AF

transgenic plants produc

. Introduction

Despite overall increases in mean daily temperatures, it isxpected that there will be an increase in the number of devas-ating spring frosts due to the erratic weather patterns associatedith global climate change (Gu et al., 2008). In April, 2007 theidwest, central and southern plains, and southeast portions of

he U.S. experienced a record breaking freezing event that causenprecedented damage to many economically important crops

n excess of 2 billion dollars (Gu et al., 2008). Since that time,everal spring frost events have caused significant losses to theruit industry. For example, 50–90% of fruit crops (grapes, peaches,

Abbreviations: AFP, antifreeze protein; DAFP, antifreeze protein from Den-roides canadensis; IBP, ice binding protein; kDa, kiloDalton; NMR, nuclearagnetic resonance; CAT, chloramphenical acetyl transferase; LT50, temperature

ethal to 50% of the individuals.∗ Corresponding author. Tel.: +1 574 631 9499.

E-mail address: Duman.1@nd.edu (J.G. Duman).

098-8472/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.envexpbot.2014.01.001

Ps, and makes suggestions for future advancements in this field of study.© 2014 Elsevier B.V. All rights reserved.

apples, cherries, etc.) were lost in the Northeast and Midwest inthe spring of 2012. In 2013, Chile experienced a late spring frostthat resulted in a 22% reduction in exportable fruit representinga loss of 800 million dollars. While efforts were made to pro-vide frost protection during all these events, most were futile.Partially to blame for this failure is our continuing lack of knowl-edge about what makes plants freeze at a particular temperatureand a lack of effective, economical, and environmentally-friendlymethods of frost protection (Wisniewski et al., 2008). Lower-ing ice nucleation temperatures and inhibiting the propagationof ice from the outside to the interior of plants is an approachto frost protection whose potential has been noted by severalauthors (Ball et al., 2002; Hacker and Neuner, 2007; Lindow,1995; Wisniewski et al., 2003, 2008, 2009). The use of highlyactive antifreeze proteins (AFPs), and possibly antifreeze glycol-ipids (AFGLs) (Walters et al., 2009, 2011), represents a logical

approach to limiting frost damage by inhibiting inoculative freezingand enhancing supercooling in freeze-sensitive, annual plants andnewly emerging plant parts on perennial plants. Transgenic plantsexpressing AFPs could also potentially extend the growing seasons

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nd expand the geographic ranges of several crops and horticulturallants.

Plant phenology, such as the timing of budbreak and the onsetf flowering and vegetative growth, is strongly controlled by cli-ate and as such has become a strong bioindicator of ongoing

limate change (Gordo and Sanz, 2010). An analysis of 29 years1971–2000) of phenological data in Europe has indicated that 78%f all leafing, flowering and fruiting records have advanced and thathe average advance of spring has been 2.5 days decade−1 (Menzelt al., 2006). A report on Mediterranean ecosystems, which includes9 perennial plant species monitored from 1943 to 2003, statedhat spring phenological events are changing more than autumnvents as the former events are more sensitive to climate condi-ions and are thus undergoing the greatest alterations (Gordo andanz, 2010). Khanduri et al. (2008) in a study of 650 temperate,lobally-distributed plant species has reported that spring-relatedhenological events have advanced 1.9 days decade−1 and autumn-elated events an average of 1.4 days decade−1. Therefore, thempact of the predicted increase in episodes of spring frosts (Gut al., 2008) will be exacerbated in many species due to the earlynset of spring growth. Additionally, Ball and Hill (2009), in a reviewf several studies, indicated that elevated atmospheric CO2 concen-rations can have a negative impact on plant cold acclimation ands a result enhance vulnerability to frost damage. Consequently, inpite of anticipated global warming the need for protecting vul-erable plants from freeze damage will continue and become evenore critical.The process by which plants actively undergo changes in gene

xpression and biochemistry resulting in an enhanced ability toithstand freezing temperatures and desiccation stress is referred

o as cold acclimation (Weiser, 1970; Wisniewski et al., 2003).echanisms associated with cold hardiness are generally divided

nto two categories: freeze avoidance and freeze tolerance (Sakaind Larcher, 1987). Plants that are cold acclimated and can with-tand exposure to subzero temperatures are principally freezeolerant or exhibit combined mechanisms of freeze tolerance inome tissues and freeze avoidance (deep supercooling) in otherissues (Kasuga et al., 2007, 2008). In contrast, many animals com-letely avoid freezing in winter, and antifreeze proteins are often

nvolved in this ability. For example, freeze-avoiding larvae of thelaskan beetle Cucujus clavipes routinely supercool to −40 ◦C ininter, and when exposed to especially cold temperatures further

dapt to avoid freezing to as low as −100 ◦C while vitrifying atemperatures near −70 ◦C by means of cryoprotective dehydration,ntifreeze proteins and glycolipids, and high molar concentrationsf glycerol (Sformo et al., 2010, 2011; Walters et al., 2009), as wells numerous other mechanisms (Carrasco et al., 2011, 2012).

Cold acclimation is a multigenic, quantitative trait that involvesiochemical and structural changes that have a dramatic effect onhe physiology of a plant (Levitt, 1980; Weiser, 1970). There is noonsensus on the number and identity of genes causally related toold acclimation. Various reports have estimated that from <100o 1000 genes are up-regulated and a similar number are down-egulated (Bassett and Wisniewski, 2009; Fowler and Thomashow,002). Cold acclimation is an inducible process requiring both lowemperature (<10 ◦C) and moderate to high light (generally above00 �mol m−2 s−1) to achieve maximum hardiness. The specificonditions required to achieve maximum cold hardiness are speciespecific. It is a dynamic process that may require days or weeks.

Because of the complex changes in physiology, metabolism,tructure, and water content associated with cold acclimation,lants that are actively growing, flowering, or breaking dormancy

ypically have little to no frost tolerance (Sakai and Larcher, 1987;

isniewski et al., 2003) and thus are very susceptible to frostamage. Therefore, current approaches to improving frost toler-nce typically involve the overexpression of a specific transcription

d Experimental Botany 106 (2014) 60–69 61

factor (e.g. CBF) controlling a cold regulon (defined as a suite ofcold-inudcible genes) or overexpression of a specific “cryoprotec-tive” gene. The former approach, however, requires the use of acold-inducible promoter to avoid the adverse effects of overex-pression of the transcription factor on growth (Wisniewski et al.,2011). While true cold acclimation requires numerous adaptations,it is still possible that less encompassing short-term approachescan be identified that are capable of protecting sensitive plants, orplant parts such as blossoms, from spring frosts, where tempera-tures reach only a few degrees below freezing. The use of antifreezeproteins obtained from freeze avoiding animals such as insects, arepotential candidates.

Antifreeze proteins (AFPs) are an important component of therepertoire of adaptations to subzero temperatures in many orga-nisms, including numerous plants. AFPs are thought to function byadsorbing onto the surface of ice crystals, blocking the addition ofwater molecules to growth sites and thereby lowering the temper-ature at which the crystal will grow (Jia and Davies, 2002; Knightet al., 1991; Raymond and DeVries, 1977; Raymond et al., 1989).Recent evidence has demonstrated that some AFPs can also affectwater structure at some distance from the actual surface of theAFP and this may also be important in the antifreeze capabilities ofthese proteins (Ebbinghaus et al., 2010, 2012; Meister et al., 2013).By conventional definition, the freezing and melting points of anaqueous sample are identical. The temperature at which a smallcrystal will melt completely if the temperature is raised slightly isthe melting point and the temperature at which the crystal willbegin to grow as the temperature is lowered slightly is the freez-ing point. However, this is not the case if antifreeze proteins arepresent. AFPs have only a small effect on the normal melting pointof water, raising it slightly (Celik et al., 2010; Knight and DeVries,1989). AFPs, however, decrease the temperature at which an icecrystal grows, defined as the hysteretic freezing point, by an aver-age of 1–2 ◦C below the melting point in fishes and 2–5 ◦C in insects,although the difference can be as much as 13 ◦C in the Alaskan bee-tle Cucujus clavipes in winter when the insect is dehydrated and theAFPs concentrated (Duman et al., 2010). This difference betweenthe melting and hysteretic freezing points is termed thermal hys-teresis (TH) and is characteristic of AFPs (DeVries, 1971, 1986). Theusual technique for determining the presence of AFPs in a sam-ple is to assay for this unique thermal hysteresis activity (DeVries,1986). The magnitude of the measured thermal hysteresis is often,depending on the AFP, inversely correlated with the size of the crys-tal(s) present in the sample being tested (Husby and Zachariassen,1980; Nicodemus et al., 2006). Consequently, the level of freez-ing protection provided by AFPs in insects to combat inoculativefreezing across the cuticle is generally greater than the measuredthermal hysteresis, because the size of the cuticular water poresthrough which external ice might propagate are much smaller thanthe size of the crystals used in the thermal hysteresis measurements(Duman et al., 2010). AFPs can also inhibit ice nucleators, therebylowering the nucleation temperature and promoting supercooling(Duman, 2001, 2002). Therefore, AFPs can promote freeze avoid-ance by inhibition of (1) inoculative freezing across the body surfaceand (2) ice nucleators (Duman et al., 2010).

Although AFPs were first found in Antarctic fish (DeVries, 1971),thermal hysteresis has been identified in numerous diverse orga-nisms including many insects (Duman, 1977, 1979a, 2001; Dumanet al., 2010), collembola (Graham and Davies, 2005; Lin et al., 2007;Zettel, 1984), spiders (Duman, 1979b; Zachariassen and Husby,1982), mites (Block and Duman, 1989), nematodes (Wharton et al.,2005), plants (Duman, 1994; Duman and Olsen, 1993; Griffith and

Yaish, 2004; Griffith et al., 1992; Huang and Duman, 2002; Simpsonet al., 2005; Smallwood et al., 1999; Urrutia et al., 1992; Worrallet al., 1998), and fungi and bacteria (Duman and Olsen, 1993;Hoshino et al., 2003; Sun et al., 1995). AFPs have been purified from

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any of these species (for reviews see DeVries, 2005; Duman et al.,010; Graether and Sykes, 2004; Griffith and Yaish, 2004). Basedn their extremely varied structures in different organisms, it islear that AFPs have evolved independently numerous times, evenithin a given taxon such as fish (Cheng and Chen, 1999; Cheng

nd DeVries, 2002; DeVries, 2005). Also, the specific thermal hys-eresis activities of different AFPs vary considerably. For example,hile highly active insect AFPs produce several degrees of thermalysteresis, in plants the activity is limited to only a few tenths ofdegree, or less. It is important to note, however, that AFPs havenly been identified in freeze tolerant plants, where the absoluterevention of freezing is not the goal. At times the only indica-ion of AFP presence in a plant is the recrystallization inhibition ofce and/or unusual ice crystal structure, both of which require only

inimal AFP activity. In spite of this low specific activity, plant AFPsre functionally significant in freeze tolerant species because ofheir recrystallization inhibition, and perhaps other abilities such asheir impact on crystal structure (Griffith and Yaish, 2004; Griffitht al., 2005; Knight and Duman, 1986). Recrystallization refers tohe process that normally occurs after the initial formation of ice. Icerystals tend to be small when they first form but over time increasen size due to the migration of water molecules from the high sur-ace free energy of small (high radius of curvature) crystals ontoarger crystals. This process is referred to as recrystallization andt can result in physical damage to cells. AFPs, even at low concen-rations, prevent recrystallization, thus promoting freeze toleranceTursman and Duman, 1995). Since all of the known AFP-producinglants are freeze tolerant, their function in plants is not to preventreezing entirely, as is the case with AFPs of freeze avoiding ani-

als. Because of the variability in specific activity and function, theerms ice-binding proteins (IBPs) or ice-structuring proteins (ISPs) areometimes used to include true AFPs, as well as those proteins fromreeze tolerant species with minimal thermal hysteresis activitynd those with only the ability to inhibit recrystallization or affecthe shape of ice crystals (Wharton et al., 2009).

In contrast to the low specific thermal hysteresis activity oflant AFPs, those found in freeze avoiding animals, especially cer-ain insects, produce thermal hysteresis of the magnitude thatould protect sensitive plants from damage resulting from typi-al spring frosts. The thermal hysteresis measured in fish serum ispproximately 1.0–2.0 ◦C (DeVries, 2005). Maximum thermal hys-eresis produced by fish AFPs is ∼2.5 ◦C, or a bit higher. Whilehis is not exorbitant, it is sufficient to protect fish since the tem-erature of seawater does not generally drop below −1.9 ◦C. Inddition to the lower specific activity of fish AFPs relative to thosef insects, there are other problems associated with the use of cer-ain fish AFPs for plant protection. The antifreeze glycoproteinsAFGPs) present in the prevalent and largely endemic Antarctic fishf the group Nototheniiformes, as well as some northern speciesuch as cods, have relatively simple protein sequences, largelyonsisting of repeating tri-peptides of alanine-threonine-alanineDeVries, 1971). Post-translational glycosylation of the threonineesidues, however, seems to rule-out the use of the genes cod-ng these proteins for use in transgenic plants since the activityf the AFGPs is dependent on the disaccharides (galactosyl-N-cetylgalactoseamine). On the other hand, a potentially positivespect of using certain fish AFPs is that, in addition to lowering thereezing temperature, type-I (winter flounder, etc.) and III (zooar-ids, etc.) AFPs and the AFGLs can provide membrane protectiont hypothermic temperatures (Kamijima et al., 2013; Tomczak androwe, 2002).

Terrestrial insects in cold climates generally experience much

ower temperatures than fish. Therefore, they have evolved morective AFPs. For example, larvae of the beetle Dendroides canaden-is from northern Indiana supercooled to approximately −20 ◦C andad a mean hemolymph thermal hysteresis of approximately 3 ◦C

d Experimental Botany 106 (2014) 60–69

during recent relatively warm winters (Nickell et al., 2013), butsupercooled to a mean of −25 ◦C exhibiting a thermal hystere-sis of 3–5 ◦C during colder winters (Duman, 1982). During thesevery cold winters it was not unusual to identify individual larvaehaving 6–9 ◦C of thermal hysteresis in their hemolymph. The AFPsresponsible for the thermal hysteresis in D. canadensis (Andorferand Duman, 2000; Duman et al., 1998; Nickell et al., 2013) aresimilar to those described in other beetles such as Tenebrio molitor(Graham et al., 1997; Liou et al., 1999), and Cucujus clavipes (Dumanet al., 2004a,b, 2010). As previously mentioned, C. clavipes fromAlaska typically supercool to −40 ◦C, and below if they are exposedto extreme low temperatures (Bennett et al., 2005; Sformo et al.,2010, 2011). Structurally, these beetle AFPs are families of 7–16 kDaproteins consisting of 12-13-mer repeating units in which certainamino acids are highly conserved. NMR and X-ray studies (Graetherand Sykes, 2004; Graether et al., 1999, 2000; Leinala et al., 2002)revealed the 3-dimensional structure of the T. molitor AFP to be aflattened cylinder formed by a right-handed �-helix. Every sixthresidue of these insect AFPs is a cysteine that forms a disulfidebridge across the interior of the cylinder to provide stability (Li et al.,1998a,b). Threonine residues adjacent to each cysteine are spacedto permit their hydroxyl groups to hydrogen bond to oxygen atomsin the ice lattice. Alternatively, they bind water molecules in anice-like fashion and these then bind to the ice. This T-C-T sequenceforms the �-helical structure that presents a flat ice-binding sur-face. Beetle AFPs bind to both the prism and basal planes of ice(Graether et al., 2000; Pertaya et al., 2008), and this may account fortheir unusually high TH activity. Additionally, Meister et al. (2013)reported that D. canadensis AFPs (DAFPs) can affect water struc-ture up to a distance of 28 A from their surface and that this playsa role in their TH activity. Certain DAFPs exhibit a self-enhancingsynergistic effect on the thermal hysteresis activity of other DAFPs(Wang and Duman, 2005). The >30 AFPs comprising the family ofDAFPs in D. canadensis are expressed in a tissue specific fashion(Duman et al., 2002; Nickell et al., 2013), and just four are rou-tinely present in the hemolymph: DAFPs-1, 2, 4, and 6. DAFP-1 and-2 enhance one another, as do DAFPs-2 and DAFP-4, and DAFPs-4and -6. In addition, a thaumatin-like protein found in D. canadensis,that lacks thermal hysteresis activity itself, also enhances the activ-ities of DAFPs-1 and -2 (Wang and Duman, 2006). This synergisticeffect has the potential to be of considerable importance in gen-erating enhanced freeze avoidance in transgenic plants expressingthe proper combination of DAFPs.

2. Transgenic AFP plants

A number of studies have produced transgenic plants express-ing AFPs from other plants, fish, and insects. Most of these did notproduce lowered freezing temperatures in the transgenic plants.In some systems, AFP expression in transgenic plants producedplants whose extracts exhibited low levels of thermal hysteresis(0.37 ◦C/mg apoplastic protein by nanoliter osmometer, Holmberget al., 2001), recrystallization inhibition in vitro (Worrall et al.,1998), or reduced electrolyte leakage in leaves after freezing (Walliset al., 1997; Wang et al., 2008; Zhu et al., 2010). Only the expres-sion of AFPs derived from beetle larvae (Huang et al., 2002; Lin et al.,2011) unambiguously resulted in increased levels of supercoolingin the transformants, however, the freezing temperatures of theplants were often not reported in other studies.

2.1. Transformation with fish AFPs

Winter flounder, Pseudopleuronectes americanus, fish typeI, AFP was infiltrated into the extracellular spaces of leavesof canola (Brassica napus), potato (Solanum tuberosum) and

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rabidopsis thalianna using vacuum infiltration. This resulted in a.8 ◦C reduction in the average freezing temperature, as measuredy differential thermal analysis, of the leaves relative to control

eaves infiltrated with cytochrome C or water (Cutler et al., 1989).he level of protection relative to controls varied with the plantype (1.8 ◦C in B. napus; 4.0 ◦C in A. thalianna. The AFP-infiltratedeaves also exhibited a decrease in the rate of ice crystal forma-ion and the amount of freezable water at various temperatures (as

easured by NMR). These results provided the impetus for severalater studies whose goal was the production of transgenic plantsroducing fish, especially winter flounder, AFPs.

The first successful reported expression of an animal AFP inlant material was in corn protoplasts (Georges et al., 1990). Alasmid that included a translational fusion of a gene encodinghe winter flounder type-I fish AFP and the N-terminal of chloram-henical acetyltransferase (CAT), along with a signal peptide, withodons based on plant preferences, and the cauliflower mosaic virus5S promoter, was introduced into the protoplasts by electropo-ation. The incorporation and expression of the AFP-cat transcriptas confirmed in protein extracts of the protoplasts by measuringAT activity and examining Western blots probed with anti-AFPnd anti-CAT antibodies. In spite of inclusion of a signal peptideequence in the afp-cat gene, only a small percentage of the fusionrotein was properly secreted into the medium while approxi-ately 75% of the protein remained in the protoplasts, presumably

ecause the signal peptide was not properly cleaved by proteases.he level of cold tolerance of the resulting protoplasts was noteported.

Hightower et al. (1991) produced transformed tomato andobacco plants expressing winter flounder AFPs. The authors syn-hesized an afa3 gene encoding a native AFP that contains threenits of the alanine rich 11 amino acid repeat from winter flounder,s well as the gene for a fusion protein (Spa-Afa5) encoding a win-er flounder based AFP with five repeats of the 11 amino acid repeatinked to a truncated Stapholococcus protein A. The constructs

ere transformed into E. coli and then introduced into Agrobac-erium tumefaciens. Plants were transformed by co-cultivation withounded leaf discs. The purpose of the study was to determine if

he recrystallization inhibition properties of the native AFP and/orhe enhanced recrystallization inhibition activity of the fusionrotein (Mueller et al., 1991) could provide some level of freeze pro-ection that might improve the post-harvest freeze/thaw qualitiesf frozen fruits and vegetables. Both afa3 and the spa-afa5 mRNAsere detected in tomato and tobacco. Only the fusion protein, how-

ver, was identified in Western blots, and only in tissue from theransgenic tomato. Recrystallization inhibition activity was mea-ured in extracts from the tomato tissue where the fusion proteinas detected. The authors did not determine whether this activityas sufficient to provide either frost protection or improve super-

ooling so as to prevent freezing in the transgenic plants, but, this isnlikely to have been the case because the low level of recrystalliza-ion inhibition reported indicates very limited antifreeze proteinctivity.

Kenward et al. (1993) also attempted expression of winterounder AFP in tobacco using the cauliflower mosaic virus 19S pro-oter and cDNA encoding for the pre-pro-AFP. Although mRNAas detected, the protein was not, unless the transgenic plantsere held at 4 ◦C for 24–48 h, after which the pro-AFP was identi-ed. The authors suggested that the proper alpha-helical secondarytructure of the flounder AFP was not maintained at high temper-tures, perhaps resulting in the breakdown of the protein. If true,his would indicate that this AFP may be not be useful for protec-

ion of frost sensitive plants because higher daytime temperaturesrior to a frost may negate any beneficial effect that may have beenchieved by the presence of the properly folded AFP. Another fac-or that may account for the low expression of the winter flounder

d Experimental Botany 106 (2014) 60–69 63

AFP in this study, however, may be the difference in codon usagebetween fish and plants.

The winter flounder AFP, with codon usage frequencies con-sistent with those of higher plants, was also used to successfullytransform potato using Agrobacterium mediated transformation(Wallis et al., 1997). Inclusion of a native signal peptide directed theexpressed protein to the apoplast. Electrolyte leakage tests (LT50)(Sukumarin and Weiser, 1972) were used to determine the efficacyof the expressed AFP. Leaves from wild-type Russet Burbank potatoand transformed lines expressing varying levels of AFP (as deter-mined by Western blots) were tested for damage after being heldfor 30 min at temperatures between −2 and −4 ◦C. Freezing wasinduced by the application of external ice. The wild-type plantsexhibited an LT50 of −2.5 ◦C while transformed lines expressingAFP exhibited a range of lower LT50 values depending on the levelof AFP production. The line expressing the most AFP exhibited anLT50 of −3.5 ◦C, indicating that the AFP provided approximately 1 ◦Cof protection relative to the wild-type. The described results wereobtained when leaves were frozen for 30 min periods. Tests werealso conducted using longer freezing times ranging from 1 to 4 h at−2.5 ◦C. The line with the most AFP expression exhibited approxi-mately 22% leakage after a one hour freeze and leakage increasedwith increased time of freezing, but 50% electrolyte leakage wasnot obtained until leaves had been frozen for four hours. The studydid not determine if the decreased electrolyte leakage in the AFPproducing transformants resulted from decreased ice nucleation, adecreased rate of ice crystal growth, or an inhibition of recrystal-lization. In other tests, in which leaves were held at temperatures of−2 to −4 ◦C without external ice nucleation, the AFP producing lineshad less electrolyte leakage, suggesting that decreased ice nuclea-tion played at least some role in these results. For example, at −4 ◦Cthe highest producing AFP line exhibited only 2.3% leakage, whilethe wild-type had 34.4%. In addition to this freeze protection, thetransgenic plants were also reported to exhibit increased toleranceto hypothermic above-freezing temperatures. At 4–6 ◦C, transgeniclines exhibited growth of new tissue and robust leaf color. Perhapsthese features could be attributed to the hypothermic protection ofmembranes provided by winter flounder type-I fish AFP (Kamijimaet al., 2013; Tomczak and Crowe, 2002).

Another study used modified winter flounder AFP genes, plusand minus a signal peptide, to transform spring wheat (Khannaand Daggard, 2006). Results were similar to those reported byWallis et al. (1997) for transgenic potato. A synthetic gene encod-ing a larger form of the winter flounder AFP was used and thiswas suggested to be responsible for the increased thermal stabilityof the expressed protein relative to an earlier study by Kenwardet al. (1993). In the study reported by Khanna and Daggard (2006),substantial amounts of the AFP accumulated in both the cyto-plasm and the apoplast when plants were grown at 14–18 ◦C. Theapoplast targeted AFP represented 1.61% of the total soluble pro-tein and 90% of the AFP was apoplastic. This is important as iceis typically initiated in the apoplast. Recrystallization inhibitionwas observed in whole plant extracts and in apoplastic fluid, indi-cating the presence and accumulation of active AFP. Electrolyteleakage tests indicated that transgenic lines expressing AFP hadimproved freeze tolerance relative to wild-type plants. This wasespecially true in the line demonstrating the highest level of AFP(measured by Western blot), with 90% of the AFP being secretedto the apoplast. While wild-type plants exhibited >50% electrolyteleakage at −5 ◦C, the highest AFP producing line exhibited only 27%leakage at −5 ◦C, 40% at −6 ◦C, and approximately 55% at −7 ◦C.Consequently, once again the best AFP producing lines provided

about 1 ◦C of frost protection relative to the wild-type. The reduc-tion in freezing injury in the transgenic lines could not be absolutelyattributed to inhibition of freezing (avoidance) or to less freezingdamage (tolerance).

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Type II fish AFP (found in sea raven, smelt and herring) con-ains five disulfide bridges, so once they are properly folded theyre more stable than type I fish AFPs. Kenward et al. (1999)sing Agrobacterium mediated transformation of tobacco leaf disks

ntroduced a gene encoding the mature sea raven type-II fish pro-ein, driven by the cauliflower mosaic virus 35S promoter. Linesith and without the tobacco mosic virus signal peptide were pro-uced in order to target the protein to either the apoplast or theytosol. The mature form of Type II fish AFP representing up to 2%f apoplastic protein was present in the transgenic tobacco lines.o accumulation was observed in the cytoplasm. Active AFP was

dentified by the presence of bipyramidal ice crystals typical ofrystal growth in the presence of this AFP, but thermal hystere-is activity was very low. Field trials to test the frost resistance ofhe transformed plants relative to wild-type plants were conductedt the Agriculture Canada Research Station in Delhi, Canada dur-ng the autumn. The transformed plants did not exhibit increasedrost resistance, however, the authors did suggest that the trans-enic AFP-producing plants could be be grown for the purpose ofarvesting type-II fish protein.

.2. Transformation with insect AFPs

The studies that expressed fish, mainly winter flounder, AFPs inransgenic plants resulted in the production of plants that were,t best, approximately 1 ◦C more freezing tolerant than wild-typelants. While this was a significant accomplishment, a greater

evel of protection is required to provide adequate levels of frostolerance in plants. The sequencing and descriptions of insectFPs with significantly greater specific thermal hysteresis activ-

ty (Andorfer and Duman, 2000; Duman et al., 1998; Graham et al.,997; Graether et al., 1999) offered an opportunity to improve onhis level of frost protection by making the transgenic plants frostvoiding.

Holmberg et al. (2001) generated the first transgenic plantsxpressing an insect AFP. A synthetic gene, based on the AFP presentn the spruce budworm, Choristoneura fumiferana, (Tyshenko et al.,997) was transformed into tobacco with a plant signal peptide andriven by the cauliflower mosaic virus 35S promoter. Success ofhe transformation was measured by the presence of appropriatelyized transcripts using RT-PCR, recrystallization inhibition activityn crude leaf homogenates and apoplastic extracts of the plants,nd most importantly, thermal hysteresis (0.37 ◦C) in the apoplas-ic fluid. The freezing response or LT50 of the transgenic tobaccoas not reported.

Huang et al. (2002) successfully produced transgenic Arabidop-is thalianna (ecotype WS-2) expressing DAFP-1 from Dendroidesanadensis. This AFP was chosen because it is the most abun-ant DAFP in the D. canadensis hemolymph and has high specificntifreeze (thermal hysteresis) activity. D. canadensis AFPs rou-inely produce a hemolymph thermal hysteresis of 5–6 ◦C (Li et al.,998a,b), and inhibit ice nucleators (Duman, 2002) and inocula-ive freezing initiated by external ice (Olsen et al., 1998). An earliertudy also indicated that DAFPs could inhibit the freeze damagen isolated cells resulting from external ice formation, probablyy preventing cytoplasmic freezing and recrystallization inhibitionTursman and Duman, 1995). Genes encoding the native DAFP-, with and without the insect signal peptide, were cloned intoxpression vectors containing the cauliflower mosaic virus 35SNA promoter and terminator. Arabidopsis was transformed viagrobacterium-mediated transformation. Transformed plants were

nitially selected for growth on kanamycin, and those with the high-

st levels of dafp-1 were identified using Northern blots and chosenor further analysis. Western blots demonstrated expression ofAFP-1, ± signal peptide, in T4 and later generation plants, although

wo bands were present on the electrophoresis gels. Because DAFPs

d Experimental Botany 106 (2014) 60–69

typically migrate on gels as if they are larger than they actuallyare, MALDI-TOFF mass spectrometry was used to better identifythe mass of the proteins in the bands. The larger band matchedthe mature DAFP, indicating that the smaller band was a partialbreakdown product, perhaps resulting from proteases in the plantshomogenates. Immunoblots of leaf apoplastic extracts showed thatonly plants expressing DAFP-1 with a signal peptide secreted themature DAFP into the apoplast. A thermal hysteresis of 0.8 ◦C wasobserved in the highest activity line, demonstatrating that thesignal peptide was recognized and cleaved in the plants. The break-down product present in the homogenates was not present in theleaf apoplast, so proteolysis was not responsible for the lower thanexpected apoplastic activity. The lines expressing DAFP-1 lackingthe signal peptide did not secrete DAFP into the apoplast but ther-mal hysteresis activity was present in homogenates, although itwas not as high as in lines expressing DAFP with the signal peptide.

To determine if there was a difference between the lines inthe temperature at which plants froze, Huang et al. (2002) usedhigh-resolution infrared thermography (Wisniewski et al., 1997)to observe the freezing process and determine the temperature atwhich freezing was initiated (Fig. 1). Although plants were frozenusing four different protocols, transgenic lines expressing DAFP-1with the signal peptide (DAFP in the apoplast) always froze at astatistically lower temperature than wild-type plants, while thosewith DAFP-1 confined to the cytoplasm did not. The four differentfreezing protocols were as follows. (1) Whole plants with exposedroots were placed in groups such that adhering soil was contiguousand freezing was initiated in the soil to insure that all plants wereequally exposed to inoculation at the same temperature. Plantswere cooled at 5 ◦C h−1. The mean freezing temperature of trans-genic plants expressing DAFP-1 with the signal peptide (DAFP inthe apoplast) was −4.6 ◦C, which was significantly lower (p ≤ 0.05)than that of the wild-type which froze at an average of −3.3 ◦C.The freezing temperature of transgenics lacking the signal peptide,and therefore lacking DAFP in the apoplast, was −3.9 ◦C which wasnot statistically different from the wild-type. In this protocol, plantfreezing was initiated by inoculation from ice in the soil and spreadfirst to the lower stem and then through the rest of the plant. (2)Plants were cut at the base, the cut stems were sealed with vac-uum grease to prevent leakage of xylem fluids, the surface of theplants were sprayed with an ice nucleating active strain (Cit7) ofPseudomonas syringae bacteria, and cooled at a rate of 5 ◦C h−1. Onceagain transgenics with DAFP in the apoplast froze at a lower tem-perature (mean temperature of −5.1 ◦C), which was significantlylower (p ≤ 0.05) than the wild-type which froze at −4.2 ◦C. In theseplants, freezing began in the leaves or stems, probably initiated bythe surface bacteria. (3) Plants were treated as in 2, except thatthey were sprayed with water that did not contain ice nucleat-ing bacteria. The results were similar to those of 2 above, exceptthat all freezing temperatures were slightly lower. Under all threeof the above freezing protocols, freezing of the plants began atfairly high sub-zero temperatures and was initiated by extrinsicnucleating agents present on the surface of the plants. The pres-ence of apoplastic DAFP-1, even at fairly modest levels of activity,produced a small, but significant, decrease in the temperature atwhich freezing was initiated. (4) The fourth cooling protocol pro-duced freezing that began at much lower temperatures. Stems weresevered at the base, mounted in vacuum grease, and cooled at5 ◦C h−1. These plants were not sprayed with water or Cit7 bacte-ria. Once again, the transgenics with apoplastic DAFP-1 froze at asignificantly lower (p ≤ 0.05) temperature (−16.6 ◦C) than the wild-type (−13.3 ◦C), an average of 3.3 ◦C colder. The transgenics with

cytoplasmic, but not apoplastic, DAFP-1 did not freeze at a signif-icantly lower temperature than the wild-type. Since freezing is astochastic event, the lower freezing temperatures observed usingthe 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 secretionto the appoplast. The gene coding the DAFP-1 protein was obtained from the beetle, Dendroides canadensis. Individual plants were removed from the soil, mounted oncardboard 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. Freezingwas 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 temperatureof 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 transgenic270-23 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 witha ◦ 16.6 ◦CT leato

atwtlrwt

signal peptide) at −17.9 C. Average freezing temperature of 340-29 plants was −he high level of supercooling indicates that the DAFP-1 may have inhibited ice nuc

bsence of high populations of extrinsic nucleators, and the facthat the plants were dry (no surface moisture) thus the plantsere able to supercool. Although the purpose of the transforma-

ion experiments was to determine if the transgenic plants froze at

ower temperatures than wild-type plants, once plants did freeze,egardless of the freezing conditions, no difference in survivorshipas observed. All plants that froze were dead seven days after

hawing.

which was significantly different (p ≤ 0.05) than the wild-type and 270-23 plants.rs present in the apoplast.

Although the results of the experiments demonstrating thattransgenic Arabidopsis expressing apoplastic DAFP-1 froze at signif-icantly lower temperatures than wild-type plants is encouraging,a degree of freeze protection greater than 1–3 ◦C will be neces-

sary to have a major impact on the ability of plants to survive asignificant frost event. The small amount of freeze avoidance(1–3 ◦C) achieved was attributed primarily to the low level of ther-mal hysteresis activity accomplished, only 0.8 ◦C at best in the

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polast, present in the transgenic plants. After these experimentsere completed, however, it was discovered that certain DAFPs,

nd certain other proteins, are able to interact with one anothero synergistically increase thermal hysteresis activity (Wangnd Duman, 2005, 2006). This suggested that transgenic plantsxpressing a combination of DAFPs with synergistic interactions,ight exhibit greater thermal hysteresis, and therefore greater

reeze protection, even if the total level of DAFP expression wasot raised.

Therefore, Lin et al. (2011) introduced two synergistic DAFPDNAs (dafp-1 and dafp-4) into the genome of Arabidopsissing Agrobacterium-mediated floral dip transformation. To gen-rate transgenic Arabidopsis expressing both DAFP-1 and -4o-transformation of multiple binary vectors containing differentenes was performed. Insect DAFP signal peptides were used to tar-et the DAFPs to the apoplast and transgenic lines expressing eitherAFP-1 or DAFP-4, and both DAFP-1 and -4 were generated. Linesith the greatest amount of thermal hysteresis were selected for

urther study. Southern blots indicated that these lines all had mul-iple copies of the transgenes, and RT-PCR confirmed that the genesere being expressed. Western blots of total soluble protein from

he lines demonstrated the presence of DAFPs, however, the levelf expression of DAFP-4 was low. The thermal hysteresis measuredn the protein extracts (50 mg ml−1 total protein) was consistent

ith the Western blots as the two lines expressing both DAFP-1 + 4ad the greatest activity (1.39 and 1.20 ◦C). The DAFP-1 line had theext highest activity (0.75 ◦C) and the DAFP-4 line did not exhibithermal hysteresis but did produce hexagonal ice crystals indicat-ng a very low level of DAFP activity. The measurment of thermalysteresis in apoplastic fluids provided similar results:

1.15 ◦C in the DAFP-1 + 4 lines and 0.75 ◦C in the DAFP-1 line.he DAFP-4 line had no activity, not even an effect on ice crytstaltructure (i.e., hexagonal crystals).

Lin et al. (2011) used the following protocol to determine theemperature at which freezing was initiated in the plants. Stemsere cut at the base, sealed with vacuum grease, placed in Petriishes in an environmental chamber, cooled, and a high-resolution

nfrared camera was used to detect freezing events. The mean freez-ng temperature of the transgenic lines expressing both DAFPs-1nd -4 was significantly lower than in wild-type plants (2.9 ◦Cower), and lines producing only DAFP-1 or DAFP-4. The freezingemperature of the lines producing only DAFP-1 was also signifi-antly lower than in the wild-type (1.8 ◦C lower), but was not inhose lines producing only DAFP-4. Tests with individual leavesxcised from these plants showed similar trends. Although theseesults were again encouraging, a greater level of freeze avoid-nce was expected in the lines producing both DAFP-1 and -4 dueo expected synergy between the two DAFPs. A greater level ofhermal hystereis in the apoplastic fluid of these lines was alsoxpected. The lower than expected level of synergy and concomi-ant thermal hysterisis activity was suspected to be the result of lowevels of expression of DAFP-4 in the transgenic lines that expressedAFP-4 alone or in conjuction with DAP-1. This premise was con-rmed by RT-qPCR. In fact, expression of dafp-1 was 116-fold and3-fold greater than dafp-4 in the two DAFP-1 + 4 expressing lines,nd when the thermal hysteresis of pure DAFP-1 and DAFP-4 wasested at ratios of 53/1 or 116/1, no synergy in thermal hysteresisctivity was observed. Consequently, the freeze protection in theransgenic plants was consistent with the levels of thermal hystere-is measured in the apoplastic fluid, as was the case in the earliertudy (Huang et al., 2002).

Wang et al. (2008) expressed an AFP from another beetle,

icrodera punctipennis, from the Xinjiang desert region of China

n tobacco using the same approach to vector construction andransformation as previously described. This AFP is similar to theFPs in D. canadensis and Tenebrio molitor beetles. Transformed

d Experimental Botany 106 (2014) 60–69

plants were identified by RT-PCR of leaf homogenates, and plantswith the highest transcript levels were selected for further analysis.The expressed AFP was localized to the cell walls of the transgen-ics using immuno-gold labeling, and Western blots demonstratedthe presence of the AFP in apoplastic fluid. Thermal hysteresis orrecrystallization inhibition tests to determine if the expressed pro-tein was active were not conducted, although it can be assumed thatat least some level of activity was present. The cold tolerance of thetransgenics relative to wild-type was tested by visual inspectionof plants and measurements of ion leakage and malondialdehyde(a measure of lipid peroxidation) release following exposure of theplants to −1 ◦C for variable periods of time up to 72 h. After 2 and3 days of exposure to −1 ◦C, wild-type plants were reported to bemore negatively affected than were the transgenics, as assessed bythe presence of more wilted leaves in the wild-type plants. Afterone day of recovery at 25–28 ◦C the transgenic plants appeared tobe fully recovered while the wild-type plants still appeared to bestressed as indicated by wilted leaves. This was evident even afterfive days of recovery. Ion leakage and malondialdehyde levels ofboth wild-type and transgenics were low after one day at −1 ◦C, butthe levels in wild-type plants rose significantly relative to the trans-genics after 2–3 days at −1 ◦C. Therefore, the presence of the beetleAFP provided some level of protection to the tobacco at this rela-tively mild, but lengthy, subzero temperature exposure. Whetherthis was due to providing freeze avoidance or increasing freezingtolerance was not determined.

Zhu et al. (2010) synthesized a spruce bud worm (C. fumifer-ana) AFP and expressed it in Arabidopsis thaliana using a plantcodon bias and a tobacco PR-S signal peptide. The expression vec-tor contained the synthetic AFP gene and a double cauliflowermosaic virus 35S promotor. Three transformed lines exhibiting thehighest levels of afp transcript were selected based on RT-PCR oftotal RNA from leaves. The levels of AFP expressed and the AFPactivities (thermal hysteresis or recrystallization inhibition) werenot determined or reported, so the claim of overexpression of theAFP was unsubstantiated. After three weeks growth at 23 ◦C underlong photoperiod, wild-type and transformed plants were trans-ferred to 4 ◦C for two days (at either long or short photoperiods)and then subjected to −20 ◦C for 30 min. Plants were then heldat 4 ◦C overnight, then transferred back to a 23 ◦C growth cham-ber. Visual inspection of the plants several days later indicatedthat while most of the wild-type plants had died, a higher levelof survival was evident in the transgenic lines, although actualnumbers were not reported. The line with the highest level of afptranscript also had the best survival and the line with the low-est level of transcript exhibited much poorer survival. Electrolyteleakage and malondialdehyde content in all of the plants increasedafter cold treatment, but the levels were significantly greater inwild-type plants than in the transformed plants. The damage in thetransformed lines was inveresly correlated with afp transcript lev-els in the three transformant lines. These results again indicatedthat expression of an insect AFP in transgenic plants amelioratedthe injury resulting from exposure of plants to freezing temper-atures. It should be noted, however, that the freezing protocolused was very atypical for freezing stress studies. Whether theplants had actually frozen during the protocol was not substanti-ated and such a short exposure (30 min) would not have guaranteedfreezing unless ice nucleation was induced at a warm, sub-zerotemperature. Therefore, whether the reduction in injury in thetransgenic plants was due to freeze avoidance (promotion of super-cooling), a reduction in the amount of physical injury to cells by thegrowth of large ice crystals being inhibited by recrystallization, oran increase in freezing tolerance (protection of cell membranes,especially the plasmalemma) could not be determined. The impact

of growing the plants under short vs. long photoperiods was notreported.

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.3. Transformation with plant AFPs (IBPs)

Given the previously mentioned low specific activity of knownlant AFPs, perhaps better described as ice binding proteins (IBPs),

t seems unlikely that expression of these proteins in frost-sensitiverop plants would result in increased freeze avoidance, and there-ore frost protection. Also, because freeze tolerance is a complexrocess involving a host of biochemical changes and adaptations,

t is improbable that expression of IBPs in frost-sensitive plantsould render them freeze tolerant. It is possible, however, thatlant IBPs could exhibit ice nucleation inhibition activity. If so,his could be of value in situations where inhibition of intrinsicce nucleators is critically important. Most plant IBPs appear to beresent in the apoplast, where ice forms in freeze tolerant plants,nd consequently these IBPs apparently do not inhibit ice nuclea-ion. Wisniewski et al. (1999), however, reported a dehydrin withhermal hysteresis activity, in the cytoplasm of acclimated peachark and xylem cells, including xylem ray parenchyma cells, whichre known for their ability to supercool to very low temperatures.

A leucine rich carrot AFP (IBP) gene, structurally similar to theolygalacturonase inhibitor protein (Worral et al., 1998) was usedo produce transformed Arabidopsis that accumulated the IBP atow temperatures (Meyer et al. (1999). AFP activity was defined ashe ability of extracts from transformed plants to affect ice crystalhape. These transgenic plants would not be expected to exhibitlower freezing temperature (i.e., increased freezing avoidance)

ince the specific thermal hysteresis activity of plant IBPs (includinghe one from carrot) are low. The plants were not tested for coldolerance.

. Conclusions and future directions

While the studies outlined in this review provide evidence thathe expression of AFPs can be used to produce more frost resistantrop plants, there is obviously considerable need for improvement.ransgenic plants with the ability to avoid freezing at temperatureselow −5 or −6 ◦C would be of great value, providing protectionrom freeze damage during the minor freezes that occur in earlypring and early autumn. Based on the results achieved in trans-enics thus far, producing this level of freezing avoidance and/orreezing tolerance by the ectopic expression of AFPS (especiallynsect AFPS) seems plausible. A few points should be kept in mind,owever, in pursuing this line of study. (1) The higher specific ther-al hysteresis activity AFPs, probably those of insects, should be

mployed. (2) A high level of expression of active proteins, tar-eted to the apoplastic fluid, is critical, as is the use of AFPs withhe ability to act synergistically in increasing thermal hysteresis.roper folding of the proteins is essential to their activity. Therefore,fp transcript levels, protein concentration, and thermal hysteresisctivity should always be determined and reported so that if opti-al results are not achieved in the transgenic plants the potential

roblem can be identified and addressed in subsequent work. (3)hen expression of the AFPs throughout the whole plant is not

ssential, the efficacy of site-specific promoters should be explored.or example, restricting AFP expression to flowers using a flowerpecific promoter could provide protection to sensitive flowers ofruit trees in spring. (4) The actual temperature at which freezings initiated in the plants being studied should be monitored, along

ith ice propagation, by infrared video thermography if posssible.his will enable the identification not only of the freezing tem-erature but also the determination of whether or not decreased

reeze injury in transgenic plants is the result of the formation ofess ice, variation in the site of formation, rate of ice propagation,tc. (5) Care should be taken to use well described, realistic, andultiple freezing regimes to test the resistance of plants. Since the

d Experimental Botany 106 (2014) 60–69 67

environmental conditions that are present during natural frosts areoften different and more complex than those used in environmen-tal chambers, field tests are eventually necessary to provide reliableproof of the ability of the plants to avoid or tolerate freezing. (6)While use of Arabidopsis or other model plants for “proof of concept”studies is often the most efficient approach, it is necessary to even-tually extend the study to the actual crop plant for which protectionis desired. Initiation of freezing, whether resulting from inoculativefreezing from the surface or from internal ice nucleators, variesbetween species. Additionally, since ice nucleation is a stochasticevent, the size or mass of the plant will have an impact on the prob-ability of a nucleation event occurring. Most crop plants are muchlarger than Arabidopsis, and therefore they may freeze at a highertemperature. Also, the developmental stage of the plant when frostprotection is required may also play a significant role since frosttolerance and/or avoidance can vary on a seasonal and develop-mental basis. In spite of these and other considerations, such as theresistance of the public to genetically engineered crop plants, it islikely that the development of transgenic plants expressing AFPswill eventually result in crops and horticulturally important plantsthat survive exposure to frosts of −5 or 6 ◦C, and perhaps lowertemperatures. Such developments will have tremendous positiveresults for growers by reducing or eliminating losses to damag-ing frosts, and expanding both the geographical range and growingperiods for crops.

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