Introduction

Potato (S. tuberosum) is an economically important world’s fourth largest food crop from Solanaceae family [1]. The potato, like other cultivated plants, can suffer from a number of abiotic stresses. Its bare and shallow root system also makes it more susceptible to these stresses [2]. Environmental abiotic constraints such as cold, heat, salinity, drought, flooding, radiation, heavy metal toxicity, etc. significantly inhibit the plant growth and cause to the serious crop losses. Among these constraints, cold stress, as chilling (<20 °C) or freezing (<0 °C), is one of the frequently exposed stresses in plants [3]. To overcome this, plants differentially regulate their genes to adapt their physiology for cold stress and to develop a cold tolerance/acclimation [4]. By using microarray technology, a large number of differentially expressed genes have been identified in Arabidopsis [5], potato [6], rice [7], wheat [8], barley [9], and oat [10] under cold acclimation/stress. DNA microarray analysis of Arabidopsis showed that cold stress (4 °C) causes the differentially expression of hundreds to thousands of cold-inducible (COR) genes, that are associated with accumulation of cryoprotectants, osmolytes, chaperones, transporters, dehydrins, and late embryogenesis abundant (LEA) proteins, and many enzymes involved in antioxidant detoxification, carbohydrate, lipid and secondary metabolite metabolism, and in biosynthesis of abscisic acid (ABA) and jasmonic acid (JA) [11, 12]. Elevated cytosolic Ca2+ is an early response to the cold stress and an important second messenger in cold stress transduction pathway, thereby, in cold acclimation development [13] (Fig. 1).

Fig. 1.
figure 1

Generic signal transduction pathway of cold stress in plants. Upon perception of cold stress by membrane receptors, a complex intracellurlar signaling cascade is activated involving secondary molecules, protein kinases, TFs etc. to develope a cold response

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Cold stress changes the membrane composition, fluidity, and protein and nucleic acids conformations and causes to accumulate small molecules such as amino acids, soluble sugars, organic acids, polyamines, and lipids [14, 15]. These molecules function as osmolytes to reduce the cellular dehydration, as compatible solutes to stabilize the membrane or as chelators to involve in detoxification [16]. Verslues et al. [17] also reported that decreased water availability, changes of accumulated molecules and cell wall properties, and induction of stress proteins are the common metabolic responses to the abiotic stresses in plants. Moreover, molecular studies have identified many TF families such as WRKY, DREB, ZF, ERF, MYB, bHLH, bZIP, etc. that extensively involving in regulation of stress-inducible gene network [18, 19].

About 30 % of Arabidopsis transcriptome was regulated by abiotic stress, in which 2409 genes have been reported to have great importance in cold, salt, and drought tolerance [20]. Over 2 % of wheat genome demonstrated more than 2-fold changes upon cold stress [21]. In chilling-stressed rice genotypes, 8484 genes have been reported to be differentially regulated [22]. In cold/drought-treated barley, 3.8 % of genes (158) were chilling specific, 2.8 % (119) were freeze-thaw specific, and 34.1 % were freeze-thaw and drought responsive [23]. In cold/salt-stressed potato, 2599 genes significantly regulated, of which 1938 were cold-induced genes [12]. Thus, identification and characterization of differentially expressed genes (DEGs) in plants constitute the basis of understanding the cold stress pathway [24]. The aim of this study was to identify the overall expression profile of genes at four time points (4, 8, 12, and 48 h) of cold-exposed (+4 °C) potato plant, with a particular emphasis on the genes related with TFs, phytohormones, lipid metabolism, signaling pathway, and photosynthesis.

Materials and Methods

The gene expression data of potato showing differential expression under different time periods of cold stress was retrieved from GEO (http://www.ncbi.nlm.nih.gov/geo/) database of NCBI. The expression data of cold stress was obtained from the series id GSE8203 by using MATLAB program. In the experiment, the TIGR10 K potato microarrays containing 15.264 complementary DNAs (cDNAs) (http://www.jcvi.org/potato/sol_ma_microarrays.shtml) were used. The data was already normalized. The boxplots displaying normalized data were provided in Supplementary Fig. 1. Researchers grown plants at 25 °C for 4–6 weeks after which cold stress was initiated by exposing the plants to 4 °C for 4, 8, 12, and 48 h. We retrieved gene expression values, which are in the form of log2, for four time points (4, 8, 12, and 4 8h). We did not include 24 h of cold treatment, since 24 h of cold treatment was performed one biological repeat. Statistically significant DEGs among sample treatments, each with two biological replicates, were identified using t test (p < 0.05) and false discovery rate of < %5. As confidence threshold for DEGs, we used a log2-fold change ≥0.6 and ≤−0.6. The ratios obtained from the microarray analysis were imported into MapMan Software [25]. Annotation and functional characterization was assigned using Stu_TIGR.m02 August07 [26]. Annotation for selected genes was confirmed by BLAST searches of the potato cDNA sequences against the TIGR potato EST database (http://www.tigr.org/) using BLAST.

Results

Identification of Chilling-Induced Genes

Log2-fold changes of ≥0.6 (up-regulated) and ≤−0.6 (down-regulated) ratios were used as confidence threshold to identify DEGs. We have identified a total of 3504 DEGs at four time points (4, 8, 12, and 48 h) (Supplementary Table S1). Of these, 1397 genes were up-regulated while 2107 were down-regulated. These genes include 419 up-regulated and 189 down-regulated genes for 4 h chilling stress, 360 up-regulated and 366 down-regulated genes for 8 h chilling stress, 356 up-regulated and 412 down-regulated genes for 12 h chilling stress, and 556 up-regulated and 1140 down-regulated genes for 48 h chilling stress. The common up- and down-regulated genes were found as 1 and 2, respectively (Supplementary Table S1) (Fig. 2).

Fig. 2
figure 2

Number of differentially expressed genes at four time points (4, 8, 12, and 48 h) of chilling-stressed potato (S. tuberosum) plant. Venn diagram a and b show the up- and down-regulated genes, respectively

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Functional Annotation of DEGs

Annotation and functional characterization of identified 3504 (1397 up; 2107 down) DEGs at four time points (4, 8, 12, and 48 h) of chilling-stressed potato plant have been done by using MapMan tool (Supplementary Table S1) (Fig. 3). MapMan allows to hierarchically organizing the list of genes into functionally similar individual BINs and subBINs to understand the biological significance of DEGs [26]. Nine hundred ninety-two up-regulated genes were annotated in 31 major BINs while 1268 down-regulated genes annotated in 32 BINs, and the remaining up- and down-regulated genes could not be assigned to any ontology.

Fig. 3
figure 3

Distribution of chilling-stressed potato up- and down-regulated genes in MapMan BINs. Blue and red chart bars show the up- and down-regulated genes, respectively. Each number at x-axis corresponds to an individual BIN code. BIN 1, photosynthesis, BIN 2 major carbohydrates, BIN 3 minor carbohydrates, BIN 4 glycolysis, BIN 5 fermentation, BIN 6 gluconeogenesis/glyoxylate cycle, BIN 7 oxidative pentose phosphate pathway, BIN 8 TCA cycle/organic acid transformations, BIN 9 mitochondrial electron transport/ATP synthesis, BIN 10 cell wall, BIN 11 lipid metabolism, BIN 12 nitrogen assimilation, BIN 13 amino acid metabolism, BIN 14 S-assimilation, BIN 15 metal handling, BIN 16 secondary metabolism, BIN 17 hormones, BIN 18 cofactor and vitamin synthesis, BIN 19 tetrapyrrole synthesis, BIN 20 stress, BIN 21 redox, BIN 22 polyamine synthesis, BIN 23 nucleotide metabolism, BIN 24 biodegradation of xenobiotics, BIN 25 C1-metabolism, BIN 26 miscellaneous enzyme families, BIN 27 RNA, BIN 28 DNA, BIN 29 protein, BIN 30 signaling, BIN 31 cell, BIN 33 development, BIN 34 transport

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Although large numbers of genes have been identified to be differentially expressed, the main focus was on the genes involved in photosynthesis (BIN 1), lipid metabolism (BIN 11), hormones (BIN 17), RNA (BIN 27), and signaling (BIN 30) pathways. In photosynthesis pathway (BIN 1), some light reaction (9 genes up; 15 genes down), Calvin cycle (5 genes up; 7 genes down), and photorespiration (1 gene up; 3 genes down) genes were differentially expressed (Fig. 4a). Lipid metabolism (BIN 11) contained fatty acid (FA) synthesis and elongation (6 genes up; 10 genes down), lipid degradation (10 genes up; 11 genes down), FA desaturation (2 genes up; 3 genes down), phospholipid synthesis (1 gene up; 3 genes down), exotics (steroids, squalene etc.) (6 genes up; 8 genes down), and lipid transfer protein (1 gene up) genes (Fig. 4b). In RNA pathway (BIN 27), transcription (4 genes up; 8 genes down), RNA binding (9 genes up; 10 genes down), processing (16 genes up; 13 genes down), and a large number of TF (75 genes up; 128 genes down) genes have been identified (Table 1 and Fig. 4c).

Fig. 4
figure 4

Differentially expressed genes (DEGs) involved in photosynthesis (a), lipid metabolism (b), RNA (c), hormone (d), and signaling (e) pathways in chilling-stressed potato. Up- and down-regulated genes indicated blue and red colors, respectively. Annotations of DEGs have been made by using MapMan

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Table 1 Expressed TF-related genes in chilling-stressed potato and their fold changes at four time points (4, 8, 12, and 48 h)
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Hormone metabolism (BIN 17) included abscisic acid (2 genes up; 5 genes down), auxin (12 genes up; 11 genes down), brassinosteroid (3 genes up; 10 genes down), cytokinin (4 genes up; 3 genes down), ethylene (5 genes up; 7 genes down), jasmonate (2 genes up; 3 genes down), gibberellin (4 genes up; 5 genes down), and salicylic acid (1 gene up) related genes (Fig. 4d). Signaling pathway (BIN 30) contained a wide range of subgroups such as receptor kinases (11 genes up; 17 genes down), MAP kinases (5 genes up; 2 genes down), calcium (12 genes up; 19 genes down), light (4 genes up; 8 genes down), G-proteins (6 genes up; 9 genes down), sugar and nutrient physiology (2 genes up; 2 genes down), phosphoinositides (1 gene up; 2 genes down), and 14-3-3 proteins (1 gene down) (Fig. 4e).

Heatmap of chilling-induced up- and down-regulated genes at four time points (4, 8, 12, and 48 h) in potato demonstrated that gene expression levels at 4, 8, and 12 h represented more similar pattern than that of 48 h. The genes have been observed to be mainly up-regulated at time points of 4, 8, and 12 h while at 48-h time point, genes inclined to down-regulate (Fig. 5 and Table 1).

Fig. 5
figure 5

Heatmap representing the expression levels of genes at four time points (4, 8, 12, and 48 h) in chilling-stressed potato. Red and green colors indicate the up- and down-regulated genes, respectively. Gene expression levels at 4, 8, and 12 h represent more similar pattern than that of 48 h

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Thus, expression levels of up- and down-regulated genes at 48-h time point were visualized by using “Metabolism overview” pathway with their putative metabolic functions (Fig. 6). At 48-h time point, plant demonstrated an inclination towards to down-regulate the genes involved in cell wall, lipid, minor CHO, light reaction, tricarboxylic acid (TCA) cycle, secondary metabolites, amino acids, and other metabolite pathways.

Fig. 6
figure 6

Metabolic overview of DEGs in 48-h chilling-stressed potato. Pathway was visualized by using “Metabolism overview” pathway of MapMan. Red and blue colors indicate the up- and down-regulated genes, respectively. For better visualization, we set the threshold 2.5 and −2.5. TCA tricarboxylic acid, OPP oxidative pentose phosphate

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Discussion

Analysis of Differentially Expressed Genes

Cold stress affects various metabolic pathways leading to the differential expression of hundreds to thousands of genes. Transcriptome analysis of three wheat varieties such as Harnesk, Paragon, and Solstice has demonstrated 1711 up-regulated and 1402 down-regulated genes upon cold stress with significant increase in expression profile [21]. In cold/drought-treated barley, 158 genes (3.8 %) were identified to be chilling specific [23]. In cold-stressed Populus simonii, 5267 genes were up-regulated while 6359 genes were down-regulated [27]. In flowering plant Anthurium andraeanum, a total of 4363 genes were reported to be significantly expressed under cold stress [28]. In tea plant, 1168 genes were up-regulated and 602 genes were down-regulated under cold treatment [29]. In this study, we have identified a total of 3504 DEGs at four time points (4, 8, 12, and 48 h) of chilling-stressed potato plant. Of these genes, 1397 were up-regulated and 2107 were down-regulated. In addition, one gene (clone name: STMGH01) was commonly up-regulated and two genes (clone names: STMGV67 and STMJF91) were commonly down-regulated for four time points. However, they are not argued in this study due to either lack of annotation or weakly annotated data. In early exposure to chilling stress, potato gives a response by mainly up-regulating its genes, and then, it demonstrates an inclination towards to the down-regulation of its genome. Overall, it may be reported that short-term chilling stress (4, 8 and 12 h) could lead the potato to up-regulate its genes related with increased synthesis of a number of cold-induced molecules or/and a shock state-related increased gene activities. However, in long-term cold stress (48 h), it lowers its metabolic activities by down-regulating its genes, highly probably to avoid further damage.

Chilling Stress-Induced Transcriptional Regulation

It is well-known that specific sets of genes, collectively known as cold-regulated (COR) genes, are differentially expressed upon cold stress. Furthermore, expression of COR genes are modulated by a number of transcription factors (TFs) [3, 14]. In this study, 75 up-regulated genes encoding TFs have been identified in chilling-stressed potato plant (Table 1). Although we have identified 75 up-regulated TF genes from 37 different families/groups, we mainly focused on the four most abundantly expressed TF families such as basic helix-loop-helix (bHLH), WRKY, CCAAT-binding (HAP3), and bZIP transcription factor families. The bHLH transcription factor family plays very diverse roles in modulation of various physiological and stress-adaptive networks in plants. Two grapevine basic bHLH transcription factors, VabHLH1 and VvbHLH1, positively modulated the CBF (C-repeat binding factor) pathway, which has an important role in cold acclimation and contributed the cold stress tolerance in Arabidopsis [30]. In a different study, six grapevine bHLH genes, including VlCEbHLH1-6 demonstrated the differential expression pattern as response to various cold temperatures [31]. The up-regulation of PtrbHLH gene enhanced the cold stress tolerance in tobacco and lemon plants while down-regulation caused to the elevated cold sensitivity in trifoliate orange [32]. The overexpression of apple MdCIbHLH1 gene conferred the cold tolerance in transgenic apple and Arabidopsis through MdCBF2 and AtCBF3 promoter, respectively. Additionally, ectopic expression of MdCIbHLH1 in transgenic tobacco enhanced the chilling tolerance [33]. Low temperatures were reported to up-regulate the bHLH148 and bHLH1 genes in rice [34, 35]. A transcription factor of ICE2 in bHLH family enhanced the tolerance to deep freezing cold in transgenic Arabidopsis [36]. Banana bHLH1/2/4 promoters were induced by cold stress and methyl jasmonate in tobacco BY2 protoplasts [37]. Furthermore, cold-related expressions of bHLH genes have been also reported in various other species. In the view of above-mentioned studies, bHLH TFs seem to play important functions in modulation of cold acclimation process in plants. In this study, we have identified the eight members of bHLH TFs with clone names such as STMHJ91, STMEK79, STMDC31, STMDE79, STMDV67, STMER91, STMHZ85, and STMCU25 at different expression folds in different time points of chilling-induced potato (Table 1). The clone STMHJ91, which showed the expression fold with 3.76, 0.81, and 2.50 at 4-, 8-, and 48-h time points, respectively, was similar to Arabidopsis AT1G69010 (AtbHLH102) gene, whose product was reported to show DNA-binding TF and protein dimerization activities in various plant tissues. The clone STMEK79 demonstrated an expression fold with 1.65 (4 h) and 1.61 (48 h) and showed similarity to Arabidopsis AT2G27230 (AtbHLH156) gene with DNA-binding TF and protein dimerization activities in guard and plant sperm cells and in primary root tips. The clone STMDC31 and STMDE79 expressed at 8-h time period with 1.70- and 1.82-folds, respectively, and showed similarity to Arabidopsis AT5G64340 (AtbHLH142) gene. The clone STMDV67 expressed at 8-h period with 0.80-folds and showed similarity to Arabidopsis AT1G72210 (AtbHLH96) gene with DNA TF and amino acid binding and protein dimerization activities in guard cells. The clone STMER91 expressed at 8- and 48-h time periods with 2.13- and 1.84-folds, respectively, and showed similarity to Arabidopsis AT5G46690 (AtbHLH71) gene. The clone STMHZ85 expressed at 12-h time period with 1.79-fold (enrichment not available). The clone STMCU25 expressed at 48-h time period with 1.69-fold and identical to Arabidopsis AT1G32640 (AtbHLH6) gene. Therefore, 8- and 48-h time periods were the points in which most of bHLH genes have been up-regulated. This may indicate that 8- and 48-h points could be critical times in induction and development of cold stress mechanisms.

In addition to bHLH family, WRKY, CCAAT-binding (HAP3) and bZIP transcription factor families have been most abundantly expressed. WRKY TF genes have been demonstrated to involve in regulation of plant growth and development and in stress responses [38]. In Arabidopsis genome, four WRKY family cDNAs have been identified as drought, cold, or high-salinity inducible genes [39]. Soybean GmWRKY21 gene conferred the cold tolerance in transgenic Arabidopsis plants [40]. Barley HvWRKY38 gene has been demonstrated to be expressed as response to low non-freezing temperature [41]. Thirteen WRKY genes were reported to be regulated only by cold, drought, and salinity in rice [42]. The hot pepper CaWRKY1 gene was found among the up-regulated cold stress genes [43]. In Populus transcriptome, 61 PtrWRKY genes were induced as response to various biotic/abiotic stresses, including cold [44]. Fifteen grapevine VvWRKY genes have been reported to show the cold stress-induced expression pattern [45]. Previous studies showed that WRKY genes play an important role in cold response pathway. We have identified four WRKY genes with clone names such as STMEG07, STMGO55, STMCN79, and STMIY73 at 4-, 8-, and 48-h time periods of chilling-stressed potato. The clone STMEG07 expressed at 4-h time period with 2.69-fold and showed similarity to tobacco WRKY4 gene with DNA-binding TF activity. The clone STMGO55 expressed at 4- and 8-h time periods with 1.58- and 1.27-folds, respectively, and demonstrated similarity to Arabidopsis WRKY12 gene. The clone STMCN79 expressed at 8-h time period with 0.91-fold and showed similarity to Arabidopsis WRKY33 gene with protein binding and DNA-binding TF activities in various tissues. The clone STMIY73 also expressed at 48-h time period with 1.18-fold and showed similarity to tobacco WRKY gene.

Another TF family identified in this study is the CCAAT-box binding (HAP3). In Arabidopsis, HAP complex consists of three different subunits, namely as HAP2, HAP3, and HAP5. Each individual HAP subunit has been shown to be involved in the regulation of many physiological processes and stress tolerance [46]. HAP2 and HAP3 subunits have been mainly reported to involve in drought or osmotic stress tolerance [47, 48]. The overexpression of HAP5A was demonstrated to confer the cold tolerance in Arabidopsis plants [49]. We have identified four CCAAT-box binding (HAP3) TF clones such as STMHL13, STMIN14, STMGF80, and STMII37 at four time points of chilling-stressed potato. The clone STMHL13 expressed at 8-h time period with 2.43-fold and showed similarity to Arabidopsis NFYB8 gene with DNA-binding TF and protein heterodimerization activities in various cell types. The clone STMIN14 expressed at 12-h time period with 2.86-fold and demonstrated a similarity to Arabidopsis AT3G58610 gene with coenzyme binding and ketol-acid reductoisomerase activities. The clone STMGF80 expressed at 48-h time point with 2.17-fold and showed similarity to Arabidopsis NFYB3 (HAP3C) gene. The clone STMII37 expressed at 48-h time point with 1.92-fold and demonstrated similarity to rice HAP3A gene.

bZIP is another TF family identified in this study with its four members. This family has been shown to regulate many physiological processes such as seed maturation, floral development, photo-morphogenesis, and stress and hormone signaling [50]. The overexpression of OsbZIP52 gene was reported to significantly increase the cold and drought sensitivity in rice [51]. Grapevine TF VvbZIP23 was demonstrated to be induced by a wide spectrum of abiotic stresses, including cold [52]. The overexpressed wheat TabZIP60 significantly enhanced the drought, salt, and cold tolerance in Arabidopsis [53]. Soybean GmbZIP44, 62, and 78 genes conferred the salt and cold tolerance in transgenic Arabidopsis [54]. We have identified four bZIP TF genes with clone names such as STMEO01, STMGP13, STMES62, and STMJO92 at three time points of chilling-stressed potato. The clone STMEO01 and STMES62 expressed at 4- and 48-h time points with 2.33- and 1.11-folds, respectively, and showed similarity to tobacco BZI-1 gene with DNA-binding TF activity. The clone STMGP13 expressed at 12-h time point with 1.20-fold and demonstrated similarity to Arabidopsis ATBZIP60 gene. The clone STMJO92 expressed at 48-h time points with 0.75-fold and showed similarity to soybean BZIP TF ATB2 gene with DNA-binding TF activity. Overall, it seems that a large number of TF genes in potato are induced as response to cold stress to either develop a cold acclimation or regulate other cold-related physiological processes.

Chilling Stress-Induced Signaling

Plants develop cold response/acclimation as a result of highly complex signaling network, but it necessitates the involvement of a number of intra- and intercellular molecules [55]. In this study, we have identified the receptor kinases, MAP kinases, calcium, light, G-protein, some sugar, and nutrient physiology related molecules, phosphoinositides, and 14-3-3 proteins in signaling pathway (BIN 30) (Fig. 4e). However, main focus will be on the protein kinases and calcium because of their primary functions in cold stress pathway. We identified two families of kinases, including receptor kinases and MAP kinases (Fig. 4e). Receptor-like kinases (RLKs) have important functions in perception and transduction of extracellular stimuli [55]. Moreover, RLKs are key regulators in plant growth and development and in stress responses [56, 57]. In Arabidopsis, more than 600 RLK-encoding genes and in rice, over 1000 RLK-encoding genes have been reported [58, 59]. In the present study, we have identified 11 up-regulated receptor kinase genes with clone names such as STMDQ19 (4-h point/3.84-fold; 48-h point/1.56-fold), STMET61 (4-h point/3.45-fold; 8-h point/0.60-fold), STMJE19 (4-h point/1.98-fold; 48-h point/1.49-fold), STMIZ38 (4-h point/1.20-fold; 8-h point/2.04-fold), STMDP55 (4-h point/0.73-fold; 48-h point/1.11-fold), STMIF31 (4-h point/0.65-fold; 48-h point/0.97-fold), STMIB37 (4-h point/3.31-fold), STMJG85 (4-h point/0.84-fold), STMJH20 (12-h point/2.34-fold), STMGC91 (48-h point/0.75-fold), STMJN32 (48-h point/0.79-fold) in chilling-induced potato. The presence of wide spectrum of chilling stress-responsive receptor kinases in potato could indicate their important functions in cold or cold-related pathways. However, three clones such as STMDQ19 (4-h point/3.84-fold), STMET61 (4-h point/3.45-fold), and STMIB37 (4-h point/3.31-fold) have demonstrated highest expression fold over 3 at 4-h time period. The clone STMDQ19 showed similarity to tomato LhirPtoB gene with ATP binding and protein serine/threonine kinase activities. The clone STMET61 was similar to Arabidopsis CLV1 gene with ATP binding, protein kinase, and receptor serine/threonine kinase binding activities. The clone STMIB37 was similar to Arabidopsis AT1G67720 gene with ATP binding and protein serine/threonine kinase activities.

In addition, mitogen-activated protein kinases (MAPKs) play important roles in downstream signaling by mediating between receptors and other intracellular signaling networks in plants [60]. Moreover, they could be also activated by various stresses simultaneously, thereby; they function as convergent points in stress transduction pathway [61, 62]. Arabidopsis genome was reported to encode nearly 60 MAPKKKs, 10 MAPKKs, and 20 MAPKs [60]. Thus, expressed MAP kinase genes in chilling-stressed potato could possibly modulate the cold stress-related transduction cascade to converge the signaling in order for developing a cold response/acclimation. We have identified five up-regulated MAP kinase genes with clone names such as STMIP25 (4-h point/0.93-fold), STMET25 (8-h point/1.93-fold), STMHH86 (12-h point/1.07-fold), STMEA92 (48-h point/0.63 point), and STMEF20 (48-h point/1.80-fold) in chilling-induced potato.

Moreover, Ca2+ functions as a second messenger in regulation of cold stress signaling pathway [63]. Upon cold stress, cytosolic Ca2+ concentration immediately rises up to a level of designated Ca2+ signatures for cold. This designated cytoplasmic Ca2+ signature is decoded by Ca2+ sensors like Calmodulins (CaM), Calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs), Calcineurin B-like proteins (CBLs), and their interacting kinases (CIPKs) to transduce the signal intracellularly [64, 65]. Therefore, differentially expressed Ca-related genes in chilling-stressed potato could have major functions in intracellular signal transduction, thereby, in development of cold acclimation. We have identified 12 calcium-related genes with clone names such as STMDV44 (4-h point/3.42-fold), STMDG68 (4-h point/2.96-fold), STMHY91 (4-h point/1.81-fold), STMCH25 (4-h point/0.97-fold), STMJH13 (4-h point/0.69-fold), STMEN67 (8-h point/2.09-fold), STMEP25 (8-h point/0.85-fold), STMID19 (8-h point/1.82-fold; 48-h point/1.02-fold), STMJA91 (12-h point/2.17-fold; 48-h point/1.12-fold), STMDM25 (48-h point/2.44-fold), STMGB13 (48-h point/0.81-fold), and STMIT08 (48-h point/0.80-fold). The clone STMDV44 showed the highest expression fold with 3.42 at 4-h time period and was similar to tomato CLB1 and Arabidopsis AT3G60950 gene with endonuclease and exonuclease activities.

Chilling Stress-Induced Hormone Regulation

An accumulating evidence shows that synergistic or antagonistic hormone cross-talks play important roles in adaptation/response to abiotic stresses [66]. Plant hormones often rapidly change the gene expression of target genes by modulating the transcriptional factors [67]. In this study, we have identified the differentially expressed phytohormone genes of abscisic acid, auxin, brassinosteroid, cytokinin, ethylene, jasmonate, gibberellin, and salicylic acid in chilling-stressed potato plant.

Endogenous abscisic acid (ABA) levels mainly increases as response to osmotic (drought, salinity) stress but some extent to cold. The exogenous ABA applications were also reported to induce a number of cold-responsive genes, but ABA role in regulation of cold-responsive genes is not clear yet [60]. However, many cold- and drought-inducible genes share common motifs such as DRE/CRT and ABRE in their promoters [68], suggesting the existence of a cross-talk between ABA-independent and ABA-dependent transduction cascades [18]. Therefore, cold stress could either directly induce ABA genes or decreased water availability resulting from cold stress indirectly induces the ABA genes. We have identified two ABA up-regulated genes with clone names such as STMEV91 (4-h point/3.97-fold) and STMHK55 (48-h point/1.72-fold). The clone STMEV91 showed 3.97 highest expression fold at 4-h time period and was similar to Arabidopsis FIP1 gene with putative vesicle transport activity. Auxin regulates many physiological processes including plant growth and development and abiotic stress response [69]. Shibasaki et al. [70] reported that cold stress affects the auxin transport pathway in Arabidopsis by inhibiting the various intracellular proteins including auxin efflux carriers. In a different study of Arabidopsis, the expression profile of ARF and Aux/IAA gene family members were demonstrated to be changed during cold acclimation [71]. Transcriptome analysis of rice revealed that at least 154 genes were auxin-induced and 50 auxin-repressed under one or more stress conditions including desiccation, salt, and cold [69]. We have identified 12 auxin-induced genes with clone names such as STMCM67 (4-h point/3.60-fold), STMCB07 (4-h point/2.02-fold; 8-h point/1.83-fold), STMEY49 (4-h point/0.85-fold; 8-h point/1.05-fold), STMJB61 (8-h point/0.99-fold), STMCF91 (8-h point/1.53-fold), STMCI37 (8-h point/0.90-fold), STMHY44 (12-h point/1.79-fold), STMEN19 (12-h point/1.15-fold), STMGE49 (12-h point/1.64-fold), STMHG73 (12-h point/0.97-fold), STMGT44 (48-h point/0.79-fold), and STMJB61 (48-h point/1.12-fold). The clone STMCM67 expressed with 3.60 highest fold at 4-h point and showed similarity to Arabidopsis ILL4 gene with IAA-Ala conjugate hydrolase and metallopeptidase activities in root, stem, and flower. Induction of a number of auxin-related genes in potato could indicate the modulation of auxin transport by cold stress. Ethylene (ET) modulates various physiological processes including germination, fruit ripening, organ abscission, pathogen, senescence, and biotic/abiotic stresses [72]. Ethylene signaling was reported to negatively regulate the freezing tolerance; increased ethylene level resulted in decreased freezing tolerance while inhibiting ethylene biosynthesis/signaling conferred the enhanced freezing tolerance in Arabidopsis [73]. A similar negative correlation was also demonstrated between ET level and freezing tolerance in Medicago truncatula [74]. However, an ET biosynthesis inhibitor 1-methylcyclopropene (1-MCP) decreased the cold tolerance in tomato, showing a positive effect on cold tolerance [75]. This indicates that ET’s role on cold tolerance could be species-dependent. However, further studies are needed to confirm whether positive or negative regulatory role of ET on cold tolerance in potato plant. We have identified five ethylene up-regulated genes with clone names such as STMHA07 (4-h point/3.17-fold), STMHQ43 (4-h point/0.92-fold), STMEA01 (8-h point/1.12-fold), STMDZ32 (48-h point/0.70-fold), and STMJH61 (48-h point/1.84-fold). The clone STMHA07 showed 3.17 highest fold at 4-h time point and was similar to tomato ACS5 gene with 1-aminocyclopropane-1-carboxylate synthase and pyridoxal phosphate binding activities. In addition, we have also identified some other phytohormone-related genes of cytokinin, brassinosteroid, jasmonate, gibberellin, and salicylic acid (Fig. 4d). In the view of above-mentioned findings, phytohormones seem to collaboratively coordinate with each other to improve the plant adaptability to cold stress.

Chilling Stress-Induced Lipid Metabolism Regulation

Plant cold acclimation involves a wide spectrum of transcriptional regulators that modulate cold-regulated (COR) genes, some of which encode lipid metabolism-related proteins and enzymes to protect the plant cell from freezing injury [76]. The primary site of freezing damage is the cellular membranes. Cold stress-related changes in membrane composition and fluidity, thereby in lipid metabolism have crucial importance in cold acclimation process [14, 15]. The content of di-unsaturated fatty acids (FAs) and phospholipids in plasma membrane increases to reduce the cold-induced membrane damage, and this leads to membrane rigidification [77]. Membrane rigidification acts as a physical signal to induce plasma membrane proteins, which then activate other downstream signaling networks to maintain membrane stability and integrity [78]. In addition, biosynthesis of desaturases was reported to be up-regulated to enhance the cold tolerance [79]. By contrast, some lipid metabolism-related molecules involved in FA synthesis and elongation, phospholipid and steroids/squalene synthesis, and lipase and lysophospholipase degradation were also reported to be down-regulated either as response to cold-related reduced growth or to preserve the cold-induced altered lipid composition [71, 77]. In this study, we have identified six up-regulated FA synthesis and elongation genes, 10 up-regulated lipid degradation genes, two up-regulated FA desaturation genes, one up-regulated phospholipid synthesis gene, six up-regulated exotics (steroids, squalene, etc.) genes, and one up-regulated lipid transfer protein gene (Fig. 4b). In lipid metabolism, FA synthesis and elongation, lipid degradation, and exotics (steroids, squalene, etc.) genes were mainly expressed. The up-regulated FA synthesis and elongation genes included with clone names such as STMGC43 (4-h point/3.25-fold), STMDB67 (4-h point/1.96-fold; 8-h point/0.70-fold), STMDQ79 (12-h point/0.77-fold), STMFA38 (48-h point/0.70-fold), STMHN19 (48-h point/0.97-fold), and STMJJ74 (48-h point/0.67-fold). The clone STMGC43 demonstrated 3.25 highest fold at 4-h time point and showed similarity to Elaeis oleifera palmitoyl-acyl carrier protein thioesterase gene with thiolester hydrolase activity. The up-regulated lipid degradation genes included the clones such as STMIU19 (4-h point/2.84-fold; 12-h point/2.01-fold), STMJP61 (4-h point/1.94-fold), STMDV25 (4-h point/0.89-fold), STMJN86 (8-h point/1.15-fold), STMHR01 (12-h point/1.59-fold), STMJG61 (12-h point/1.40-fold), STMIG79 (12-h point/1.81-fold), STMIT79 (12-h point/1.90-fold), STMGW07 (48-h point/2.22-fold), and STMIH07 (48-h point/3.09-fold). The clone STMIH07 demonstrated the highest expression fold with 3.09 at 48-h time period and showed similarity to Arabidopsis PLA2-ALPHA gene with calcium ion binding and phospholipase A2 activities. The up-regulated exotics (steroids, squalene, etc.) genes included the clones such as STMJA61 (8-h point/1.17-fold), STMHL68 (8-h point/2.51-fold), STMDB31 (12-h point/0.89-fold), STMDH37 (12-h point/2.36-fold), STMGX37 (48-h point/1.75-fold), and STMIF80 (48-h point/1.03-fold). Therefore, it seems that genes involved in chilling-induced lipid metabolism and their expression patterns could lead the potato to alter its membrane composition, thereby, to transduce the downstream signaling network to develop a cold response/acclimation.

Chilling Stress-Induced Photosynthetic Regulation

Photosynthesis has crucial importance as being a universal stress sensor in green plants. Photosystem I (PS I) and II (PS II) and Rubisco are thought to be primary stress sensors in the chloroplast. The stress-related changes in these sensors cause to generate stress signals like production of reactive oxygen species (ROSs), change of sugar levels, change of redox reactions from photosynthetic electron transport system, and energy imbalance, which accordingly lead to the metabolic/molecular reprograming of stress adaptation [80, 81]. The photosynthetic genes were previously reported to be down-regulated as response to cold stress [82]. A number of genes in light signaling have been also demonstrated to be down-regulated in medium to long-term cold exposure; however, short-term exposure mainly up-regulated the genes, probably resulting from a temporary “shock state” before cold adaptation [11]. We have identified nine up-regulated and 15 down-regulated light reaction genes in chilling-stressed potato (Fig. 4a). The up-regulated light reaction genes included the clones such as STMEA37 (4-h point/3.27-fold), STMEY61 (8-h point/1.19-fold), STMJB01 (8-h point/1.48-fold), STMCH07 (48-h point/2.69-fold), STMCO92 (48-h point/0.62-fold), STMCS92 (48-h point/0.74-fold), STMCX85 (48-h point/1.12-fold), STMEU31 (48-h point/1.96-fold), and STMGQ55 (48-h point/0.85-fold). It seems that as low temperatures caused to oxidative damage, plants may have minimized this damage by down-regulating the light reactions genes, which otherwise could increase the oxidative damage. Abiotic stress factors can also change the rates of primary photochemical reactions and reduce the enzyme activities of Calvin cycle [80]. Low temperatures were also reported to inhibit electron transport by altering thylakoid lipids and to significantly decrease the enzymatic reaction rates involved in C, N, and S reduction [83]. We identified that five Calvin cycle genes were up-regulated and seven down-regulated, while one photorespiration gene were up-regulated and three down-regulated in chilling-stressed potato (Fig. 4a). This implicates that cold stress may have reductive effects on enzyme activities in Calvin cycle and photorespiration, resulting in down-regulated gene activities. The up-regulated Calvin cycle genes included clones such as STMES37 (4-h point/2.36-fold; 12-h point/3.21-fold), STMCP14 (4-h point/1.14-fold), STMCB56 (8-h point/0.67-fold), STMCP20 (8-h point/1.91-fold), and STMIY49 (12-h point/1.00). The clone STMES37 showed 3.21 highest expression fold at 12-h time point and was similar to tomato ribulose bisphosphate carboxylase/oxygenase activase gene with ATP binding activity. In addition, one photorespiration gene with clone name STMCE67 was up-regulated at 4-h time point with 1.19-fold.

In conclusion, we have identified the overall expression profile of genes at four time points (4, 8, 12, and 48 h) of chilling-exposed (+4 °C) potato plant, with a particular emphasis on DEGs. A number of genes with diverse biological functions have been found to be differentially expressed in chilling-stressed potato; however, main focus was on the genes related with transcription factors, phytohormones, lipid metabolism, signaling, and photosynthesis. We believe that findings of this study will be a valuable theoretical knowledge in terms of understanding the chilling-induced tolerance mechanisms in cultivated potato species as well as expanding their cultivation to different climatologic areas. In addition, identified genes could be used as potential candidate genes in engineering and for developing the cold stress-resistant food crops.