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Article

Physiological and Metabolomics Analyses Revealed That Overexpression of CBL-Interacting Protein Kinase 23 Accelerate Tuber Sprouting in Potato

by
Fang Zhou
,
Fengjuan Wang
,
Xing Zhang
,
Yifei Lu
,
Bi Ren
,
Shimin Yang
,
Liming Lu
and
Liqin Li
*
College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 342; https://doi.org/10.3390/horticulturae11040342
Submission received: 23 February 2025 / Revised: 16 March 2025 / Accepted: 17 March 2025 / Published: 21 March 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

:
The potato (Solanum tuberosum L.) plays an important role in ensuring global food security. Potato tubers store abundant nutrients and are also reproductive organs. The adjustment of tuber sprouting plays a vital role in timely sowing and improving tuber product quality. CBL-interacting protein kinases (CIPKs) exert an important function in the entire life cycle of plants and in coping with stress. In our present study, we found that the StCIPK23 expression level increased during storage and that overexpression of StCIPK23 can accelerate tuber sprouting. Physiological assays indicated that overexpressing StCIPK23 altered carbohydrate metabolism and antioxidant-related enzyme activities during storage. Starch branching enzyme (SBEI) gene expression was upregulated, while sucrose synthase (SS), 3-phosphoglyceric phosphokinase (PGK), and glyceraldehyde-3-phosphate dehydrogenase 1 (GAPC1) gene expression were downregulated in StCIPK23-overexpressing potato. High gibberellin (GA) content and low abscisic acid (ABA) content were also detected in transgenic tubers. We conducted metabolomics analysis on bud eyes, and the results showed a total of 94 differential metabolites were found. Among them, 61 metabolites were increased, the top three metabolites were coumaryl alcohol, glutathione and quercetin–glucoside–glucoside–rhamnoside. Our results suggest that StCIPK23 is a positive regulator of potato tuber sprouting.

1. Introduction

Potato (Solanum tuberosum L.) is the fourth most produced crop worldwide after rice, wheat, and corn. The tuber is the most important economic and reproductive organ in potato. Owing to its high nutritional value: enriched with starch, protein, and other compounds, potato is considered an important crop for food supply and nutrition security [1]. Sprouting is one of the vital factors that contribute to the timely sowing and long-term storage of potatoes, and with the start of sprouting, tubers turn into source organs promoting bud growth [2]. The premature sprouting of tubers causes commercial problems, resulting in quality losses and reduced marketability [3]. In addition, both insufficient and excessive sprouting of tubers prior to planting can cause potato loss, ultimately resulting in reduced yield and productivity [4,5]. Understanding the molecular mechanisms of potato tuber sprouting has significant implications for potato planting and yield.
Research has also suggested that potato plants overexpressing the inorganic pyrophosphatase (PPase) gene sprout earlier than WT tubers do, while antisense transgenic lines sprout two weeks later than WT tubers [6]. Deng et al. reported that the overexpression of snakin-2 (StSN2) inhibits potato tuber sprouting by negatively modulating lignin biosynthesis and hydrogen peroxide accumulation [7]. Further research revealed that StSN2 interacts with the brassinosteroid signalling suppressor StBIN2 to delay tuber sprouting [8]. Calcium (Ca2+) is a ubiquitous second messenger for plant growth and development. The CBL-interacting protein kinase (CIPK) family is a class of serine/threonine (Ser/Thr) protein kinases. CIPKs are an indispensable protein family in the Ca2+-mediated plant signalling pathway and play a significant role in plant development and resistance to biotic and abiotic stresses [9]. In potatoes, 27 StCIPK sequences have been identified, and StCIPK18 was reported to positively regulate potato drought stress response and defence by improving the ability of plants to clear ROS [10]. Similarly, StCIPK10 overexpression can increase potato tolerance to drought and osmotic stress and increase both the ability to scavenge reactive oxygen species and the content of corresponding osmoregulatory substances [11]. Transcriptome analysis revealed that three CIPKs (OsCIPK13, OsCIPK14, and OsCIPK17) play critical roles in the early germination of rice seeds [12]. Several studies have also shown that overexpression of StCIPK23 can promote the growth of potato seedlings under both low potassium and low nitrogen conditions [13,14].
Zhang et al. reported that the nitrogen content is closely related to the sprouting time of potato tubers [15]. However, whether StCIPK23 can regulate potato tuber sprouting is still unknown. In this study, the StCIPK23 expression level increased with increasing storage time, and elevated StCIPK23 levels correlated with accelerated tuber sprouting. Overexpression of StCIPK23 could alter carbohydrate metabolism and antioxidant-related enzyme activities in tubers. Meanwhile, four gene expression levels were evaluated in StCIPK23-overexpressing potato. Gibberellin (GA) content and abscisic acid (ABA) content were altered in transgenic tubers. Our research provides a foundation for the study of potato sprouting mechanisms and the creation of breeding materials.

2. Materials and Methods

2.1. Experimental Design

In order to study the function of StCIPK23 in tuber sprouting, three transgenic lines of tuber overexpressing StCIPK23 were harvested, and then put into depository at 20 ± 2 °C for storage experiment, while wild-type tuber (E3 potato cultivator) were used as controls. After 30 days of storage, the sprouting phenotype and starch granule morphology of tubers were observed. Meanwhile, the bud eye region of the tubers was taken to determine the substance content in glycometabolism. Hydrogen peroxide (H2O2), lignin, GA, and ABA content were also measured. The expression levels of relevant genes were detected by real-time fluorescent quantitative PCR. At last, the content of important metabolites and metabolic pathways were analysed using metabolomics methods in tuber (Figure 1).

2.2. Potato Planting and Tuber Harvesting

Sterile robust seedlings of the same height were selected from both the transgenic and wild-type lines for the hardening-off treatment [14]. First, the tissue culture bottles were moved to a greenhouse maintained at 20 ± 2 °C. The bottles were opened, and 2 cm of sterile water was added for 5 days. Subsequently, the seedlings were transplanted into pots with a diameter of 24 cm, containing a mixture of nutritious soil (grass charcoal soil:perlite = 4:1) after washing the agar from the roots. After 2 weeks of cultivation, the seedlings were transferred to the experimental base at Sichuan Agricultural University (Chengdu, China), where the light intensity ranged between 300 and 800 μmol m−2 s−1m and the temperature varied from 18 °C to 25 °C. The harvested tubers were then allowed to heal for 10 days. Sixty potato tubers (including three transgenic lines and wild-type tuber) of similar size were selected and stored in a cardboard box at 20 ± 2 °C under dark conditions, the storage room was well-ventilated, and the humidity was maintained between 85 and 90%.

2.3. Measurement of Physiological Indicators and Enzyme Activity

After 30 days of storage, the bud eye region was obtained according to previously described methods [7]. Five grams of tubers were placed separately in 2 mL centrifuge. Afterwards, the tubers were ground into a fine powder with liquid nitrogen for subsequent experiments. For starch content analysis, a 1.0 g sample of tuber was ground in 50 mM HEPES-NaOH buffer (pH 7.4), and supernatant was obtained after centrifuging for 10 min at 8000× g. Then, 250 µL of anthrone buffer was added to 50 µL of the supernatant and incubated in a 95 °C water bath for 10 min. The absorbance was set at 620 nm according to the method described previously [16]. The contents of D-fructose, D-galactose, and glucose were tested using high-performance liquid chromatography (HPLC) as method reported [17]. Samples were ground into a fine powder and then extracted twice with 80% ethanol at 60 °C for 24 h. The extract samples (2 mL) were dried and resuspended in 1 mL of ultrapure water. A Waters HPLC system equipped with a 250 mm × 4.6 mm Varian Microsorb-MV 100-5 aminocolumn (Varian, Palo Alto, CA, USA) and mobile phase (75% acetonitrile) was employed to analyse 20 µL of the samples.
The hydrogen peroxide content was determined using a previously described method [18]. A 1.0 g sample of tuber was ground with 5 mL of trichloroacetic acid (TCA; 0.1% w/v) and then centrifuged at 12,000 rpm for 15 min. The supernatant (0.5 mL) was collected and mixed with 0.5 mL of potassium phosphate buffer (10 mM, pH 7.0) and 1 mol/L potassium iodide (KI). The absorbance was measured at 390 nm. The lignin content was determined using a previously described method [19]. A 5.0 g sample of tuber was ground with 5 mL of 95% ethanol. Then, 95% ethanol and an ethanol:n-hexane (1:2, V/V) solution were used to wash the bottom sediments. Next, 1 mL of 25% acetyl bromide in glacial acetic acid was put into the sediments after drying. The mixture was then kept at 70 °C for 30 min. Subsequently, 0.1 mL of hydroxylamine hydrochloride (7.5 M) and 2 mL of glacial acetic acid were put into the mixture. The mixture was centrifuged for 15 min at 4000 rpm to collect upper liquid. Then, 0.5 mL of the supernatant, and 4.5 mL of glacial acetic acid were mixed. 280 nm was used to measure absorbance.
The catalase (CAT) and superoxide dismutase (SOD) activities in the tubers were measured using previously published methods [20]. For CAT activity measurement, 1.0 g of tuber sample was ground in phosphate buffer (pH 7.8). Then, the mixture was centrifuged for 15 min at 4000 rpm to obtain the supernatant, which was mixed with 0.1 mol/L hydrogen peroxide and incubated for 10 min at 30 °C. The reaction was terminated by adding 10% H2SO4, and the absorbance was measured at 560 nm. For SOD activity measurement, 1.0 g of tuber sample was ground in phosphate-buffered saline (pH 7.8) and centrifuged at 1000 rpm for 20 min to collect the supernatant. The samples were incubated with 0.05 mol/L phosphate-buffered saline, 130 mM methionine, 750 µM nitroblue tetrazolium, 100 µM EDTA-Na2, 20 µM riboflavin, and 0.25 mL of H2O under 4000 Lux for 20 min. The absorbance was measured at 560 nm. The amylase activity and SBE activity were determined as previously described [21,22]. Sucrose synthase activity and SPS activity were also determined according to previously described methods [23,24].

2.4. Measurement of Gibberellin and Abscisic Acid Content

A 1 g bud eye region from a tuber was ground in liquid nitrogen for analysis of gibberellin and abscisic acid content. Ten volumes of acetonitrile solution and 8 μL of internal standard mother liquor were added. The mixture was extracted overnight at 4 °C, centrifuged at 12,000× g for 5 min, and the supernatant was collected. A total of 35 mg of C18 filler was added, and the mixture was vigorously shaken for 30 s and then centrifuged at 10,000× g for 5 min, after which the supernatant was collected. The mixture was blown dry with nitrogen and dissolved in 400 μL of methanol for high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) analysis [25].

2.5. Real-Time Fluorescence Quantitative PCR

A 0.1 g bud eye region from each tuber was ground in liquid nitrogen for total RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was subsequently acquired using a reverse transcription kit (Servicebio, Wuhan, China), and real-time fluorescent quantitative PCR was performed using the SYBR® Green Premix Pro Taq HS qPCR Kit on a 7500 Real Time PCR System (Life Technologies, Carlsbad, CA, USA). The cycling conditions were as follows: (1) an initial denaturation at 95 °C for 30 s, followed by (2) 40 cycles consisting of 95 °C for 15 s and 60 °C for 30 s. Potato elongation factor 1 alpha-like was used as the internal reference gene. The 2−ΔΔCt method was used to calculate gene expression. The sequences of primers used, including StCIPK23, StSbeI, StSS, StGAPC1, and StPGK, are listed in Supplementary Table S1.

2.6. Metabolomics Analysis

Weigh 0.1 g of tuber powder and dissolve it in 0.6 mL of 70% methanol extract; Vortex six times, centrifuge (at a speed of 10,000× g for 10 min), extract the supernatant, filter the sample through a microporous membrane (0.22 µm pore size), and store it in an injection bottle for ultra performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) analysis. Six duplicate samples were used for UHPLC–MS/MS. Using a T3 C18 1.8 µm chromatographic column, mobile phase A is ultrapure water (with 0.04% acetic acid added), and phase B is acetonitrile (with 0.04% acetic acid added); flow rate of 0.35 mL/min; column temperature 40 °C; and the injection volume is 4 µL. The temperature of electric spray ion source (ESI) is 550 °C, and the mass spectrum voltage is 5500 V. After analysis, metabolites with fold change ≥ 2 and fold change ≤ 0.5 were selected. Meanwhile, p-value ≤ 0.05, and the OPLS-DA model ≥ 1.

2.7. Statistical Analysis

For all data in this study, at least three biological replicates were performed for each sample. Statistical software programs (Excel 2019 and SPSS 14.0) were used to analyse the data. The data are shown as the means ± SE (n = 3), with n representing the number of biological replicates. The F test and one-way ANOVA were used to determine the significance of differences between samples at p ≤ 0.01 and/or p ≤ 0.05.

3. Result

3.1. StCIPK23 Overexpression Accelerates Potato Tuber Sprouting

To investigate the relationship between StCIPK23 expression level and the tuber sprouting process, we extracted RNA from tubers and carried out qRT–PCR. The results revealed that StCIPK23 was expressed at the lowest level after harvest (0 days). With prolonged storage at room temperature, the expression level of StCIPK23 significantly increased, peaking at 40 days (Figure 2A). Three independent StCIPK23-overexpressing transgenic lines (with different gene expression levels) were obtained in previously published articles [14]. The potato storage experiments suggested that the three StCIPK23-overexpressing transgenic tubers sprout earlier than wild-type tubers do at 30 days of storage (Figure 2B), indicating that StCIPK23 can regulate potato tuber sprouting.

3.2. Overexpressing StCIPK23 Alters Glycometabolism During Tuber Sprouting

In our study, to elucidate the effects of StCIPK23 on carbohydrate metabolism in transgenic tubers, four physiological indices were determined at 30 days of storage. The starch content was approximately forty percent lower in StCIPK23-overexpressing tubers than in WT tubers. In contrast, D-fructose, D-galactose, and glucose content were markedly higher in StCIPK23-overexpressing transgenic lines (Figure 3). These data indicate that StCIPK23 overexpression promotes starch breakdown during storage.

3.3. Overexpressing StCIPK23 Alters Enzyme Activities in Tuber Sprouting

We detected the activities of four enzymes at 30 days of storage, and our results suggest that starch branching enzyme activity and sucrose synthase activity were elevated, and that amylase activity and sucrose phosphate synthase activity were decreased in StCIPK23-overexpressing potato tubers (Figure 4). Consequently, it is reasonable to believe that overexpressing StCIPK23 in potato tubers has a significant effect on carbohydrate metabolism during tuber sprout growth.

3.4. Overexpressing StCIPK23 Alters the Antioxidant System and Lignin Contents

Both H2O2 and lignin are important factors promoting tuber growth. We analysed the H2O2 and lignin contents of potato tuber buds after 30 days of storage. These results indicate that overexpressing StCIPK23 promotes the accumulation of H2O2 and lignin. Moreover, our study revealed that in StCIPK23-overexpressing potato tubers, superoxide dismutase (SOD) activity increased by more than sixty-six percent, especially in OE line 1. SOD activity also increased by nearly seventy-eight percent, and CAT activity decreased by more than thirty-two percent (Figure 5). These changes may play a vital role in maintaining high levels of H2O2 and lignin in potato tuber buds.

3.5. Overexpressing StCIPK23 Affects GA and ABA Content

The potato sprouting process is controlled by the complex interactions of hormones. To evaluate whether StCIPK23 influences potato sprout growth, we measured gibberellin (GA) and abscisic acid (ABA) content. The results showed that the GA content was largely increased in the transgenic lines. However, the ABA content decreased by more than thirty-three percent (Figure 6). These findings suggest that StCIPK23 positively regulates tuber sprouting by elevating the GA/ABA ratio.

3.6. Overexpressing StCIPK23 Alters Gene Expression Levels During Tuber Sprouting

The gene expression level of starch branching enzyme I (SBEI), sucrose synthase (SS), 3-phosphoglyceric phosphokinase (PGK), and glyceraldehyde-3-phosphate dehydrogenase 1 (GAPC1) were closely related to tuber sprouting [21]. So, we carried out a qRT-PCR assay to determine the expression of four genes in tuber buds after 30 days of storage. Compared with WT tubers, the SBEI expression level increased by greater than sixty percent in StCIPK23-overexpressing potato tubers, whereas the expression of SS, PGK, and GAPC1 decreased by more than forty percent (Figure 7).

3.7. Overexpressing StCIPK23 Alters Starch Granules During Tuber Sprouting

We extracted starch from tubers stored for 30 days and observed the starch granules using scanning electron microscopy. Results found that the starch granules from the wild type are mostly egg-shaped or oval-shaped, with smooth surfaces and intact granules, while the starch granules in the transgenic sample (OE3) show roughness, with many filamentous structures, and exit small starch granules (Figure 8). It is speculated that overexpression of the StCIPK23 tuber alters the structure of starch granules, thereby promoting tuber sprouting.

3.8. Overexpressing StCIPK23 Alters Metabolite Levels During Tuber Sprouting

We conducted metabolomic analysis on bud eyes stored for 30 days, and the results showed that compared with the control, a total of 94 differential metabolites were found in the transgenic sample (OE3), of which 61 were upregulated. Coumaryl alcohol, glutathione, and quercetin–glucoside–glucoside–glucoside–rhamnoside were the top three metabolites with upregulated multiples. A total of 33 metabolites were found to be downregulated, with limonin, phlorizin, and chrysoeriol-7-O-rutinoside ranking in the top three downregulated multiples (Table 1). All metabolite data are shown in Supplementary Table S2. KEGG enrichment of metabolism indicates that the pathways enriched in the top four are flavonoid biosynthesis, flavone and flavonol biosynthesis, galactose metabolism, fructose and mannose metabolism (Figure 9). In conclusion, our results indicate that flavonoid and small molecule sugars play important roles in the tuber sprouting process.

4. Discussion

Sprouting is a significant stage of the typical life cycle of a potato tuber [26]. This complex process is accompanied by metabolic changes, as well as physiological changes, enzymatic reactions, hormone regulation and gene expression changes [27,28]. In our current study, the qRT-PCR results indicated that the expression of StCIPK23 increased within forty-eight days (Figure 2A). This expression pattern was similar to that of auxin response factor 6 in the tuber sprouting process [29]. However, two dormancy-associated genes, delay of germination 1 (DOG1) and subtilisin-like proteases (SLPs), were expressed at low levels in tubers during sprouting, possibly indicating the onset of sprouting [30]. These findings indicate that many functional genes participate in the complex potato sprouting process. In the current study, storage experiments revealed that StCIPK23 overexpression accelerated tuber sprouting (Figure 2B). On the contrary, overexpressing StPP2A significantly delays tuber sprouting in cold storage [31]. It was speculated that StPP2A altered the ABA signalling pathway in sprouting. The relationship between StCIPK23 and ABA deserves further research in tuber sprouting.
Previous studies have shown that sucrose transport into tuber buds is a precondition for triggering tuber sprouting and that a low sucrose content is vital for controlling starch mobilisation [32]. Both starch degradation and sucrose biosynthesis are closely linked to the sprouting of tubers [33]. During potato tuber sprouting, starch breakdown prevails over starch synthesis, leading to soluble sugar formation and meeting the nutritional and energy demands of bud growth [34]. In our study, we primarily determined four physiological indices. The starch content was reduced, and the D-glucose, D-fructose, and galactose contents were increased in StCIPK23-overexpressing tubers over that of WT tubers (Figure 3). A similar result was reported in a study related to the crucial rice seed germination regulator a pyruvate kinase (OsPK5), where the glucose, soluble sugar, fructose and sucrose contents increased in the rice seeds of Ospk5 mutants [35]. Ethylene responsive factor 1 (TERF1) from tomatoes promoted seed germination through carbon metabolism, and the sucrose and glucose contents in over-expressing TERF1 seeds were markedly taller than those in control [36]. These results indicate that overexpressing StCIPK23 can promote starch degradation and soluble sugar synthesis during tuber sprouting.
To investigate whether StCIPK23 overexpression is correlated with changes in enzyme activity, the activities of four enzymes related to starch mobilisation or sucrose biosynthesis were measured. SBE activity and SS activity increased in StCIPK23-overexpressing tubers, whereas amylase and SPS activity decreased (Figure 4). Zhang et al. found that the expression of potato α-amylase (StAmy23) in tubers was significantly reduced after sprouting [37]. Interestingly, silencing StAmy23 expression in tubers induced delayed sprouting [38]. The speculated reason is that amylase activity varies among different varieties and during different sprouting phases [39]. Under water scarcity conditions, a decrease in sucrose phosphate synthase (SPS) activity could reduce sucrose synthesis and increase starch synthesis in tubers [40]. This may be one of the adaptations to stress. Dai et al. found that exogenous ethylene could inhibit tuber sprouting by enhancing SPS activity, which is consistent with our results [41]. This indicates that SPS plays a role in regulating the sucrose-starch balance during sprouting.
In potato tubers, an increase in H2O2 content and a reduction in catalase (CAT) activity result in rapid sprouting [42,43]. In our study, StCIPK23-overexpressing tubers presented a greater reduction in CAT activity than WT tubers (Figure 5). Zhu et al. found the opposite result, overexpressing StSnRK1.1 accelerated tuber sprouting, but CAT activity increased in tubers of transgenic lines [44]. A cation peroxidase (StOD42) was found to improve POD and ɑ-amylase activity to promote tuber sprouting [45]. The respiratory burst oxidase homolog (RBOH) family played vital roles in ROS production. In Arabidopsis, CBL1/9CIPK26 complexes accelerated ROS synthesis by phosphorylating NADPH oxidase (RBOHF) [46]. Beltrán et al. found that overexpression of a universal stress protein (SlRd2) reduced ROS synthesis compared to control. Further research has suggested that SIRd2 can be phosphorylated by SlCIPK6 and that SlRd2 interacts with SlCIPK6 to decrease reactive oxygen species (ROS) output in Arabidopsis [47]. Therefore, we infer that overexpressing StCIPK23 also alter the ROS signalling pathway in tuber sprouting, but it is a valuable task to determine the regulated protein by StCIPK23 in the future.
In many plants, the balance between ABA and GA is the primary regulator of dormancy, and GA facilitates the process from dormancy to germination [48]. AtCIPK26 interacts with ABI1, ABI2, and ABI5 in the ABA signalling pathway, thereby promoting seed germination [49]. Kumar et al. reported that ABA accumulation reaches its maximum at the onset of tuberification and dormancy, whereas a decrease in ABA is a key factor in determining the interruption of dormancy [50]. Similarly, in our study, the ABA level in StCIPK23-overexpressing plants was lower than that of WT plants, and the GA content increased (Figure 6). Therefore, we hypothesised that StCIPK23 functions similarly to ZmCIPK32 in Zea mays, because ZmCIPK32 encourages seed germination by modulating GA signals under stressed conditions [51].
The expression levels of four carbohydrate metabolism-related genes in the transgenic lines were evaluated via qRT-PCR. Compared with the WT tubers, StCIPK23-overexpressing tubers presented higher SbeI expression levels, whereas sucrose synthase, PGK and GAPC1 expression levels were lower (Figure 7). This is similar to the function of IbAGPaseS, as overexpression of IbAGPaseS increased starch branching enzyme I levels and induced the downregulation of sucrose synthases I and II [52]. In contrast, potato tubers overexpressing Snakin-2 presented lower SbeI expression levels and higher expression levels of sucrose synthase, PGK and GAPC1 expression [21].
The particle morphology of starch plays an important role in studying the structure, properties, and processing of starch. We found the starch granules in the transgenic sample (OE3) show roughness, with many filamentous structures, and exit small starch granules compared to WT (Figure 8). Different starch biosynthetic pathways can influence granule morphology. Therefore, inhibiting the expression of starch synthase in potatoes may lead to changes in granule morphology and an increase in phosphate content [53]. Xu et al. reported that overexpression of a potato glucan, water dikinase, regulated starch granule morphology and amylose content [54]. Hence, we hypothesised that overexpressing StCIPK23 could alter granule morphology by affecting the activity of starch-related enzymes.
Our metabolomics results indicate that flavonoid biosynthesis, flavone and flavonol biosynthesis, phenylpropanoid biosynthesis and galactose metabolism pathway were significantly enriched (Figure 9). Recent metabolomic studies have shown that flavonoids and coumarins play important roles in potato sprouting [55]. In our study, we detected 27 types of flavonoids (15 upregulated, 12 downregulated) and 4 types of upregulated coumarins in StCIPK23 overexpressing tubes (OE3), indicating that these two types of substances are commonly present during tuber sprouting and are not specific to the specific variety. Meanwhile, zeatin biosynthesis pathways were enriched in our study. Recent research identified zeatin biosynthesis in the sprouting stage in both long-dormant and short-dormant potato varieties [56]. Zeatin likely functions similarly to cytokinins, which have been reported to promote tuber sprouting by activating bud growth [57]. Therefore, more research is urgently needed to explain the molecular mechanisms by which zeatin regulates sprouting. Additionally, our assay unveiled that two vital clusters—cysteine and methionine metabolism and amino acid biosynthesis—were also enriched. These results suggest that numerous amino acids are synthesised to meet the demands of sprouting [55].
In this study, we found the content of mannitol and sorbitol was increased in StCIPK23 overexpressing tubes. Zhu et al. found that sorbitol immersion promotes the synthesis of lignin in potato tuber wounds [58], and lignin was reported to play an important regulatory role in tuber sprouting [7]. At the same time, four types of chlorogenic acid and its derivatives were found to increase, some studies have shown that these substances can affect Arabidopsis seed germination [59] and accumulate in segmented potatoes under different altitudes region [60]. So, it is speculated that chlorogenic acid methyl ester plays a positive regulatory role in the sprouting process. Subsequent research will focus on how StCIPK23 regulates the synthesis of these important metabolite molecules in the tuber sprouting process.

5. Conclusions

CIPKs are vital protein kinases in plants that participate in regulating various physiological responses and stress tolerance. In this study, we first reported that the StCIPK23 expression level continuously increased during the 40-day storage period. Storage experiments revealed that overexpression of StCIPK23 accelerated tuber sprouting. A working model for the StCIPK23 overexpression line was summarised during sprouting (Figure 10). First, overexpression of StCIPK23 altered the carbohydrate metabolism pathway, for example, by enhancing enzyme activities and gene expression of related starch branching enzymes and sucrose synthase. Meanwhile, overexpression of StCIPK23 promoted starch degradation and increased the content of three monosaccharides. Second, the GA/ABA ratio and ROS pathway were also altered during sprouting. At last, a total of 94 metabolites were determined by metabolomic analysis. Among them, the content of eight metabolites was significantly upregulated in StCIPK23 overexpression tubers. Therefore, these important metabolites and StCIPK23 expression can be regarded as markers for potato breeding materials during the dormancy period.
Currently, there are two main challenges. First, gene-editing materials for the StCIPK23 gene are not yet available. Second, to meet production needs, low-temperature storage experiments needed to be conducted. In the future, storage experiments will be carried out at both room temperature (20 ± 2 °C) and low temperature (12 ± 2 °C) using StCIPK23 overexpression lines and gene editing materials. In China, a storage experiment at room temperature (20 ± 2 °C) was suitable for potatoes planted in three seasons within a year. Future work will also address the development of varieties resistant to sprouting or the artificial regulation of sprouting time using genetic engineering methods. To elucidate more functions of StCIPK23, the next step is to explore the effects of StCIPK23 overexpression on tuber yield and quality in experimental fields under different agricultural conditions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11040342/s1, Table S1: Primers sequences used in this study; Table S2: Information of all metabolites were listed.

Author Contributions

L.L. (Liqin Li) and F.Z. designed the experiments and wrote the first draft; F.W., F.Z., and X.Z. finished the experiments; B.R. and Y.L. analysed the data; S.Y. and L.L. (Limin Lu) revised and edited the draft. All authors have read and agreed to the published version of the manuscript.

Funding

The study was funded by the College Student Innovation Training Program of Sichuan Agricultural University (202310626017).

Data Availability Statement

The authors confirm that data generated in this study are available in the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. The schematic diagrams of all experiments.
Figure 1. The schematic diagrams of all experiments.
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Figure 2. StCIPK23 is involved in the tuber sprouting process. (A) StCIPK23 expression pattern in different storage times. (B) Observation of sprouting time in transgenic tubers. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05. WT, wild type. OE1–OE3, StCIPK23 transgenic tubers.
Figure 2. StCIPK23 is involved in the tuber sprouting process. (A) StCIPK23 expression pattern in different storage times. (B) Observation of sprouting time in transgenic tubers. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05. WT, wild type. OE1–OE3, StCIPK23 transgenic tubers.
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Figure 3. Changes in glycometabolism indices in transgenic tubers. (A) Starch content; (B) D-fructose content; (C) D-galactose content; and (D) glucose content. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
Figure 3. Changes in glycometabolism indices in transgenic tubers. (A) Starch content; (B) D-fructose content; (C) D-galactose content; and (D) glucose content. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
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Figure 4. Changes in the enzyme activity in transgenic tubers. (A) Amylase activity; (B) starch branching enzyme activity; (C) sucrose synthase activity; (D) sucrose phosphate synthase activity. Significance levels for differences between potato lines are represented by different letters, with a p-value of less than 0.05.
Figure 4. Changes in the enzyme activity in transgenic tubers. (A) Amylase activity; (B) starch branching enzyme activity; (C) sucrose synthase activity; (D) sucrose phosphate synthase activity. Significance levels for differences between potato lines are represented by different letters, with a p-value of less than 0.05.
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Figure 5. Changes in the antioxidant system and lignin content. (A) H2O2 content; (B) lignin content; (C) catalase activity; (D) superoxide dismutase activity. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
Figure 5. Changes in the antioxidant system and lignin content. (A) H2O2 content; (B) lignin content; (C) catalase activity; (D) superoxide dismutase activity. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
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Figure 6. Changes in gibberellin and abscisic acid content in transgenic tubers. (A) GA content; (B) abscisic acid content. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
Figure 6. Changes in gibberellin and abscisic acid content in transgenic tubers. (A) GA content; (B) abscisic acid content. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
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Figure 7. Changes in gene expression in transgenic tubers. (A) Starch branching enzyme I; (B) sucrose synthase; (C) glyceraldehyde-3-phosphate dehydrogenase I; (D) 3-phosphoglyceric phosphokinase. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
Figure 7. Changes in gene expression in transgenic tubers. (A) Starch branching enzyme I; (B) sucrose synthase; (C) glyceraldehyde-3-phosphate dehydrogenase I; (D) 3-phosphoglyceric phosphokinase. The significance level for differences between potato lines is represented by different letters, with a p-value of less than 0.05.
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Figure 8. Scanning electron microscope pictures of starch granules. (A) Wild type. (B) OE3, StCIPK23 transgenic tubers, magnification 1000×.
Figure 8. Scanning electron microscope pictures of starch granules. (A) Wild type. (B) OE3, StCIPK23 transgenic tubers, magnification 1000×.
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Figure 9. KEGG enrichment of metabolite in tuber. Note: The horizontal axis symbolises the rich factor of pathway, and the vertical axis symbolises name of pathway and the appearance of the points. The redder the colour, the more significant the enrichment. The size of the dots represents the enriched differential metabolites number.
Figure 9. KEGG enrichment of metabolite in tuber. Note: The horizontal axis symbolises the rich factor of pathway, and the vertical axis symbolises name of pathway and the appearance of the points. The redder the colour, the more significant the enrichment. The size of the dots represents the enriched differential metabolites number.
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Figure 10. A work model of the role of StCIPK23 plays in potato sprouting. Note: The red and green colours in each column indicate upregulation and downregulation, number indicates metabolites number.
Figure 10. A work model of the role of StCIPK23 plays in potato sprouting. Note: The red and green colours in each column indicate upregulation and downregulation, number indicates metabolites number.
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Table 1. Partial metabolite information is listed.
Table 1. Partial metabolite information is listed.
IndexCompoundsClassLog2FCp Value
mws0921p-Coumaryl alcoholPhenolic acids10.602.6594 × 10−5
pme1086Glutathione reduced formAmino acids and derivatives6.061.4914 × 10−4
Lmmp002334Quercetin-glucoside-glucoside-rhamnosideFlavonols5.915.2036 × 10−5
mws1077ScopolinCoumarins4.686.1833 × 10−7
Hmdp0021696-Hydroxy-7-methoxy-coumarinCoumarins3.971.7789 × 10−12
pmb0550Cyanidin 3-O-glucoside (Kuromanin)Anthocyanins3.469.6507 × 10−7
pme1261PantothenolSaccharides and Alcohols3.251.8434 × 10−2
Hmcp001757Quercetin-O-rhamnoside-O-Hexoside-O-rhamnosideFlavonols3.174.4403 × 10−5
Lmhp008763LysoPE 16:1 (2n isomer)LPE3.147.7189 × 10−4
mws1639IsofraxidinCoumarins2.689.9598 × 10−9
mws0036HesperidinDihydroflavone−1.989.1199 × 10−9
mws0055TangeretinFlavonols−2.015.7735 × 10−1
mws0043NobiletinFlavonoid−2.015.2729 × 10−2
pme0001Hesperetin 7-O-neohesperidoside (Neohesperidin)Dihydroflavone−2.021.1439 × 10−7
mws0052BaicalinFlavonoid−2.172.8922 × 10−10
pme0460EpicatechinFlavanols−2.402.0138 × 10−7
pmp001252p-CoumaroyltyraminePhenolamine−3.543.5747 × 10−1
pme3544LimoninOthers−3.591.6466 × 10−4
mws2118PhlorizinChalcones−3.931.6868 × 10−14
pmb3002Chrysoeriol-7-O-rutinosideFlavonoid−11.254.4212 × 10−13
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Zhou, F.; Wang, F.; Zhang, X.; Lu, Y.; Ren, B.; Yang, S.; Lu, L.; Li, L. Physiological and Metabolomics Analyses Revealed That Overexpression of CBL-Interacting Protein Kinase 23 Accelerate Tuber Sprouting in Potato. Horticulturae 2025, 11, 342. https://doi.org/10.3390/horticulturae11040342

AMA Style

Zhou F, Wang F, Zhang X, Lu Y, Ren B, Yang S, Lu L, Li L. Physiological and Metabolomics Analyses Revealed That Overexpression of CBL-Interacting Protein Kinase 23 Accelerate Tuber Sprouting in Potato. Horticulturae. 2025; 11(4):342. https://doi.org/10.3390/horticulturae11040342

Chicago/Turabian Style

Zhou, Fang, Fengjuan Wang, Xing Zhang, Yifei Lu, Bi Ren, Shimin Yang, Liming Lu, and Liqin Li. 2025. "Physiological and Metabolomics Analyses Revealed That Overexpression of CBL-Interacting Protein Kinase 23 Accelerate Tuber Sprouting in Potato" Horticulturae 11, no. 4: 342. https://doi.org/10.3390/horticulturae11040342

APA Style

Zhou, F., Wang, F., Zhang, X., Lu, Y., Ren, B., Yang, S., Lu, L., & Li, L. (2025). Physiological and Metabolomics Analyses Revealed That Overexpression of CBL-Interacting Protein Kinase 23 Accelerate Tuber Sprouting in Potato. Horticulturae, 11(4), 342. https://doi.org/10.3390/horticulturae11040342

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