Abstract

   Potato (Solanum tuberosum L.) is an important food crop, but low
   temperature affects the potato growth and yield. In this study, the
   expression level of StBBX14 was significantly increased over 1 h and
   then gradually decreased under cold stress. The subcellular
   localization of the StBBX14 protein took place in the nucleus. The
   OE-StBBX14 transgenic lines showed less leaf damage and significantly
   lower electrolyte leakage compared with the WT under cold stress,
   indicating that the overexpression of StBBX14 in the potato enhanced
   the cold resistance. A transcriptome analysis showed that a total of
   2449 and 6274 differentially expressed genes were identified in WT-1 h
   and WT-12 h, respectively, when compared with WT-0h. A Gene Ontology
   enrichment analysis revealed that photosynthesis, cell wall, thylakoid,
   transcription regulator activity, oxidoreductase activity and
   glucosyltransferase activity were significantly enriched in OE-StBBX14
   and WT. A total of 14 distinct modules were generated by a WGCNA
   analysis based on all differentially expressed genes (DEGs). Four major
   modules with cold-related genes were isolated. RT-qPCR analysis showed
   that the expression patterns of eight DEGs were consistent between the
   qPCR and RNA-seq. These findings illustrate that the StBBX14 played an
   important role in cold stress in potato and provided a data basis for
   the genetic improvement of cold resistance traits of potato.

   Keywords: potato, cold resistance, BBX gene, overexpression,
   transcriptome, WGCNA

1. Introduction

   Cold stress is one kind of abiotic stress, which can severely impair
   growth, development, and yield, while also imposing substantial
   environmental burdens [[32]1]. To withstand these challenges, plants
   have evolved intricate response mechanisms. Transcription factors (TFs)
   play a crucial role in regulating the stress response and present
   promising targets for enhancing crop performance [[33]2]. BBX (B-Box)
   proteins are a group of zinc-finger transcription factors or regulators
   with B-box domains or CCT domains [[34]3]. The B-box structural domain
   in the proteins encoded by the BBX gene family is highly conserved.
   Although the B-box domains exhibit similar structural and sequence
   features across different BBX proteins, their functions can vary
   [[35]4]. The BBX protein family plays a pivotal role in light
   regulation, encompassing photomorphogenesis, flowering processes, and
   pigment accumulation [[36]5]. The overexpression of CmBBX8 promotes
   flowering under both long- and short-day conditions. UV-B-induced
   anthocyanin biosynthesis is regulated by MdBBX22, which directly
   interacts with MdHY5 (long hypocotyl 5) and promotes anthocyanin
   biosynthesis in apple [[37]6]. VvBBX44 represses the expressions of
   VvHY5 and VvUFGT (UDP glucose flavonoid 3-O-glucosyltransferase) and
   impedes anthocyanin biosynthesis in grape [[38]3]. MdBBX37 inhibits
   anthocyanin biosynthesis in light signaling, which interacts with
   MdMYB1 and MdMYB9 [[39]7]. PpBBX16 serves as a positive regulator for
   light-induced anthocyanin accumulation that activates anthocyanin
   biosynthesis-related genes in pears via PpHY5 activation [[40]8]. It
   was demonstrated that BBX11 loss of function leads to the significant
   elongation of hypocotyls in mutant seedlings under red-light and
   long-daylight conditions [[41]9]. Furthermore, BBX24 functions as a
   negative regulator by controlling the post-transcriptional activity of
   HY5 to regulate photomorphogenesis [[42]10].

   Many studies indicate that the BBX gene family is also involved in
   response to cold stress. The BBX transcription factor VvZFPL in grape
   introduced into Arabidopsis thaliana increases the plant’s resistance
   to low temperatures, high salinity, and drought [[43]11]. Similarly,
   the expression of CmBBX24 in Chrysanthemum is modulated by GA4/7, and
   suppressing this gene reduces the plant’s tolerance to cold and drought
   [[44]12]. Among various cold-signaling pathways, the C-receptor binding
   factor (CBF) pathway stands out as the most extensively studied and
   crucial regulatory mechanism in plants [[45]13]. In apples, MdBBX37
   binds to the promoters of MdCBF1 and MdCBF4, activating their
   transcription, which also interacts with MdICE1, boosting its ability
   to enhance the transcription of MdCBF1 and, in turn, improves the cold
   tolerance [[46]7]. In tomato, overexpressing SlBBX17 enhances the cold
   tolerance mediated by the CBF pathway [[47]14]. Additionally, BBX7 and
   BBX8, which act downstream of HY5, are known to positively influence
   the freezing tolerance by regulating the expression of COR genes
   [[48]15]. It was shown that the loss of SlBBX31 function correlates
   with diminished cold tolerance in tomatoes and SlBBX31 modulates the
   expression of several ERF transcription factors in response to cold,
   including CBF2 and DREBs [[49]16].

   The potato (Solanum tuberosum L.) is one of the four most important
   food crops in the world. However, existing cultivars of potato prefer
   cold climates but lack frost tolerance, exhibit sensitivity to low
   temperatures, and have an inability to acclimate to cold conditions,
   where even brief exposure to low temperatures can significantly reduce
   potato yields [[50]17]. Several cold-responsive genes, including SAD
   [[51]18] and ADC1 [[52]19] were identified and characterized in potato.
   The overexpression of the S. commersonii SAD gene significantly
   enhanced freeze tolerance in Zhongshu 8 [[53]20]. A previous study
   demonstrated that the upregulation of ADC1 expression elevated the
   putrescine content and conferred improved freezing tolerance in potato
   [[54]19]. However, the molecular mechanism of cold resistance in potato
   remains unclear.

   In this study, the expression and function of the StBBX14 gene were
   analyzed. To further explore the molecular mechanism underlying the
   roles of StBBX14 in regulating potato freezing tolerance, wild-type and
   OE-StBBX14 seedlings were treated with 2 °C for 0 h, 1 h, and 12 h to
   identify StBBX14-regulated COR genes by RNA-sequencing (RNA-seq). The
   different expression genes in OE-StBBX14 plants were validated by
   reverse-transcription quantitative real-time PCR (RT-qPCR) analyses.
   The findings provide new insights into the molecular mechanisms
   underlying plant cold tolerance and pave the way for molecular breeding
   strategies aimed at developing more resilient potato varieties.

2. Results

2.1. Characterization of BBX14 in Potato Plants

   BBXs play important roles in the plant response to cold stress
   [[55]14,[56]15]. Transcriptome analysis found that StBBX14 was
   differently expressed under cold stress in S. commersonii (cold
   resistance) and S. cardiophyllum (cold sensitive). To investigate the
   StBBX14 gene response to cold stress in potato, the gene expression
   level of StBBX14 was examined by qRT-PCR. The results show that the
   expression level of StBBX14 was significantly increased in 1 h and then
   gradually decreased under cold stress ([57]Figure 1A). The coding
   sequence of StBBX14 contains 1209 bp and encodes a protein of 402 amino
   acids. Phylogenetic analysis found that the StBBX14 had the highest
   homology to the SlBBX14 from Solanum lycopersicum L. ([58]Figure 1B).
   To investigate the subcellular location of the StBBX14 protein, the CDS
   of StBBX14 was cloned into the pAN580 vector under the CaMV35S promoter
   to construct an in-frame fusion protein plasmid 35S::pAN580-BBX14-GFP.
   This result suggests that the StBBX14 protein is mainly localized in
   the nucleus of tobacco ([59]Figure 1C).

Figure 1.

   [60]Figure 1
   [61]Open in a new tab

   Molecular characterization of StBBX14. (A) The gene expression of
   StBBX14 was identified after the 2 °C cold treatment for different
   times; Student’s t-test with * represented p < 0.05 and *** represented
   p < 0.001 were used to indicate significant differences. The
   phylogenetic analysis of the StBBX14 protein. (B) The phylogenetic tree
   was calculated using the maximum-likelihood method by MEGA 7.0
   software. Bootstrap values of 1,000 replicates for each branch are
   shown. The protein sequences from tomato (Sl, Solanum lycopersicum L.),
   Arabidopsis (At, Arabidopsis thaliana), apple (Md, Malus domestica),
   rice (Os, Oryza sativa L.), and pear (Pb, Pyrus bretschneideri). (C)
   Subcellular localization of StBBX14 in tobacco protoplasts.

2.2. Overexpression of StBBX14 in Potato Enhanced Cold Resistance

   To investigate whether StBBX14 had a role in the regulation of cold
   stress, the CDS of StBBX14 was placed under the control of the
   cauliflower mosaic virus CaMV 35S promoter, and the construct was
   introduced into potato for the generation of stable StBBX14-OE potato
   lines. The transcript levels of StBBX14 OE-3, OE-7, and OE-17 plants
   were over 30 times higher when compared with the wild type (WT)
   ([62]Figure 2A). The cold resistance of 30-day-old transgenic lines and
   WT grown in soil pots was used to assess StBBX14’s role in potato cold
   tolerance. The electrolyte leakage experiment results displayed that
   the three transgenic lines showed significantly lower electrolyte
   leakages at −2 °C, −4 °C, and −6 °C compared with the WT ([63]Figure
   2B). The transgenic lines and the WT displayed no obvious morphological
   differences under optimal growth conditions. Three overexpression
   lines, along with WT plants, were subjected to −2 °C for 4 h and
   recovery for 3 days without cold acclimation. The wild type (WT)
   suffered from more severe plant damage than the three transgenic lines.
   The three transgenic plants had some wilted leaves ([64]Figure 2C,D).
   The electrolyte leakage results were in agreement with the observed
   phenotype. These results suggest that StBBX14 positively regulates the
   cold resistance in potato.

Figure 2.

   [65]Figure 2
   [66]Open in a new tab

   Overexpression of StBBX14 increased the cold resistance. (A) The gene
   expressions of StBBX14 in CK, OE-StBBX14-3, OE-StBBX14-7, and
   OE-StBBX14-13 plants. (B) The electrolyte leakages of genotypes at the
   indicated temperatures. (C) Representative plants of the CK and StBBX14
   overexpression transgenic lines were photographed before exposure to
   the freezing treatment and after being subjected to −2 °C for 4 h (D),
   followed by 3 days of recovery under normal conditions. Student’s
   t-test was performed (* represented p < 0.05, ** represented p < 0.01
   and *** represented p < 0.001). Each error bar represents the standard
   error (SE) calculated from three replicates. Means denoted by the same
   letter were not significantly different at p < 0.05 based on Student’s
   test.

2.3. StBBX14 Activated the Expression of Multiple Cold-Responsive Gene

   To investigate the role of StBBX14 in regulating freezing tolerance,
   RNA-seq was conducted on OE-StBBX14 and WT under normal and
   low-temperature conditions (2 °C for 0, 1, and 12 h). A total of 798
   million raw reads were generated in eighteen libraries. After filtering
   out the low-quality reads and adapter sequences, 756 million clean
   reads were obtained ([67]Table S1). The clean reads were mapped to the
   potato reference genome and the mapping rate ranged from 79.1–82.4%.
   Compared with WT-0h, totals of 2449 ([68]Table S2) and 6274 ([69]Table
   S3) differentially expressed genes (DEGs) were identified in WT-1h and
   WT-12h, respectively. Totals of 1432 ([70]Table S4) and 7545 ([71]Table
   S5) DEGs were detected in OEBBX14-1h and OEBBX14-12h when compared with
   OEBBX14-0h, respectively ([72]Figure 3C,D). It was showed that more
   specific DEGs were detected after 12 h for either the WT or the
   OEStBBX14. In all, 945 and 3526 overlapping DEGs were identified in
   WT-1h versus vs. WT-12h and WT-12h vs. OEBBX14-12h ([73]Figure 3A,B).
   Totals of 448 and 735 overlapping DEGs were obtained in WT-1h vs.
   OEBBX14-1h and OEBBX14-1h vs. OEBBX14-12h ([74]Figure 3C,D).

Figure 3.

   [75]Figure 3
   [76]Open in a new tab

   The numbers of DEGs in WT-1h vs. WT-12h (A), WT-12h vs. OEBBX14-12h
   (B), WT-1h vs. OEBBX14-1h (C), and OEBBX14-1h vs. OEBBX14-12h (D).

   The Gene Ontology (GO) enrichment analysis revealed that
   photosynthesis, cell wall, thylakoid, transcription regulator activity,
   oxidoreductase activity, and glucosyltransferase activity were
   significantly enriched in the DEGs of both OEBBX14 and the WT. The GO
   terms related to the peptide metabolic process and calmodulin binding
   were significantly enriched in OEBBX14. The cellular carbohydrate
   metabolic process and FAD binding were significantly enriched in WT.
   The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
   analysis revealed that the KEGG pathways and photosynthesis and plant
   hormone signal transduction were significantly enriched in the DEGs of
   both OEBBX14 and the WT. The MAPK signaling pathway was significantly
   enriched in OEBBX14.

2.4. WGCNA Analysis

   To identify the key genes associated with cold stress in potato, a
   WGCNA analysis was performed to construct the co-expression networks
   based for all the DEGs in OEBBX14 and the WT. A total of 14 distinct
   modules were generated ([77]Figure 4). In the WT, the red module (r =
   0.98, p = 5 × 10^−4) was significantly correlated with WT-0h. In the
   OEBBX14, the green (r = 0.91, p = 1 × 10^−2), magenta (r = 0.99, p = 2
   × 10^−4), and brown (r = 0.94, p = 6 × 10^−3) modules were highly
   associated with OEBBX14-0h, OEBBX14-1h, and OEBBX14-12h, respectively.

Figure 4.

   [78]Figure 4
   [79]Open in a new tab

   Module–sample relationships in potato under cold stress by weighted
   gene co-expression network. Each row denotes a module eigengene gene.
   Each cell comprises the corresponding correlation and p-value.

   In the red module (477 genes), the putative functions of these DEGs
   were enriched in the GO categories ‘transcription regulator activity’,
   ‘DNA-binding transcription factor activity’, and ‘lyase activity’
   ([80]Figure 5A, [81]Table S6). The DEGs in the red module were highly
   expressed in WT-0h, including Stphytochrome interacting factor 5
   (StPIF5 K[ME] = 0.99), ABSCISIC ACID INSENSITIVE 5 (StABI5, K[ME],
   0.9), mitogen-activated protein kinase kinase 4 (StMAPKK4, K[ME],
   0.94), ethylene response factor 3 like (StERF3-like, K[ME], 0.9), and
   ethylene response factor 60 (StERF60, K[ME], 0.88). The ‘green’ module
   comprised 771 DEGs that were highly expressed in OEBBX14-0h ([82]Figure
   5B, [83]Table S7), including five ethylene responsive element binding
   factor, heat shock factor 24 (StHSF24, K[ME,] 0.95), and dehydration
   response element B1A (StCBF3, K[ME], 0.75). The GO terms related to the
   ‘DNA-binding transcription factor activity’ and ‘transcription
   regulator activity’ were the most enriched. The GO terms associated
   with ‘response to auxin’ and ‘response to endogenous stimulus’ were
   significantly enriched in the magenta module. All 90 DEGs in the
   magenta module were highly expressed in the samples of OEBBX14-1h,
   where 19 BRI1-associated receptor kinase, glutathione S-transferase F11
   (StGST1, K[ME], 0.96), and glutathione S-transferase (StGST1-like,
   K[ME], 0.8) were highlighted ([84]Figure 5C, [85]Table S8). In the
   brown module (1596 DEGs), the genes were significantly enriched in
   ‘nucleoside-triphosphatase activity’ and ‘ADP binding’.
   ETHYLENE-INSENSITIVE3-like (StEIL1, K[ME], 0.96), dehydration response
   element B1A (StCBF2, K[ME], 0.98), xyloglucan endotransglycosylase 23
   (StXTH23, K[ME], 0.86), B-box type zinc finger family protein 4
   (StBBX4, K[ME], 0.95), and a total of nine MAPK genes in the brown
   module were highly expressed in the samples of OEBBX14-12h ([86]Figure
   5D, [87]Table S9).

Figure 5.

   [88]Figure 5
   [89]Open in a new tab

   Gene expression patterns of four modules: (A) red module, (B) green
   module, (C) magenta module, and (D) brown module. The bar graph of
   eigengene expression displays the eigengene value calculated from the
   singular value composition for each module.

2.5. RT-qPCR Validation of Gene Expression

   To validate the hub gene expression patterns in response to cold
   stress, eight DEGs were selected for RT-qPCR analysis ([90]Figure 6).
   Compared with the WT, the StADC1, StCBF1, StCBF2, StCBF3, StSAD1, and
   StSAMDC2 genes were significantly increased in OE-StBBX14-1h and
   OE-StBBX14-12h. Compared with the WT, the StCOR27 and StXHT23 genes
   were significantly increased in OE-StBBX14-1h and decreased in
   OE-StBBX14-12h. The expression patterns of the eight DEGs were
   consistent between the qPCR and RNA-seq, indicating the reliability of
   the transcriptome sequencing results.

Figure 6.

   [91]Figure 6
   [92]Open in a new tab

   The expression levels of eight DEGs at 0 h, 1 h, and 12 h after the 2
   °C cold treatment in the WT and OE-StBBX14. Student’s t-test test with
   * represented p < 0.05, ** represented p < 0.01 and *** represented p <
   0.001 were used to indicate significant differences.

3. Discussion

   With global warming, extreme temperatures have become more prevalent.
   As an important abiotic stress, low-temperature stress was proved to
   affect the growth and development of crops. Plants adapt to
   low-temperature stress through changes in their morphology, physiology,
   biochemistry, and metabolic regulation [[93]20]. Potato cultivars are
   not resistant to low-temperature frost, so it is important to explore
   the cold resistance gene of S. commersonii, namely, ScSAD, transformed
   in the cultivated potato variety Zhongshu 8 to significantly enhance
   the freezing tolerance of Zhongshu 8 [[94]18]. Previous results showed
   that the SaADC1 gene functioned in the cold-acclimated freezing
   tolerance of potato by promoting putrescine accumulation [[95]19].
   SaCBL1-like was exhibited to confer freezing tolerance via the CBF
   regulon in potato [[96]17]. The overexpression of ScAREB4 promotes
   freezing tolerance and functions as a downstream transcription factor
   of ABA signaling [[97]21]. Although some genes related to potato cold
   resistance were functionally verified, the molecular mechanism of
   potato cold resistance has not been fully understood.

   The BBX gene was demonstrated to play a pivotal role in plant cold
   tolerance [[98]14,[99]15]. In Arabidopsis, a total of 32 BBX family
   genes were identified [[100]22]. Previous research indicated that the
   CRYPTOCHROME2 (CRY2)-COP1-HY5-BBX7/8 module plays important roles in
   the regulation of blue-light-dependent cold acclimation in Arabidopsis
   [[101]15]. MdBBX37 bound to the MdCBF1 and MdCBF4 promoters to activate
   their transcription and MdBBX37 cold resistance in apple [[102]7]. A 27
   bp InDel in the promoter of the SlBBX31 is significantly associated
   with cold tolerance in tomato [[103]16]. Although 30 BBX family genes
   were detected in potato [[104]23], it is still necessary to further
   explore the gene function of the BBX gene family in potato. In this
   study, overexpression of the StBBX14 gene displayed less leaf damage
   and significantly lower electrolyte leakage compared with the WT under
   cold stress, indicating that StBBX14 increased the potato’s cold
   resistance capabilities. Previous studies suggested that the CBFs play
   key roles during cold acclimation in plants [[105]24,[106]25]. Ectopic
   AtCBF1 overexpression exhibited improved cold acclimation in transgenic
   S. commersonii [[107]26]. When comparing the ectopic overexpression of
   StCBF1 and ScCBF1 in Arabidopsis, ScCBF1 showed much more tolerance to
   freezing than StCBF1 [[108]27]. In this study, the expression levels of
   StCBF1, StCBF2, and StCBF3 were significantly increased in
   OE-StBBX14-1h and OE-StBBX14-12h. The overexpression of SlBBX17
   positively regulates cold tolerance through a CBF-dependent process in
   tomato [[109]14]. It was found that BBX29 is a negative regulator of
   cold stress by ABA- and CBF-independent pathways in Arabidopsis
   [[110]28]. Taken together, the BBX gene can regulate low temperature
   expression depending on a CBF-independent or -dependent pathway in
   plants.

   The RNA-seq technique was employed to compare gene expression profiles
   between OE--StBBX14 and the WT under cold stress. The DEGs were
   detected in 1 h and 12 h in either the WT or OE-StBBX14. A total of 14
   distinct modules were generated by a WGCNA analysis. Five modules were
   highly correlated with the cold stress. In the red module, StPIF5,
   StABI5, and StMAPKK4 were highly expressed in WT-0h. The results imply
   that PIF1, PIF4, and PIF5 negatively regulate the cold stress in
   Arabidopsis [[111]29]. MaABI5 in the ABA signaling pathway is involved
   in cold tolerance by interaction with MaC3HC4-1 in banana [[112]30].
   Compared with the wild type, the overexpression of SaMKK2 was increased
   the expression of CBF1/2/3 under cold stress [[113]31]. The cytochrome
   P450, MYB, and WRKY gene family were highly expressed in WT-1h. The
   transcription factors play key roles in plant responses to cold
   stresses. The apple MdMYB308L positively regulated cold tolerance by
   binding to the promoters of MdCBF2 [[114]32]. A total of 143
   differentially expressed cytochrome P450 genes were identified under
   cold stress in tomato [[115]33]. The overexpression of VaWRKY33
   enhanced the cold tolerance in grape calli with lower low-temperature
   exothermic values than the empty vector calli [[116]34]. StCBF3 and
   StHSF24 were highly expressed in OE-StBBX14-0h in the ‘green’ module.
   In S. commersonii, all four CBF genes were cold responsive, with CBF1
   and CBF3 being the most actively responsive under both acclimated and
   nonacclimated conditions [[117]35]. The PpHSFA4c-mediated HSF-HSP and
   ROS pathways promote fruit cold resistance in peach [[118]36]. The
   StGST1 and StGST1-like were highly expressed in the samples of
   OE-StBBX14-1h in the magenta module. The BoGSTU19, BoGSTU24, and
   BoGSTF1 genes were highly expressed at 6 h and 1 h in the cold-tolerant
   and cold-susceptible lines of B. oleracea [[119]37]. In the brown
   module, StEIL1, StCBF2, StXTH23, StBBX4, and a total of nine MAPK genes
   in the brown module were highly expressed in the samples of
   OE-StBBX14-12h. In upland cotton, XTH22 positively regulated the
   response to cold stress [[120]38]. These results provide abundant data
   to explore the molecular mechanism of potato’s response to cold stress.

4. Materials and Methods

4.1. Materials and Growth Conditions

   The low-temperature-sensitive potato cultivar ’Desiree’ was used in
   this study. The Désirée variety of potato has light yellow flesh and
   light red skin. The tubers of the Désirée variety are medium-large in
   size, oblong in shape, with shallow eyes. They have red skin and yellow
   flesh. The plants are tall and erect, the leaves flat, slightly
   divided. Many reddish-purple flowers appear in summer and form fruits.
   Désirée is a semi-late to late variety. It is then harvested 145 days
   after sowing, when the foliage has completely withered. Initially, the
   cultivar was inoculated on an MS medium supplemented with 3% sucrose
   and 0.7% agar and cultured in an incubator maintained at 22 ± 2 °C,
   with a light intensity of 2400 Lux and a photoperiod of 16 h/8 h. After
   four weeks of in vitro culture, the plants were transferred to the
   field. The potato tubers were harvested 12 weeks post-transplantation
   for subsequent analyses.

4.2. Sequence and Phylogenetic Analysis of StBBX14

   The amino acid sequences of BBX family proteins from various plant
   species were retrieved from monocot and dicot libraries, including the
   StBBX14 gene (ID: Soltu.DM.03G034030.1) from the potato genome
   database. Sequence alignments were performed using DNAMAN 6.0 software.
   Phylogenetic trees were constructed using the maximum likelihood method
   with MEGA 7.0 software, set to 1000 bootstrap replicates to assess the
   reliability of the inferred phylogeny.

4.3. Subcellular Localization Analysis

   The coding sequence of StBBX14 was PCR-amplified and cloned into the
   pAN580-linker vector and tagged with GFP. This construct was
   transformed into tobacco protoplasts, and GFP fluorescence was observed
   under a Nikon C2-ER laser confocal microscope (Nikon, Tokyo, Japan)
   using an excitation wavelength of 488 nm [[121]39].

4.4. Frost Resistance Determination

   To assess the frost tolerance of the transgenic plants, the electrolyte
   leakage from the leaves was measured. Relative leaves of the second and
   third compound leaves were taken, and the assay method was described in
   a previous paper [[122]18]. The ion leakage was expressed as the ratio
   of electrolyte leakage from frostbitten tissue (R1) to the electrolyte
   leakage after autoclaving (R2). The relative electrolyte leakage rate
   was calculated using R1/R2 × 100%. In addition, LT50 (half-lethal
   temperature) was calculated based on the electrolyte leakage to measure
   the freezing resistance.

4.5. RT-qPCR Analysis

   We extracted the total RNA using the Plant Total RNA Kit (OMEGA,
   Norcross, GA, USA). First-strand cDNA was synthesized using the Reverse
   Transcription Kit TRUEscript RT MasterMix (XinBaiJi Biotech, Nanjing,
   China). First-strand cDNA was synthesized using the BlasTaqTM 2X qPCR
   MasterMix (Abm, Vancouver, BC, Canada) on a Bio-RadCFX96machine
   (Bio-Rad Laboratories, Hercules, CA, USA) for RT-qPCR. Calibration and
   standardization were performed with the internal reference base
   β-tubulin2. Three replicates were set up for each biological sample,
   and the relative expression of genes was calculated using the 2^−ΔΔCt
   method.

4.6. Plasmid Construction and Genetic Transformation

   Homologous recombination was used to ligate the vector pFGC1008
   linearized with Kpn I and Sac I. The overexpression vector plasmid was
   constructed and subsequently transformed into Agrobacterium rhizogenes
   GV3101 by the heat-shock method. The low-temperature-sensitive variety
   ‘Desiree’ was used for the Agrobacterium-mediated transformation.
   Genetic transformation of potato miniature tubers followed the method
   described in a previous paper [[123]18]. To test the effectiveness of
   the transgenic plants, PCR amplification with the correct size was used
   to identify positive plants. qRT-PCR was used to further detect the
   gene expression of StBBX14 in the transgenic lines. The transgenic
   potatoes were planted in the field, and the WT plants were cultured
   under the same conditions as a control. After 30 days, they were
   subjected to low-temperature treatment at −2 °C for 4 h for phenotypic
   characterization. The primers used for amplification and qRT-qPCR are
   provided in [124]Table S10.

4.7. Transcriptome Analysis

   The 4-week-old wild-type (WT) potato and StBBX14 overexpression lines
   were treated with a 2 °C cold treatment for 0 h, 1 h, and 12 h. Each
   treatment with 3 biological replicates underwent RNA sequencing. Reads
   with splice adapters, unidentifiable base information, and low-quality
   reads were removed and filtered so that clean reads from each sample
   were aligned to the potato reference genome. The FPKMs (Fragments Per
   Kilobase of transcript Per Million Fragments) indicate the number of
   fragments per kilobase per million reads, where the sequencing depth
   and gene length were corrected according to the FPKMs [[125]40]. HISAT2
   v2.0.5 software was used to analyze the differentially expressed genes
   (DEGs) in the wild-type and transgenic plants under the low-temperature
   treatment. FDR ≤ 0.05 and log2|(FoldChange)| ≥ 1 were used as
   thresholds for significant differential gene expression. The
   differential gene sets were analyzed by clusterProfiler (3.8.1)
   software for the GO function enrichment analysis and KEGG pathway
   enrichment analysis to determine the biological functions and pathways.

4.8. Weighted Correlation Network Analysis (WGCNA)

   The outlier samples were filtered by expression matrix correlation, and
   the gene co-expression network was constructed by the R package WGCNA
   4.0.3 [[126]41]. The automatic network building function was employed
   to acquire co-representation modules, and the correlation between
   modules and processing was calculated to obtain the eigenvalues of each
   module.

4.9. Statistical Analysis

   The data were subjected to statistical analysis using Student’s t-tests
   or one-way analysis of variance, and all graphs were generated using
   the Prism 9.5 software package. The results are presented as the mean ±
   standard deviation. Significant differences between means were
   indicated by asterisks (* p < 0.05, ** p < 0.01 and *** p < 0.001).

5. Conclusions

   In this study, the overexpression of StBBX14 in potato enhanced the
   cold resistance. The Gene Ontology (GO) enrichment analysis revealed
   that photosynthesis, cell wall, thylakoid, transcription regulator
   activity, oxidoreductase activity, and glucosyltransferase activity
   were significantly enriched in the DEGs of both OEBBX14 and WT. A total
   of 14 distinct modules were generated by a WGCNA analysis. Four major
   modules with cold-related genes were isolated. RT-qPCR analysis showed
   that the expression patterns of eight DEGs were consistent between the
   qPCR and RNA-seq. These findings illustrate that the StBBX14 plays an
   important role in cold stress in potato and provides a data basis for
   the genetic improvement of cold resistance traits of potato.

Acknowledgments