Genomics and Functional Genomics of Winter Low Temperature Tolerance in Temperate Fruit Crops

Abstract

Winter low temperature tolerance is the result of genotype interaction with environmental cues influencing plant development and metabolic activity in preparation for sustained low temperatures and freezing conditions. In deciduous fruit trees the phenology of acclimation and dormancy processes are well documented and recent advances in functional genomic analyses are beginning to map the molecular mechanisms underlying the physiological and biochemical responses resulting from inductive short photoperiod and/or low temperatures. There are many commonalities between herbaceous annual plant acclimation and perennial plant acclimation; however, the ability to survive sustained periods of low temperature, extreme low temperatures and the presence of ice within tissues are not common in annual model plant systems. In addition, subzero temperature tolerance mechanisms vary with tissue type and age and seasonal and climatic conditions; thus, defining the phenotypes and underlying genetic control of these dynamic and complex quantitative traits is both time and land intensive. Rapidly increasing genomic sequences and databases of transcriptomic, proteomic and metabolomics data sets, coupled with existing mapping studies and new genotype by sequencing and single nucleotide polymorphism (SNP) marker analyses provide the opportunity to dissect winter survival and elucidate its genetic basis. This review explores recent genomic and functional genomic analyses that are contributing to a greater understanding of the molecular mechanisms regulating low temperature response and freezing tolerance in temperate woody plants. Developments are providing the opportunity to more quickly and precisely link responses to genes and gene variations underlying low temperature response and tolerance in temperate fruit crops.

Keywords:

• cold hardiness
• environmental stress
• gene expression
• apple
• blueberry
• grapevine
• peach

I. WINTER SURVIVAL IN WOODY TEMPERATE FRUIT CROPS

Subzero freeze events in late spring and early fall and winter minimum temperatures limit the sustainable production of fruit tree crops. Orchard location, cultivar selection and cultural practices have been used extensively to minimize production losses; however, freezing injury is a continuing problem for perennial crop production in cold winter climate regions. Freezing tolerance is an active trait, responsive to environmental fluctuations and it is genotype dependent for the timing of acclimation and dormancy, genetic potential for mid-winter freezing tolerance, rate of deacclimation, ability to reacclimate and timing of budbreak (Stushnoff, 1972). Therefore, winter survival is a multi-trait phenomenon that is impacted by photoperiod, temperature and water and nutritional status; all of which can interact differentially with genotype and tissue developmental stages (Fennell, 2004; Gusta and Wisniewski, 2012; Palonen and Buszard, 1997; Stushnoff 1972). Progress in selecting sustainable cultivars requires identifying critical phenotypes and susceptible tissues as well as critical environmental events (early fall freeze, mid-winter ultimate low temperature, deacclimation events). Breeding and selection of adapted cultivars is hindered not only by the complexity of the trait, but also by long generation time, heterozygosity and inbreeding depression, large plant size, rootstock interactions as well as fruit quality traits; all of which confound precise phenotyping and genetic analysis. Therefore advances in identification of trait characters and their genomic basis are needed to promote marker identification and marker assisted selection for improved winter survival in temperate fruit trees.

Survival of subzero temperatures depends on the woody plants ability to acclimate in response to low temperature and or photoperiod; promoting physical and biochemical changes in multiple tissues, which will allow the plant to avoid ice nucleation in tissues or compartmentalize ice in locations where it does not cause injury (Ashworth, 1982; 1992; Gusta and Wisniewski, 2012; Pearce, 2001; Wisniewski et al., 2004). It should be noted that freezing tolerance and avoidance traits within a given species are not static, rather they are dynamically influenced by stage of development, intrinsic and extrinsic nucleators and environmental conditions (Gusta and Wisniewski, 2012; Pearce 2001). Tolerating the presence of ice in tissue apoplast and cellular dehydration as water moves from the cell to ice in the extracellular spaces allows many plant tissues to tolerate lower temperatures without injury (Levitt 1980). In apple (Malus), the bark and phloem tissue tolerate extracellular ice formation while the xylem parenchyma cells tolerate subzero temperatures by supercooling as freezing of the xylem parenchyma results in tissue death (Ashworth, 1992; Gusta et al., 2009; Quamme et al., 1973). Supercooling limits the strain of freeze dehydration, although progressive dehydration can occur with extended subzero temperatures. It also limits the range of plants using this mechanism to regions with winter low temperatures above the homogenous nucleation temperature or greater (Ashworth and Wisniewski, 1991; George et al., 1974; Quamme, 1976). Development of the ability to supercool depends on low temperature and/or photoperiod induced acclimation responses that promote reduction in water content, changes in cellular biochemistry and the development of barriers to ice propagation from the stem into the bud tissue (Ashworth, 1982; Ashworth and Wisniewski, 1991; Burke and Stushnoff, 1978; Jones et al., 2000; Gusta and Wisniewski, 2012, Levitt, 1980). In grapevine (Vitis species) and some Prunus species, the overwintering flower or compound buds supercool and adjacent stem tissue may tolerate extracellular freezing (Ashworth, 1992; Fennell, 2004; Mathers, 2004; Kader and Proebsting, 1992; Quamme et al., 1995). The ability to supercool depends on tissue water content, shoot morphology, the stage of bud development and absence of intrinsic and extrinsic nucleators. It is a dynamic characteristic and will change in response to climatic conditions and dormancy status of the plant. The factors that determine ice initiation and location are frequently overlooked in genomic analysis of woody plant systems and careful experimental planning is needed to ensure functional analysis of relevant freezing tolerance characteristics (Arora and Rowland, 2011; Ashworth, 1984; Gusta and Wisniewski, 2012). Therefore, to improve the understanding of the genomic mechanisms contributing to the physical and biochemical changes involved in winter survival requires temporal studies and systems biology approaches (from a holistic rather than a reductionist perspective) that address specific tissues in the context of the whole plant and its responses to natural environmental conditions (Dhanaraj et al., 2007).

Table 1 Fruit tree genome assemblies and databases

Species/Genome		First assembly		Genome assembly	Gene	Major assembly
Publication	Family	release	2n	size Mb	Model	locations
Vitis vinifera Jaillon et al., 2007	Vitaceae	2007	38	486.2	27,791	http://urgi.versailles.inra.fr/cgi-bin/gbrowse/vitis_12x-pub/ http://genomics.cribi.unipd.it/Grape_Genome
Carica papaya Ming et al., 2008	Cariaceae	2008	18	372	24,746	http://asgpb.mhpcc.hawaii.edu/papaya/
Prunus persica Arús et al., 2012	Rosaceae	2010	16	227.2	28,702	http://www.rosaceae.org/peach/genome
Malus × domestica Velasco et al., 2010	Rosaceae	2010	34	742.3	57,386	http://genomics.research.iasma.it/gb2/gbrowse/apple/ http://www.rosaceae.org/
Fragaria vesca Shulaev et al., 2011	Rosaceae	2010	14	240	34,809	http://www.strawberrygenome.org/
Theobroma cacoa Agrout et al., 2011	Sterculiaceae	2010	10	326.9	37,885	http://www.cacaogenomedb.org/
Citrus clementine*	Rutaceae	2011	18	370	35,796	ICGC http://www.citrusgenome.ucr.edu/*
Citrus sinensis* Xu et al., 2012**	Rutaceae	2011/2012	18	367	29,445	ICGC http://www.citrusgenome.ucr.edu/*
Pyrus bretschneideri Wu et al., 2013	Rosaceae	2012	34	512	42,812	http://peargenome.njau.edu.cn/

*Genome sequence assembly was released prior to publication of the genome summary article. **Second assembly published.

Table 2 Genome-wide gene family analyses

Gene Family	ID	Role	Species	Expression data*	Reference
Heat Shock transcription factor	Hsf	Transcription factor, Stress response	Apple (M. domestica)	Gene expression in developing flowers and fruit	Giorno et al., 2012
Dehydrin	MdDHN	Stress response	Apple (M. domestica)	Cold and drought induced gene expression	Liang et al., 2012
Dehydration responsive element binding	MdDREB	Transcription factor, Stress response	Apple (M. domestica)	Cold, drought, ABA and heat induced gene expression	Zhao et al., 2012
APETELA2/Ethylene Response factor	AP2/ERF	Transcription factor, Growth and development, Stress response	Grape (V. vinifera)	Tissue specific microarray data	Licausi et al., 2010
SET DOMAIN GROUP	SDG	Epigenetic regulators	Grape (V. vinifera)	Tissue specific gene expression of subset of genes	Aquea et al., 2011
MIKC^c-type MADS Box	MADS	Growth and development, Signal transduction	Grape (V. vinifera)	Tissue specific gene expression	Díaz-Riquelme et al., 2009
Dehydrin	DHN	Stress response	Grape (V. vinifera)	Cold, drought, and heat induced gene expression	Yang et al., 2012
Auxin/indoleacetic acid	Aux/IAA	Growth and development	Grape (V. vinifera)	Salt and drought gene expression data, berry development	Çakir et al., 2013

*Genome-wide family analysis includes publications with referenced published expression datasets.

In spite of the complexity of woody plant systems, an atlas is emerging for some of the physiological and molecular responses to environmental cues that drive plant low temperature acclimation and deacclimation processes (Arora et al., 2003; Gusta and Wisniewski, 2012, Guy, 2003; Janska et al., 2010; Obata and Fernie, 2012; Theocharis et al., 2012; Welling and Palva, 2006). This review will focus specifically on genomic resources and recent functional genomic analyses that are furthering the understanding of winter survival of temperate tree fruit crops, with an emphasis on apple (Malus), blueberry (Vaccinium), grapevine (Vitis) and peach (Prunus).

II. GENOMIC RESOURCES

The number of flowering plant genome sequences and tools for functional genomics has increased dramatically in the decade since the first plant genome sequence assembly (Arabidopsis thaliana, 2001) was released. In 2007, genome assemblies of an inbred of Pinot Noir and the heterozygous Pinot Noir (V. vinifera) cultivar were published, making these two genomes the first fruit crop genomes and grapevine the fourth vascular plant species genome sequence to be released (Table 1) (Jaillon et al., 2007; Velasco et al. 2007). Preceding the grapevine genome release, markers, expressed sequence tags (ESTs) and tools such as VitisGeneChip (Affymetrix) were made publically available thus providing resources to initiate systems biology research (Adam-Blondon, et al., 2004; Goes da Silva et al., 2005). This was quickly followed by the development of visualization tools for the large transcriptomic, proteomic or metabolomic (omic) datasets (Grimplet et al., 2009; 2011; http://PlexDB.org). Availability of the genome sequence assemblies, ESTs and proteomes has promoted rapid identification of gene families. Several gene families that have been associated with abiotic stress or growth and development have been described very recently (Table 2). Genome-wide analysis of gene families with expression analysis, in differential environmental conditions, for relevant tissues and stages of development have aided the functional annotation of genomes and provided phenotype information needed for discovery of genetic mechanisms.

In the five years since the first grapevine genome sequence assembly was released, 29 new vascular plant genomes have been published and many more assemblies are in progress (Michelmore, 2012). The vast majority of these genomes are crop plants and several are highly valuable fruit tree crop species making comparative genomic analyses of important crop traits more accessible (Table 1). The fruit crop or relative species are diploids with relatively small genomes making them more amenable to sequencing. However, the fruit trees are highly heterozygous and have a long generation time, which has made genetic analysis and advancement in selection for quantitative traits like cold tolerance onerous. DNA markers and linkage maps of apple, peach, grapevine and other fruit species have long been published, but availability of genomes has made it possible to more fully integrate genetic maps and identify gene variation between genotypes. Genome publications and international public and consortium databases provide links not only to genome sequences, but also for genomic and bioinformatics tools and transcriptomic, proteomic, metabolomics and microRNA data sets, thereby enhancing opportunities for genome annotation improvement and comparative and functional genomic analyses.

III.  FUNCTIONAL GENOMICS OF ACCLIMATION RESPONSES FOR WINTER SURVIVAL

Acclimation for winter seasons are cued by short photoperiod and/or low temperature and over a century of observations and studies in natural temporal or controlled environment conditions have identified changes in plant biochemistry and physiology related to the ability of plants to tolerate low and freezing temperatures. Acclimation processes are marked by a reduction in water content, fluxes in calcium, modifications in membranes, metabolic reprogramming, accumulation of carbohydrates, amino acids, proteins and secondary metabolites and changes in cell wall structure (Ashworth, 1992; Fennell, 2004; Gusta and Wisniewski, 2012; Guy, 2003; Levitt, 1980; Smallwood and Bowles, 2002; Theocharis, 2012; Welling and Palva, 2006; Wisniewski et al., 2009). Correlation of these biochemical changes with freezing tolerance has provided many gene candidates for functional gene analysis; however the complexity of winter survival indicates that, rather than single gene analysis, targeted and holistic analysis are needed to precisely determine the genetic mechanism involved in winter survival. Functional genomic tools have been increasingly applied to acclimation and dormancy studies (Table 3) in fruit trees in the last decade and characteristics specific to species and woody plant systems are emerging.

Table 3 Functional genomics studies of low temperature and short photoperiod response in fruit trees

Method	Treatment(s)	Tissues**	Species	Reference*
EST sequencing	Warm temperature, low temperature (5°C, 24 h) and water deficit	leaf, bark, xylem	Apple (Malus x domestica)	Wisniewski et al., 2008
EST sequencing	Cold acclimated	flower bud	Blueberry (Vaccinium spp.)	Dhanaraj et al., 2004
EST sequencing	Cold acclimation through deacclimation	flower bud	Blueberry (Vaccinium corymbosum)	Rowland et al., 2012
cDNA microarray	Seasonal and cold room acclimation studies	flower bud	Blueberry (Vaccinium corymbosum)	Dhanaraj et al., 2007
EST, cDNA microarray, subtractive hybridization	Cold acclimated and non-acclimated	flower bud	Blueberry (Vaccinium corymbosum)	Rowland et al., 2008
EST sequencing	Low temperature and short photoperiod	Bark	Peach (Prunus persica)	Bassett et al., 2006
EST sequencing	Short photoperiod endodormancy induction	Bark	Peach (Prunus persica)	Jimenez et al., 2010b
EST sequencing	Seasonal endodormancy, and ecodormancy		Peach (Prunus persica)	Leida et al., 2010
Proteomic 2-D DiGE	Low temperature and short photoperiod	Bark	Peach (Prunus persica)	Renaut et al., 2008
Proteomic 2-D gel	Seasonal paradormancy endodormancy, ecodormancy and dormancy relesease	flower bud	Apricot (Prunus mume)	Zhuang et al., 2012
microRNA	Low temperature treatment of vegetative bud and leaf at growing temperature	vegetative bud	Peach (Prunus persica)	Barakat et al., 2012
cDNA microarray	Low temperature acclimation and chilling fulfillment, endodormant through ecodormant	compound bud, 5 time points	Grape (Vitis riparia)	Mathiason et al., 2009
GrapeGen a520510F array	Temporal seasonal development, paradormancy through dormancy release	compound bud, 7 time points	Grape (V. vinifera)	Dííaz-Riquelme et al., 2012
NimbleGen Vitis	Temporal seasonal development, paradormant, endodormant	compound bud	Grape (V. vinifera)	Fasoli et al., 2012

*Low temperature acclimation or short photoperiod studies are included in table.**Some studies analyzed multiple treatment conditions and tissues; however, only the tissues receiving low temperature/acclimation or short photoperiods are listed in tissue column.

A.  Apple

Apple acclimates in response to low temperature, but not photoperiod, to induce acclimation and dormancy (Heide and Prestrud, 2005). The acclimated apple tree tolerates equilibrium freezing in phloem and bark tissues while the xylem parenchyma cells supercool (Ashworth and Wisniewski, 1991; Quamme et al., 1973). A comparison of gene expression in apple leaf, bark and xylem tissues during early acclimation to low temperature and water deficit identified about 1000 genes (∼65% known function) differentially expressed between tissues and low temperature and water deficit treatments (Wisniewski et al., 2008). In low temperature acclimated apple bark and xylem, genes related to energy, cell growth and development, protein metabolism and defense related were up-regulated relative to control; and photosynthesis, metabolism and transport were down-regulated relative to warm control. Most notable was a strong up-regulation of dehydrin genes at low temperatures in the bark and xylem but not in the leaves. A recent genome-wide survey identified 12 dehydrin genes in apple and four of these (MdDHN2, MdDHN4, MdDHN6 and MdDHN8) increased dramatically in leaf tissue upon exposure to low temperature (Table 2) (Liang et al., 2012). The MdDHN2 showed a strong similarity to the dehydrin previously identified in apple bark under low temperature induction and bark and buds under midwinter conditions (Garcia-Bañuelos et al., 2009; Wisniewski et al,. 2008). Dehydrins have been shown to increase during low temperature acclimation and decreased water content in a variety of woody species and this associative data suggest a potential role in winter survival mechanisms (Artlip et al., 1997; Sarnighausen et al., 2004; Welling et al., 2004; Wisniewski et al., 1996, 2006).

B.  Peach and Apricot

Peach (Prunus persica) responds to decreasing photoperiod and/or temperature to initiate dormancy and acclimation. A study in peach bark designed to separate low temperature acclimation and short photoperiod dormancy induction specific responses found dehydrins (PpDhn1 and PpDhn3), chitinase and thaumitin transcripts specifically upregulated in low temperature while heatshock protein transripts were up-regulated in short photoperiod treatments (Bassett et al., 2006). Suppression subtractive hybridization of cDNA libraries for seasonally induced endodormant and ecodormant buds from low and high chill requiring peach cultivars produced 364 ESTs, 101 contigs and identified genes specific to endodormancy, ecodormancy and the low chill genotype (Leida et al., 2010). Genes up-regulated in buds during dormancy included metabolism, stress and defense and signaling and transcription (Late embryo abundant (LEA), Dormancy associated MADs box (DAM), NAC domain and AP2/EREBP). Genes upregulated during the ecodormant phase included a large number of peroxidases. Thus, there was a distinct difference in transcriptomes corresponding to the dormancy dehydration and chilling fulfillment in the two cultivars with changes in transcription related to dehydration and protection being the most relevant during acclimation and winter phase of dormancy.

Discovery of an ever-growing peach genotype has advanced genetic analysis of peach tree dormancy and cold acclimation induction. The evergrowing peach (P. persica evg) genotype does not respond to short photoperiods for induction of dormancy and acclimation and is less freezing tolerant than the deciduous peach (Arora et al., 1992). Suppression subtractive hybridization of short photoperiod induced cDNA libraries from wild type (WT) and evg peach buds was used to identify 177 ESTs that produced 106 contigs (Jimenez et al., 2010b). Analysis of the expression of 23 of these genes in WT and evg, during short photoperiod treatments, indicated three patterns of expression, two of which appear to be associated with dormancy induction. A group of genes were upregulated quickly upon transition to short photoperiod and subsequently declined at two weeks of short photoperiod (signaling/transcription, transport and defense). Another group was upregulated later in short photoperiod, but continued to increase in expression for eight weeks (including: LEA, PR proteins, DAM and metallothioneins). These patterns indicate signaling responses resulting in reprogramming of the metabolism as well as sustained gene expression are associated with dormancy maintenance.

Proteomic analysis of peach bark from low temperature and/or short photoperiod treatments indicated that low temperature had the greatest effect on proteome expression with 25 peptide spots more abundant and six less abundant relative to 25°C treatment (Renaut et al., 2008). Short photoperiod specific expression included one peptide spot of higher abundance and seven peptide spots of lower abundance. It is noteworthy that short photoperiod and low temperature, which promote the best acclimation response, showed additive effects, resulting in 36 more abundant and three less abundant peptide spots. The low temperature specific increased abundance peptides were related to stress response (dehydrin and PR-protein), energy, amino acid, lignin metabolism, cytoskeleton organization and defense mechanisms. Dehydrins are suggested, but not conclusively shown, to have a protective role in cells under freeze dehydrated conditions. Similarly PR-proteins such as glucanases and thaumitin-like proteins have been reported to have antifreeze properties that impact the location and growth of ice crystals (Griffith et al., 2005). Changes in transcript and protein abundance of the dehydrins and PR-proteins during the acclimation of bark tissues, which tolerate equilibrium freezes, provide support for a potential role in mitigating freeze dehydration stresses; however their precise function remains to be determined (Bassett et al., 2002; Wisniewski et al., 1996, 1999; 2006; Welling and Palva, 2006).

Proteomic analysis of apricot (Prunus mume) flower buds showed an increased abundance of 32 protein spots in endodormant buds subjected to low temperature (Zhuang et al., 2012). Similarities between peach bark and apricot floral buds include stress response and defense, energy metabolism and cell structure related (Renaut et al., 2008). Further, analysis of microRNAs in low temperature acclimated peach buds and actively growing leaves identified several highly abundant microRNAs in buds that have been suggested to target floral development genes; however, this study did not include non-acclimated floral bud therefore it is difficult to determine whether there was a true abundance of these negative regulators (Barakat et al., 2012). The negative regulatory role of many microRNAs requires that studies include the same tissue exposed to differential temperature or photoperiod and for microRNA and transcriptome analysis to identify relevant microRNAs and their potential targets.

C.  Blueberry

An analysis of 600 ESTs each from non-normalized cold acclimated and non-acclimated blueberry (Vaccinium corymbosum) floral buds showed a greater abundance of beta amylase, dehydrin, early light-inducible protein and senescence-associated protein transcripts in cold acclimated buds (Dhanaraj et al., 2004). A cDNA microarray containing 2500 elements was subsequently used to compare cold acclimation in field conditions with controlled environment conditions (Dhanaraj et al., 2007). Studies indicated that freezing tolerance of the blueberry floral bud was greater in the field after prolonged acclimation than in the controlled environment conditions (Rowland et al., 2008). While dehydrins, cell wall protein, LEAs and early-light inducible protein were abundant in both conditions, a greater number of genes were induced in controlled environment and it was noted that many of these were suppressed in the field conditions, perhaps in part due to fluctuating temperatures, temperature extremes or other environmental factors. Genes (in particular, auxin-repressed protein and protein kinase PINOID), which had not previously been identified as cold induced in Arabidopsis, were found to be cold induced in both field and controlled environment acclimated floral buds. Cold induced gene expression specific to the field or controlled environment acclimation were found, as were differences in gene expression between the non-acclimated plants in field or controlled environment. Genes related to light stress, photosystem IID1, photosystem II CP 47 and early light-inducible protein, were specifically up-regulated in the field in contrast to controlled environment. The greater abundance of gene expression between controlled environment and field environment are a caution and may result from shock-like conditions in controlled environment rather than the more gradual temperature reduction and diurnal cycling, however, it should also be noted that additional environmental cues may play a role in dampening gene expression in the field. Therefore, it is important to cross verify controlled environment systems biological studies with natural environmental conditions to address the holistic plant responses.

D.  Grapevine

Grapevines acclimate in response to photoperiod and temperature and tolerate midwinter subzero temperatures by extracellular freezing in stem tissues and supercooling of bud tissue (Fennell, 2004; Victor et al., 2010). Under short photoperiod and cold acclimation transcripts related to responses to external stimuli and PR-proteins such as chitinases and thaumitin-like proteins were up-regulated during early cold acclimation (Mathiason et al., 2009). Sustained chilling resulted in down regulation of the bud transcriptome; however, many transcripts related to response to external stimuli and reactive oxygen species were maintained or increased during the transition to ecodormancy. A transcriptome analysis of temporal/seasonal bud development from paradormancy to ecodormancy showed the greatest differential gene expression during bud initiation phase, with moderate levels during endodormancy and transition to ecodormancy and lowest levels during mid-winter (∼1500, 800, 900 and 200 genes, respectively) (Díaz-Riquelme et al., 2012). While specific analysis in relation to low temperature response were not conducted, it was noted that during the endodormant phase of bud development transcripts related to temperature stress response, starch catabolism, ABA catabolism, CCAAT family transcription factor, HSP-mediated protein folding and stilbenoid biosynthesis were up-regulated and gene pathways related to photosynthesis, primary and secondary metabolism (fatty acid, carbohydrate, cell wall and flavonoid biosynthesis and cell cycle) were down regulated. Similarly, a tissue atlas of gene expression in fifty-two grapevine tissues identified gene expression related to seasonal programming of tissue development (Fasoli et al., 2012). When gene expression was clustered by organ, the transcriptomes of bud and woody stem tissues were most closely related. Genes related to these mature/woody tissues (stem and bud) were enriched in protein metabolic processes, translation, cellular processes and response to external stimuli, with a high level of expression of dehydrin and metallothioneins. Coexpression analysis indicated a small group of genes (105) that were related to tissue maturation in stem and bud. Bud specific expression identified 384 genes, predominately signaling proteins, transcription factors and transporters, with 29 specific to paradormant and endodormant buds and 92 that were expressed from paradormancy through budburst. Tissue specific genes are useful in functional analysis and it is well established that tissue maturation is a key component of grapevine freezing tolerance; however, acclimation requires coordinated reprogramming within the whole plant so caution should be exercised in using only tissue specific genes as candidates for freezing tolerance studies (Fennell and Hoover, 1991; Howell and Shaulis, 1980). The identification of coexpressed genes between bud and woody stem tissues does provide potential paths for elucidating plant responses to environmental stimuli that promote cold acclimation as well as development of barriers to ice nucleation of the grapevine bud.

IV.  TRANSCRIPTIONAL REGULATION OF LOW TEMPERATURE RESPONSE

A.  Calcium Signaling

Deciduous trees respond to low temperature and/or short photoperiod to induce cold acclimation and that short photoperiod and low temperature are synergistic in many species promoting a greater freezing tolerance than low temperature (Fennell, 2004; Arora et al., 2003; Tanino et al., 2010). Analyses of low temperature responses are well defined in the model herbaceous plant (Arabidopsis thaliana) and cold acclimation processes in woody fruit trees show many commonalities with Arabidopsis. In model systems, low temperature responses are mediated through calcium and reactive oxygen signaling, which results in a cascade of cold mediated transcription regulation that promotes development of protective proteins and metabolites (Figure 1) (Theocharis et al., 2012; Zhu et al.,2007).

Figure 1 Schematic of signaling network involved in low temperature and/or photoperiod acclimation. Calcium signaling mediates activation of transcriptional cascades involving CBF or ZAT12 (Theocharis et al, 2012; Thomashow, 2010). The expression of CBFs is negatively regulated by PIF4 and PIF7 in long photoperiods. Short photoperiods repress expression of PIF4 and PIF7 promoting an increase in freezing tolerance. It should be noted that the exact mechanisms underlying repression of CBF and PIF4 and PIF7 in response to photoperiod still need to be resolved and may not be the only photoperiod responsive mechanism involved in acclimation processes.

In mulberry buds, increased calcium levels were noted in the cytosol and nucleus in response to decreased day length and dormancy induction and theses levels declined in midwinter (Jian et al., 2000). Similarly, decreasing temperature promoted an influx of calcium into cytosol and nuclei in apricot buds (Wang et al., 2008). In grapevine, calcium signaling related transcripts were shown to be upregulated in leaves in response to a short-term low temperature exposure (Tattersall et al., 2007). In addition to calcium fluxes as signals, calcineurin B-like proteins (CBL) are suggested to interact with CBL-interacting protein kinases (CIPK) to “decode” calcium signaling and promote spatially specific cellular response (Batistič et al. 2010). Recently, an apple CBL- interacting protein kinase (MdCIPK6L) was shown to be induced by calcium and cold temperature (4°C) and promoted enhanced freezing tolerance in Arabidopsis, thus supporting the role of calcium and CIPK in low temperature signal transduction (Wang et al., 2012). Further emphasizing the role of calcium signaling, a calcium regulated calmodulin-binding transcription activator (CAMTA), that has been shown to bind to a C-repeat binding factor (CBF) gene promoter, and a calcium/calmodulin-regulated receptor-like kinase (CIPK) were shown to be involved in increased freezing tolerance of Arabidopsis (Doherty et al., 2009; Yang et al., 2010). Thus, a low temperature network is suggested to be regulated by calcium and/or kinase/phosphatases (Theocharis et al., 2012). Additionally, it is suggested that a calcium-calmodulin CAMTA complex may directly regulate members of the AP2 domain-containing transcription factors, specifically CBF1 and CBF2, also called dehydration-responsive element (DRE) binding proteins (DREB1) or low temperature response elements (LTREs) (Thomashow, 2010). The CBFs are generally accepted as a regulatory hub of cold responsive genes (COR) during herbaceous plant cold acclimation; however, the CAMTA role still needs to be established (Theocharis et al., 2012; Thomashow, 2010). In addition, there is substantial evidence in Arabidopsis for a parallel cold induction pathway through a zinc finger protein (ZAT12), which plays a role in high light and may play a role in the high light and low temperature responses through the ROS signaling pathway (Doherty et al., 2009; Davletova et al., 2005; Theocharis et al., 2012; Vogel et al., 2005).

B.  Inducer of CBF Expression (ICE)

The transcription factor inducer of CBF expression (ICE) acts upstream of the CBFs and is a constitutively expressed basic helix-loop-helix transcription factor (bHLH) that is rapidly activated by low temperature, through sumoylation and phosphorylation (Theocharis et al., 2012; Thomashow, 2010). It has been shown that ICE1 is a major regulator of CBF3 and overexpression of ICE1 has been shown to increase freezing tolerance in Arabidopsis (Miura et al., 2011; Thomashow, 2010). An apple cold-induced bHLH gene (MdCIbHLH1), similar to AtICE1 and AtICE2, was shown to increase in expression within three hours of exposure to 4°C (Feng et al., 2012). Upregulation of MdCIbHLH1 was also followed by an increased expression of the five apple CBFs (MdCBF1, MdCBF2, MdCBF3, MdCBF4 and MdCBF5) during the low temperature exposure and the MdCIbHLH1was shown to bind to an AtCBF3 promoter MYC4 site. When MdCIbHLH1was constitutively expressed in Arabidopsis all three AtCBFs were upregulated. In apple, the MdCIbHLH1 bound to three MdCBF2 promoter MYC recognition regions, but did not bind to MdCBF1, MdCBF3, MdCBF4 or MdCBF5. Ectopic expression in apple callus and tissue cultures resulted in increased expression of MdCBF1, MdCBF2, MdCBF3, MdCBF4 and MdCBF5 and maintained viability of transgenic cells at low temperature, indicating that the MdCIbHLH1 plays a similar role to AtICE1 in mediating CBF regulation at low temperature.

C.  CBF/DREB1 Expression

The CBF/DREB1 genes, a sub-family of the APETALA2/ETHYLENE RESPONSE Factor (AP2/ERF) transcription factors, have been characterized as a regulatory hub in freezing tolerance in herbaceous plant systems (Theocharis et al., 2012; Thomashow, 2010). CBF/DREB1 homologs have also been identified in fruit trees and show similar responses to environmental stimuli as the Arabidopsis CBFs.

Sour cherry (Prunus cerasus L., PcCBF1) and strawberry (Fragaria x ananassa Duchesne, FaCBF1) were up-regulated in their respective leaves at 4°C and ectopic expression of FaCBF1 in strawberry increased leaf freezing tolerance (Owens et al., 2002). Similarly, a sweet cherry (Prunus avium) CBF ortholog (PaCBF) increased freezing tolerance in Arabidopsis (Kitashiba et al. 2004). A CBF found in highbush blueberry (V. corymbosum, BB-CBF) was expressed at higher levels in a cold tolerant northern cultivar than in southern cold sensitive cultivar during early cold acclimation stage (Polashock et al., 2010). In addition, overexpression of BB-CBF in a southern cold sensitive cultivar resulted in increased leaf and floral bud freezing tolerance at nonacclimating temperatures. Two almond CBFs (PdCBF1 and PdCBF2) were shown to bind to DRE elements and to be rapidly induced by low temperature. Induction of PdCBF1 and PdCBF2 was higher during the early dark period of the photoperiod cycle suggesting both photoperiod and temperature regulation (Barros et al., 2012a, b). In apple, which acclimates and enters dormancy solely in response to low temperatures, apple CBFs (MdCBF1, MdCBF2) increased between 30 minutes and 2 hours in response to low temperature (Heide and Prestrud, 2005; Wisniewski et al., 2011). In addition, an apple DREB/CBF gene, with similarity to the Arabidopsis DREB2B, was up-regulated by cold, drought, heat and ABA within 2 hours and increased expression for 8 to 24 hours (Table 2) (Zhao et al., 2012). In apple rootstock (Malus baccata) DREB1/CBF (MbDREB1) was rapidly up-regulated by cold, drought, salinity and ABA in several tissues and was most strongly up-regulated in stem and leaves at 4°C (Yang et al., 2011). The MbDREB1 promoter region contained an ICE1-like and an ABA responsive element (ABRE) binding site. Cold, drought and ABA stimuli promoted induction of a chimeric GUS gene (MbDREB1 promoter). In Arabidopsis, expression was localized to the nucleus and overexpression of MbDREB1 increased freezing tolerance (Yang et al., 2011). Analysis of the CBF/DREB1 pathway in grapevines has identified four CBF genes in V. vinifera and V. riparia that are up-regulated in response to low temperature (Tillett et al., 2012; Xiao et al., 2006, 2008). Overexpression of VvCBF4, in the hybrid grapevine rootstock Freedom, increased freezing tolerance and reduced growth (Tillett, et al., 2012). In contrast to VrCBF1, overexpression of VrCBF4 resulted in sustained expression of all CBFs (days rather than hours) and a greater increase in freezing tolerance and greater drought tolerance (Siddiqua and Nassuth, 2011). In addition, overexpression of three V. vinifera CBFs and a zinc finger protein (VvZFPL) increased cold tolerance in Arabidopsis and promoted the expression of a CIPK7 (Takuhara et al., 2011; Kobayashi et al., 2012). These studies all indicate that CBFs are present in fruit trees and respond to low temperature in a similar way to what has been shown in the Arabidopsis model.

D.  CBF/DREB1 Regulated Gene Expression

The CBF/DREB1 proteins bind to the C-repeat/dehydration-responsive element (CRT/DRE) of target COR genes and promote increased freezing tolerance (Theocharis et al., 2012; Thomashow, 2010).

In non-acclimated transgenic grapevines overexpressing VvCBF4, 59 transcripts (47 upregulated and 12 downregulated) were differentially expressed in relation to control plants (Tillett et al., 2012). These included transcripts for stress-responses, lipid metabolism and cell wall structure. A comparison of the non-acclimated grapevines overexpressing VvCBF4 with Arabidopsis overexpressing multiple CBF/DreB1 or Populus overexpressing AtCBF1 indicated 13 and 14 genes, respectively, with patterns of expression in common between grapevine, Populus and Arabidopsis. Of note are stress responsive, calcium-binding proteins and cell wall–related genes indicating that the CBF mediated responses are highly conserved across herbaceous and woody species. A test of VrCBF1 and VrCBF4 overexpression on endogenous Arabidopsis COR (AtCOR15a, AtRD29A, AtCOR6/KIN2 and AtCOR47) and development genes (AtGA2ox3, AtRGL3, AtFLC and AtICE1) indicated that both CBFs enhanced expression of COR genes and the AtRD29A (Siddiqua and Nassuth, 2011). Thus this suggests a role in both ABA-independent and ABA-dependent low temperature response pathways; however, the interaction of the CBF with a basic leucine zipper domain transcription factor (bZIP) remains to be shown to support its role in the ABA-dependent pathway. Further, differential response was noted among the development genes with greater expression of AtGAox7 in VrCBF1 lines and expression of AtRGL3 only detected in VrCBF4 lines. The flowering locus C gene (AtFLC) and At1ICE1 were up-regulated in both VrCBF1 and VrCBF4 transgenic lines. The ICE1 gene promotes CBF3 expression resulting in an increased freezing tolerance in several species. However, overexpression of VrCBF1 or VrCBF4 in Arabidopsis also enhanced ICE1 expression suggesting a potential CBF and ICE1 interaction involved in maintaining CBF3 expression (Siddiqua and Nassuth, 2011).

The most well documented cold and CBF regulated genes are the dehydrin gene family which contain the DRE/CRT/LTRE regulatory element in their promoter. Several studies have shown an increased abundance of dehydrin transcripts and proteins accumulating in woody plant buds and bark during induction of dormancy and cold acclimation (Arora et al., 1997; Arora and Wisniewski, 1994, 1996; Garcia-Bañuelos, et al., 2009; Muthalif and Rowland, 1994; Winiewski et al., 2006, 2008; Xiao et al., 2006; Yamane et al., 2006). In blueberry, constitutive expression of BB-CBF resulted in a high level of expression of two dehydrin genes and a galactinol synthase under non-acclimating conditions (Walworth et al., 2012). Further the almond PdCBF1 and PdCBF2 were shown to bind DRE elements and promoted a ­≥ twofold increase in expression of an almond dehydrin gene PdDHN1 and expression of an ABA-dependent stress-induced gene (AtRD29A) (Barros et al., 2012b). The almond CBFs were induced more strongly at the end of the photoperiod. When low temperature occurred in the dark period peak expression occurred earlier than when cold temperature occurred in the light period. In apple, overexpression of MbDREB1 upregulated the expression of COR15a and rd29B, further supporting the suggestion that DREB1 may be active in both ABA-dependent and ABA-independent pathways (Yang et al., 2011). Insertion of a peach CBF (PpCBF1) in an apple rootstock increased freezing tolerance in non-acclimated and cold acclimated plants over the wild type and resulted in high level of expression of an apple dehydrin (MdDhn1) in stems and leaves of the transgenic plant (Wisniewski et al., 2011). In apple, dormancy and cold acclimation are normally induced by low temperature not photoperiod (Heide and Prestrud, 2005). However, overexpression of the PpCBF1 in apple plants increased freezing tolerance and promoted induction of dormancy under shortphotoperiods alone, without a low temperature inductive stimulus (Wisniewski et al., 2011). The induction of photoperiod sensitivity in apple by PpCBF1, is novel and suggests interplay between genes regulating cold acclimation and photoperiod induction of dormancy. This is very noteworthy as it has long been recognized that in many woody plants there is a synergistic effect of short photoperiods and low temperature in the development of cold acclimation (Fennell, 2004; Arora et al., 2003; Tanino et al., 2010).

Recently it has also been shown that freezing tolerance in Arabidopsis can be induced by short photoperiod (Dong et al., 2011; Harmer et al., 2000). Short photoperiod resulted in increased expression of CBF1, CBF2 and CBF3 at 8 hours after dawn (zeitgeber time 8, ZT8) and upregulated expression of cold regulons AtCOR15 and AtGOL3 (Lee and Thomashow, 2012). It was also noted that short photoperiod and low temperature resulted in greater freezing tolerance in Arabidopsis as has been shown for woody plant systems and that concordant expression of PIF4 and PIF7 were required to repress acclimation in long photoperiods. The role of short photoperiod was uncovered using a double mutant of the phytochrome-interacting factor, a bHLH transcription factor, (pif4 and pif7). The pif4 and pif7 mutant eliminated repression of CBF pathway under long photoperiods resulting in increased CBF transcription levels and an increased freezing tolerance, similar to freezing tolerance in wild type plants under short photoperiod (Lee and Thomashow, 2012). Therefore, the short photoperiod induction of freezing tolerance and synergistic responses of photoperiod with cold temperature that has long been observed in woody plants may be mediated in part through the CBF pathway (Figure 1). Further analysis of the genes such as the peach CBF for an evening element and related DNA regulatory motifs should be conducted and may explain the photoperiod responsiveness observed by Winiewski et al., (2011) in the overexpressing PpCBF1 apple rootstocks.

Both photoperiod and temperature are factors affecting the entrainment of the circadian clock. The circadian clock senses and resets the clock in response to diurnal light and temperature cycles. A change in photoperiod resets the clock and subsequently the metabolism and physiology of the plant promoting induction of dormancy and acclimation responses (Cook et al., 2012; Espinoza et al., 2010; Lindlőf, 2010). Circadian regulation through circadian clock associated 1 (CCA1) and LATE HYPOCOTYL (LHY) exert positive regulation on CBF cold induction resulting in maximum freezing tolerance (Dong et al., 2011). In chestnut (Castanea sativa), low temperature disrupts the circadian oscillations resulting in a high levels of constant expression of TIMING OF CHLOROPHYLL a/b binding protein expression 1 (TOC1/PRR1), LHY1, and LHY2 and PSUEDO-RESPONSE REGULATOR-genes (PRR5, PRR7, and PRR9) (Ibanez et al., 2008). These responses further emphasize that there is an interchange between photoperiod and cold temperature on circadian rhythms that impacts the CBF hub regulatory pathway.

E.  Dormancy Associated MADS Box Gene Expression

Dormancy associated MADS Box genes (DAM) have been associated with the photoperiod and low temperature induction of dormancy in peach and apricot and it is noted that prolonged cold temperature suppresses their expression (Li et al., 2009; Sasaki et al., 2011). Thus the DAM genes are thought to play a role in the induction, maintenance as well as release from dormancy during the winter period. Growth cessation and leaf abscission do not occur in the evergreen peach mutant (evg) when exposed to short photoperiod and /or low temperatures (Rodriguez et al., 1994). Sequence analysis of the evg loci indicated the evg peach was missing 6 DAM genes (DAM1, DAM2, DAM3, DAM4, DAM5, and DAM6) (Bielenberg et al., 2008). Characterization of the DAM genes in field and controlled short photoperiod grown wild type peach indicated that five genes (DAM1, DAM2, DAM4, DAM5, and DAM6) were photoperiod responsive, with DAM6 expression showing the highest level of up-regulation upon exposure to short photoperiod. DAM1 and DAM2 were expressed at the time of dormancy induction in field conditions and DAM5 and DAM6 were most highly expressed in mid-winter, suggesting a role in dormancy maintenance. In addition, DAM5 and DAM6 expression was found to be higher in high chill requiring cultivars (Jiménez et al., 2010). The DAM3 appears to be low temperature regulated and decreases throughout the winter season and may play a role in dormancy release (Bielenberg et al., 2009). The role of DAM genes in dormancy release is further supported by work of Yamane et al. (2011) which showed that prolonged exposure to low temperature decreased expression of DAM5 and DAM6 and the decreased expression correlated with increased budbreak potential. Thus differential responsiveness of the DAM genes under controlled photoperiod and warm temperatures, in contrast to field conditions, suggests that they differ in potential freezing tolerance induction and dormancy release roles.

F.  Epigenetic Regulation

Epigenetic regulation is emerging as an important integrator of the environmental influences on plant growth and development. Vernalization, the cold induction of flower development, has been shown to be regulated by histone modification of chromatin (Ahmad et al., 2010; Pin and Nilsson, 2012). In chestnut, expression of two histone genes involved in chromatin remodeling, histone monoubiquitinase (HUB2) and histone acetyltransferase (GCN5L), was greater at dormancy induction than at dormancy release (Santamaria et al., 2011). In peach, decreasing levels of DAM6 expression are associated with chilling fulfillment and dormancy release (Jimenez et al., 2010a; Leida et al., 2011; Yamane et al., 2011). Investigation of histone modifications of DAM6 in low and high chill peach cultivars indicated specific chromatin modifications correlated with the down regulation of DAM6 and the progression from bud dormancy through dormancy release (Leida et al., 2011). This suggests that sustained low temperature represses DAM6 expression through histone modification of chromatin. This response is similar to the low temperature induced histone modifications that results in the repression of Flowering locus C, a MADS-box transcription factor, expression of which prevents flowering transitioning prior to vernalization (Ahmad et al., 2010). While epigenetic responses are apparent in low temperature mediated plant developmental processes their role in cold acclimation processes remains to be determined.

V. GENETIC ANALYSIS OF WINTER SURVIVAL

It is well established that acclimation and freezing tolerance are under genetic control and tissue and species specific (Ashworth, 1984; Gusta and Wisniewski, 2012; Kader and Proebsting, 1992; Owens, 2005). While the complexity of winter survival has limited detailed genetic analysis, early studies in apple, peach and blueberry, testing different components of the trait (photoperiod response, dormancy, acclimation, chilling requirement and timing of budbreak) indicate trait heritability and suggest additive variance (Cain and Anderson, 1980; Fear et al., 1985; Fejer, 1976; Mowry, 1964; Watkins and Sangelo, 1970).

Intraspecific hybridization of fruit crop species divergent in freezing tolerance and backcrossing to improve quality are frequently used to introgress winter survival traits and improve cultivar sustainability in adverse environments. Analyses of blueberry F1 and testcross populations, derived from blueberry species divergent in freezing tolerance (Vaccinium darrowi and V. caesariense, freezing sensitive (−13°C) and freezing tolerant (−20°C)) provided the opportunity to dissect inheritance patterns (Arora et al., 2000). Analysis of the F1 populations indicated that lower freezing tolerance characteristic is a partially dominant trait in blueberries with no significant maternal influence. Analysis of the generation means indicated additive gene effects of a small family of genes. Dehydrins have been observed to increase in buds during seasonal transition into dormancy and cold acclimation, therefore a candidate dehydrin (bbDHN1) was mapped in the testcross populations and compared with freezing tolerance of the populations (Panta et al., 2004; Rowland et al., 2004). Inheritance of freezing tolerance was not correlated with the bbDHN1 marker used; however, this represents the mapping of only one gene in a small population against a quantitative trait.

Chilling is required to transition bud and cambial tissues from endodormancy to ecodormancy in preparation for resumption of growth in spring. In Prunus species, chilling requirement plays a role in potential injury as a result of late winter and early spring freezing temperatures. Therefore, chilling fulfillment and budbreak are considered major factors in freezing tolerance. An F2 mapping population from a selfed F1 (generated from low and high chill requiring parents) was phenotyped for chilling requirement, heat requirement (growing degree hours) and bloom date (Fan et al., 2009). Twenty QTL were identified, including one major QTL for chilling requirement and two for bloom date. The remainder co-localized with other traits. One of the co-localized groups was identified in the region previously identified for the deciduous peach (EVG). Similar to peach, apricot is susceptible to freezing damage in late winter and early spring resulting in reduced yields (Olukolu et al., 2009). In apricot, chilling requirement segregated within a mapping population generated from low and high chill genotypes and 12 QTL were identified, with several localized to similar QTL as previously described for peach (Fan et al. 2009). Gene candidates associated with the QTL include the MADS-box transcription factors, a mitogen activated protein kinase 7 (MPK7), and abscisic acid insensitive 3 (ABI3) genes. The recent publication of the peach genome assembly will aid in further identification of genes and genetic variation underlying these QTL.

In grapevine, response to photoperiod promotes early growth cessation, dormancy and acclimation processes and promotes synergistic increase in freezing tolerance upon cold acclimation (Fennell, 2004; Fennell and Hoover, 1991; Schnabel and Wample, 1987). An F2 mapping population from a selfed F1 (generated from V. riparia, very freezing tolerant and highly photoperiod responsive and hybrid wine grape Seyval, moderate freezing tolerance and low photoperiod responsive) was monitored for growth cessation and lateral emergence in controlled environment and field conditions (Garris et al., 2009). Different QTL were observed for growth cessation and critical photoperiod response in the two environmental conditions. A major QTL on linkage group 13 explained 80–90% of phenotypic variation for controlled environment conditions with non-inducing low temperatures. In contrast, a QTL on linkage group 11 explaining 85 to 94% of phenotypic variance was observed in field conditions. The differences between environments suggest the presence of non-photoperiodic cues for induction of growth cessation in the field. These results are in sync with the observations that both photoperiod and low temperatures play a role in cold acclimation processes.

Changing climate with increased warming trends impacts the timing of budbreak and flowering which can increase potential for freezing injury. Analysis of budbreak and flowering time was conducted in 120 progeny of a Reisling x Gewurztraminer cross (V. vinifera) (Duchêne et al., 2012). Three phenotypes related to thermal time were examined and six QTL were identified for the traits of degree days for chilling requirement to budbreak, budbreak to flowering and flowering to veraison. Two main QTL explaining 11 and 12% of phenotypic variance were identified for budbreak on linkage group 4 and 19 and two main QTL explaining 16 and 27% of the phenotypic variance were found for flowering time. Candidate genes for the budbreak QTL were identified by comparison of genes within the loci with previous bud chilling transcriptomic analysis (Mathiason et al., 2009). These comparisons identified a glutathione S-transferase and a WRKY transcription factor underlying the QTL loci. Analysis of the QTL loci for flowering time indicated nine genes linked to flowering including known flowering time gene VvFT and a zinc finger Constans-like gene (VvCOL2). The availability of transcriptomic expression datasets and genome sequence provide the opportunity to quickly identify relevant candidate genes underlying QTL.

These studies indicate that genetic variation for traits related to winter survival exist in most fruit crops and/or related wild species. However, the need to select for freezing tolerance at the same time as other quantitative traits (fruit quality and production characteristics) intensifies the challenge for the breeder. Finally, decreased genotyping costs provide the opportunity for high density SNP discovery allowing the development of suites of SNP markers and increasing potential marker definition without the need to construct linkage maps (Elshire et al., 2011). Plant gene expression is also controlled by regulatory regions that fall outside of the coding DNA and genome-wide SNP discovery provides the ability to identify SNPs in all regulatory regions for marker assisted breeding. Thus the availability of genome, genotype by sequencing and SNP analysis and transcriptomic, metabolomics and proteomic expression profiles from relevant tissues and environmental conditions provide the enhanced opportunities for dissecting complex traits like winter survival.

VI.  OPPORTUNITIES

Development of genomic and functional genomic resources is providing strong platforms to use in addressing, in a holistic fashion, the multifaceted trait of woody plant winter survival. The ability to use transcriptomic, proteomic and metabolomics expression data in conjunction with inheritance studies present new opportunities for determining the molecular and genetic basis of cold tolerance in fruit trees. Recent genetic analysis of metabolites in apple fruits identified a metabolite QTL (mQTL) for phenolic compounds (Khan et al., 2012). It is known that acclimation responses result in major shifts in metabolism (Janska et al., 2010) and through-put capacity in metabolite analysis is increasing steadily; therefore use of metabolite profile as phenotypes provide a similar opportunity for dissecting genetic mechanisms, as using transcriptome profiles guided candidate gene identification in budbreak QTL (Duchêne et al., 2012). Similarly proteogenomics, expressed protein tags from mass spectrometric analysis of proteins, can provide opportunities for protein QTL (pQTL) and greater depth in expression profiling as well as genome annotation. Finally any study of the genomic components of winter survival must consider that the ability of the plant to survive winter conditions, involves response to multiple environmental cues and that the presence or avoidance of ice includes physiological, morphological and biophysical factors. Thus studies must take into consideration water interactions with cellular components and molecules, intracellular communication, interactions between tissues and kinetics of ice formation and duration of freeze for complete systems biology analyses to dissect the underlying genomic components.

ACKNOWLEDGMENTS

Support was received from the South Dakota State University Agricultural Experiment Station.