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Article

Overexpression of CBL-Interacting Protein Kinases 23 Improves Tolerance to Low-Nitrogen Stress in Potato Plants

by
Feiyun Huang
,
Yifei Lu
,
Zi Li
,
Lang Zhang
,
Minqiu Xie
,
Bi Ren
,
Liming Lu
,
Liqin Li
* and
Cuiqin Yang
*
College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(5), 526; https://doi.org/10.3390/horticulturae10050526
Submission received: 22 February 2024 / Revised: 28 April 2024 / Accepted: 10 May 2024 / Published: 19 May 2024
(This article belongs to the Section Developmental Physiology, Biochemistry, and Molecular Biology)
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Abstract

:
CBL-interacting protein kinases (CIPKs) play important regulatory roles in plant growth development and abiotic stress tolerance. However, the biological roles of these genes in response to low-nitrate (LN) stress in potato plants have not been determined. Here, we reported that StCIPK23 was expressed mainly in roots and leaves. StCIPK23 was located mainly in the cell membrane, nucleus, and cytoplasm. Further research suggested that, compared with wild-type (WT) plants, StCIPK23-overexpressing plants were taller and had significantly greater nitrate and ammonium nitrogen contents under LN stress. StCIPK23 overexpression can increase StAT, StNRT2.1, StNR, StGS1-3, and StGOGAT expression levels in StCIPK23 transgenic seedlings compared to those in WT plants under LN stress. The results of yeast two-hybrid and luciferase complementation imaging experiments suggested that StCIPK23 could interact with StCBL3. Real-time reverse transcription–PCR revealed the StCIPK23 expression level peaked at 6 h and the StCBL3 expression level peaked at 9 h in the roots under LN stress. In conclusion, we found that StCIPK23 and StCBL3 form a complex to regulate the expression of key genes in the nitrogen metabolism pathway to improve LN tolerance in potato plants.

1. Introduction

Potatoes have been among the four important food crops in recent years. Potatoes are commonly grown in a great number of nations and areas worldwide. The potato is also a staple and represents an important food for people in developing countries, especially in regions of northern China [1]. Abiotic stressors are becoming more frequent and persisting longer as the global warming process accelerates, which causes potato yields and quality to decrease globally [2]. Nitrogen has a significant impact on potato growth, tuber yield, and tuber quality. However, excessive nitrogen consumption can exert negative effects on the environment and increase manufacturing costs; as a result, enhancing potato nitrogen usage efficiency (NUE) is a sustainable solution for these problems [3]. Advanced efforts have been undertaken to improve NUE in model plants, such as Arabidopsis thaliana [4] and Oryza sativa [5], through molecular and physiological approaches. In potato plants, phosphatase 2C, high-affinity nitrate transporters, sugar transporters, proline-rich proteins, bHLH transcription factors, superoxide dismutase, GDSL esterase lipase, etc., were identified as candidate genes for enhancing NUE by genome-wide RNA-sequencing analyses [6].
Plants have gradually formed low-affinity transport systems (LATS) and high-affinity transport systems (HATS) to cope with changes in nitrate concentrations in the external environment. When the concentration of NO3− exceeds 1 mmol/L in the soil, LATS plays a major role in the transport of 3−. When the concentration of NO3− is below 1 mmol/L, HATS plays a major role in the transport of NO3− [7]. Many studies have found nitrate (NO3−) and ammonium (NH4+) to be the two main nitrogen-absorbing forms for plants [3]. At present, some vital genes that take part in N metabolism have been revealed; they are nitrate transporter, ammonium transporter, nitrate reductase, nitrite reductase, glutamine synthase, glutamate synthase, etc. [8]. So far, nitrate transporters have been divided into four major families, namely NPF (NRT1/PTR), NRT2, chloride channel protein family (CLC), and slow anion homologous genes (SLAC/SLAH). NRT/NPF and SLAH transporters are responsible for NO3− import in roots; on the other hand, ammonium transporters are involved in NH4+ uptake. In Arabidopsis, higher NRT1.1 expression in shoots than in roots can promote seedling growth under LN stress [9], suggesting NRT1.1 plays a vital role in N metabolism.
Within cells, calcium (Ca2+) is considered to play an important regulatory role in many physiological reactions and developmental processes. Ca2+ signals are decoded and transmitted through Ca2+ sensing proteins, leading to the response of various intracellular signaling systems to environmental changes. Ca2+ regulates multiple NRT/NPFs and SLAH transporter proteins for NO3− uptake and transport [10]. As a Ca2+ sensor, calcineurin B-like protein (CBL) can sense Ca2+ signals in stress signals, enabling plants to adapt to environmental changes [11]. CBL-interacting protein kinase (CIPK) is a plant-specific gene family containing NAF/FISL and a protein phase interaction motif. The NAF motif is an essential site for binding to CBL [12]. As a Ser/Thr protein kinase, CIPK has been systematically identified in plants [13]. For example, OsCIPK3 and OsCIPK7 expression increases in response to cold stress, and transgenic plants that overexpress (OE) OsCIPK3 and OsCIPK7 exhibit cold tolerance traits [14]. Overexpression of three CIPKs (TaCIPK2, TaCIPK23, and TaCIPK27) increased drought tolerance in Arabidopsis [15].
CIPKs interact with CBL proteins and bind to each other to create a complex that interacts with specific target proteins, helping plants respond to a variety of abiotic challenges [16]. The CBL-CIPK signaling network has been demonstrated to play a significant role in stress responses to conditions such as high pH, high salt concentration, low potassium concentration, cold temperature, dehydration, and abscisic acid exposure [17]. TaCIPK23 can interact with TaCBL1 on cell membranes, and TaCIPK23 overexpression can strengthen wheat germination and drought resistance [18]. In pepper, CaCIPK13 can interact with four CaCBLs (CaCBL1/6/7/8) in the plasma membrane to play a positive function in cold tolerance by increasing the expression level of cold-related genes [19]. Ma et al. reported that 27 CIPK genes were found in potato plants, and overexpressing the StCIPK10 gene improved resistance to osmotic stress and drought [20]. Later research suggested that overexpression of StCDPK28 potato plants showed high activities of CAT, SOD, and POD under osmotic and water deficiency stress [21]. Similarly, Yang et al. reported that StCIPK18 knockout plants decreased proline contents and antioxidant enzyme activities compared with the wild type under drought stress. Y2H and a bimolecular fluorescent complimentary experiment show StCIPK18 and StCBL4 can interact at the protein level [22].
In potato plants, the overexpression of StCIPK23 seeding enhanced low potassium tolerance [23], so we want to explore the function of StCIPK23 in low-nitrogen stress. In this study, the potato StCIPK23 gene was cloned. StCIPK23 was located mainly in the cell membrane, nucleus, and cytoplasm. Compared with wild-type (WT) plants, StCIPK23-overexpressing plants were taller and had significantly greater nitrate and ammonium nitrogen contents under low-nitrogen (LN) stress. StCIPK23 overexpression can increase the levels of five key genes in StCIPK23 transgenic seedlings compared to those in WT plants under LN stress. such as ammonium transporter 1 (StAT), high-affinity nitrate transporter 2.1 (StNRT2.1), nitrate reductase (StNR), glutamine synthetase (StGS1-3), and glutamate synthase 1 (StGOGAT). Further research via yeast two-hybrid (Y2H) and luciferase complementation imaging (LUC) experiments showed that StCIPK23 interacts with StCBL3. The qRT–PCR showed that the StCIPK23 expression level peaked at 6 h and the StCBL3 expression level peaked at 9 h in the roots. StCIPK23 and StCBL3 exhibited similar expression patterns in roots, stems, and leaves under LN stress. The results of this study will provide a reference for further research into the CBL-CIPK regulatory network in potato plants under LN stress, and StCIPK23 can be used for breeding to increase nitrogen use efficiency in the future.

2. Materials and Method

2.1. Potato Transformation and Growth Conditions

The coding region of StCIPK23 (PGSC gene ID: Soltu.DM.02G002430.1) was obtained by reverse transcription–polymerase chain reaction. Then, the recombinant vector (StCIPK23-PBI1221) was transferred into Agrobacterium tumefaciens GV3101 to infect the WT (E3 potato cultivar). Positive clones were obtained using the PCR method. For transgenic experiments, first, microtubers 12–20 weeks old were cut into slices, then immersed in the Agrobacterium tumefaciens suspension for 5–15 min. The tuber slices were incubated in a medium after drying. After 36 h of co-cultivation in the dark at 24 °C, tuber slices were moved onto the regenerating medium to induce regenerated shoots. In vitro-generated transgenic potato plants were grown in solid MS (Murashige–Skoog) media, adding 50 mg/mL kanamycin. The growth conditions in the artificial growth chamber were 23 ± 2 °C with a photoperiod of 16 h light/8 h dark. The detailed gene transformation method was performed according to a previously described method [24]. The primers used are listed in Table S1.

2.2. Hydroponics of Potato Seedlings and Nitrogen Content Measurement

The potato seedlings of the WT and three StCIPK23 transgenic lines (growing on MS media for 20 days) were hydroponic. Each material includes 60 seedlings. One N-sufficient solution was added with 1 mM Ca (NO3)2; the other was added with 0.1 mM Ca (NO3)2. The solution formula is reported in [25]. The growth conditions in the artificial growth chamber were 23 ± 2 °C with a photoperiod of 16 h light/8 h dark. Seedlings from hydroponics at different time points were collected for N content measurement and gene expression level analysis. Nitrate and ammonium content were analyzed using corresponding kits (G0440W, G0410W) produced by Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China) [26].

2.3. Bioinformatics Analysis

The NCBI database (https://www.ncbi.nlm.nih.gov (accessed on 9 September 2023)) was used to analyze the conserved structural domain of the StCIPK23 protein. The StCIPK23 protein was analyzed physicochemically using the ExPASy-ProtParam tool (https://web.expasy.org/protparam/ (accessed on 15 September 2023)). The online website NetPhos-3.1 (https://services.healthtech.dtu.dk/services/NetPhos-3.1/ (accessed on 15 September 2023)) was used to predict the phosphorylation sites of the StCIPK23 protein. MEGA 11 (https://www.megasoftware.net/ (accessed on 11 September 2023)) software was used to construct a phylogenetic tree of the StCIPK23 protein with 19 other CIPK23 proteins, including JcCIPK23, MeCIPK23, SoCIPK23, HuCIPK23, JrCIPK23, ZjCIPK23, MdCIPK23, PmCIPK23, VrCIPK23, ArCIPK23, HaCIPK23, LsCIPK23, SiCIPK23, NtCIPK23, NsCIPK23, NaCIPK23, CaCIPK23, SICIPK23, and SpCIPK23. The online analysis website MEME (https://meme-suite.org/meme/ (accessed on 11 September 2023)) was used to analyze the motif contained in 20 CIPK 23 proteins.

2.4. Quantitative Real-Time PCR

To check the expression pattern of genes, the seedlings were treated for 0 h, 0.5 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h after LN treatment. Before performing the qRT–PCR experiment, the plant total RNA was extracted using an extraction kit (Sangon Biotech Co., Ltd., Shanghai, China). The quality of the total RNA was examined, then tansferred into cDNA with a reverse transcription kit (Wuhan Servicebio Technology Co., Ltd., Wuhan, China). The iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) was used for the qRT–PCR analysis. Then, a 7500 Real Time PCR System (Life Technologies, Carlsbad, CA, USA) was performed according to the manufacturer’s instructions. The expression levels of StCIPK23, StCBL3, StAT, StNRT2.1, StNR, StNiR, StGS1-3, and StGOGAT were counted using the 2−∆∆Ct method. Elongation factor 1 alpha-like (EF1αL) expression was used as an internal control. The primers are listed in Table S1.

2.5. Subcellular Localization Analysis

StCIPK23 was inserted into the vector pCAMBIA2300-35S-EGFP using the SpeI and BamH1 restriction sites. Tobacco plants (Nicotiana benthamiana L.) aged 4 to 6 weeks were selected for subcellular localization analysis. Agrobacterium tumefaciens GV3101 was cultured overnight at 28 °C in Agrobacterium rhizogenes liquid medium (YEB) with shaking at 250 rpm until A600 = 0.5~1.0. Healthy tobacco leaves were infected with Agrobacterium tumefaciens suspension in the back, then put in the dark for 24 h at 25 °C and kept under light for another 48 h. Three independent leaves were injected for each independent experiment. A laser scanning confocal microscope was used to check the fluorescence of the leaf. The wavelength of GFP excitation is 395 nm.

2.6. Yeast Two-Hybrid Analysis

The coding region of the StCBL3 (PGSC DM.03G022500.1) gene was inserted into the pGADT7 vector using the NdeI and BamHI enzymes. Similarly, the coding region of the StCIPK23 gene was inserted into the pGBKT7 vector via the NdeI and BamHI restriction sites. The recombinant vectors were subsequently transformed into yeast AH109 cells with the PEG/LiAc method. First, the successfully transformed colonies were selected on SD-Leu-Trp-deficient media at 30 °C for 3 d. Positive clones were obtained using the PCR method with target gene primers. Then, the deficient medium SD-Trp-Leu-His-Ade containing 5-bromo-4-chloro-3-indolyl-α-D-galactoside (X-α-Gal) was used to evaluate the interaction of StCIPK23 with StCBL3. In the experiment, pGADT7 + pGBKT7 was used as a negative control, and AtCBL1 + AtCIPK23 was used as a positive control. The experiments were repeated three times.

2.7. Luciferase Complementary Imaging Assay

The recombinant StCIPK23-JW772 and StCBL3-JW772 vectors were subsequently transformed into Agrobacterium tumefaciens GV3101. Agrobacterium tumefaciens GV3101 was cultured overnight at 28 °C in Agrobacterium rhizogenes liquid medium (YEB) with shaking at 250 rpm until A600 = 0.5~1.0. Nicotiana benthamiana leaves (approximately 4 to 6 weeks old) were infected in the back with Agrobacterium tumefaciens suspension. Three independent leaves were injected for each independent experiment; subsequently, the injected leaves were cultured in darkness at 25 °C for 24 h to determine the LUC using a chemiluminescence imaging system (Tanon 5200 Multi, Shanghai, China); the exposure intensity was adjusted according to the results of the experiment. In the experiment, nLUC + cLUC, StCIPK23-nLUC + cLUC, and StCBL3-cLUC + nLUC were used as negative controls.

2.8. Statistical Assay

For the data analysis, statistical software programs (Excel 2019 and SPSS 14.0) were used. The data in this study were obtained from three biological replicates. The data are shown as the mean ± SE (n = 3). and n represents the biological replicates. The significance of differences between samples was assayed using the F test and one-way ANOVA at p ≤ 0.01 and/or p ≤ 0.05.

3. Results

3.1. StCIPK23 Phylogenetic Relationships

Bioinformatics analysis revealed that the length of the coding region of StCIPK23 (PGSC gene ID: Soltu.DM.02G002430.1) was 1368 bp, encoding 455 amino acids. To determine the homology of StCIPK23 with CIPK23 in plants, StCIPK23 was compared with CIPK23 from 19 different plants via MEGA11 (Supplemental Table S2). The phylogenetic tree showed that the 20 selected plants evolved into three clades, with StCIPK23 conserved, consistent with the evolutionary relationships of the species. Among them, the three closest evolutionary relatives to StCIPK23 (Solanum tuberosum, XP_006361546.1) were CaCIPK23 (Capsicum annuum, XP_016558593.1). SlCIPK23 (Solanum lycopersicum, XP_ 004231541.1) and SpCIPK23 (Solanum pennellii, XP_015066690.1) (Figure 1A). To further understand the structural features of these 20 different plant CIPK23, the motif structures of all CIPK23 were analyzed through the MEME online website (Figure 1B). We found that many CIPK23 contained the full 12 motifs except for JcCIPK23 (Jatropha curcas, XP_012072157.1) and MeCIPK23 (Manihot esculenta, XP_021599119.1), which did not contain motif 12.

3.2. Subcellular Localization of StCIPK23

The pCAMBIA2300-EGFP-StCIPK23 fusion vector was constructed to identify the StCIPK23 location in tobacco leaf cells. StCIPK23 was transiently expressed in tobacco leaf cells by Agrobacterium-mediating methods. The results from the laser confocal scanning microscope showed that StCIPK23 was expressed mainly in the cell membrane, nucleus, and cytoplasm, similar to the EGFP control vector (Figure 2). This finding suggested that the location of this protein was nonspecific.

3.3. StCIPK23 Overexpression Improves Tolerance to Low-Nitrogen Stress

To explore the function of StCIPK23 in potato plants, we assessed its expression level in the root, stem, leaf, flower, tuber, and bud. The qRT–PCR results showed that the relative expression level of this gene was greatest in the roots, stems, and leaves and lowest in the tubers (Figure 3A). After that, we obtained StCIPK23 transgenic potato plants (OE-1, OE-2, and OE-3), and the expression level of the StCIPK23 gene in three transgenic lines was greater than that in the WT, according to the qRT–PCR results (Figure 3B). To study the responses of the transgenic lines under LN stress, seedlings of StCIPK23 transgenic lines were cultured in LN solid media for 20 days. We found that the WT and overexpression StCIPK23 plants had similar heights under N-sufficient conditions, but the transgenic plants had taller heights than the WT plants for 20 days under LN stress (Figure 3C,D). Further analysis suggested that both the nitrate and ammonium nitrogen contents of the transgenic plants were higher than those of the WT after LN stress treatment (Figure 3E,F). These findings indicate that StCIPK23 enhances LN tolerance by promoting N absorption and updating.

3.4. StCIPK23 Adjusted the Expression Level of Genes in Nitrogen Metabolism

To explore the function of StCIPK23 in nitrogen metabolism, we analyzed the expression levels of six nitrogen metabolism-related genes (StAT, StNRT2.1, StNR, StNiR, StGS1-3, StGOGAT) under LN stress. The results of qRT–PCR suggested that StAT, StNRT2.1, StNR, StGS1-3, and StGOGAT were significantly upregulated in the StCIPK23 transgenic lines compared to the WT at the 0 h point, but there was no difference in the expression of nitrite reductase (StNiR) in the WT or StCIPK23 transgenic lines. After 6 h of LN stress, the expression levels of StAT, StNRT2.1, NR, and StGS1-3 were greater in the StCIPK23 overexpression line than in the WT. But StGOGAT and StNiR expression were lower in the StCIPK23 overexpression lines than in the WT (Figure 4). We speculated that high expression levels of N metabolism-related genes help potato plants withstand LN stress.

3.5. Interaction between StCIPK23 and StCBL3

Our seeding transcriptome data (unpublished) from potato plants subjected to LN stress for 24 h indicated that StCBL3 expression obviously increased after LN stress, so we wanted to evaluate the correlation between StCIPK23 and StCBL3. First, the interaction between StCBL3 and StCIPK23 was assessed using Y2H (Figure 5A). Next, the StCIPK23-nLUC and StCBL3-cLUC fusion vectors were transiently expressed in Nicotiana benthamiana leaves. As a result, a strong luminescence signal was detected only in regions containing StCIPK23-nLUC and StCBL3-cLUC, but there was no luminescence signal in the negative control (Figure 5B). Based on the above results, these two proteins interact with each other.

3.6. StCIPK23 and StCBL3 Share the Same Low-Nitrogen Response Pattern

To study the expression patterns of StCIPK23 and StCBL3 under LN stress, we collected samples of potato roots, stems, and leaves at various intervals within 24 h of LN stress. First, the StCIPK23 expression level peaked at 6 h, and the StCBL3 expression level peaked at 9 h in the roots; the expression level of two genes had decreased to the lowest level at 24 h (Figure 6A,B). StCIPK23 and StCBL3 expression levels increased rapidly in the stem, peaking at 9 h. The expression level of StCIPK23 gradually increased before 9 h. The expression level of StCBL3 decreased obviously at 0.5 h (Figure 6C,D). StCIPK23 and StCBL3 expression levels increased gradually in the leaves, peaking at 9 h, then rapidly declining at 12 and 24 h (Figure 6C,F). So, qRT–PCR revealed that StCIPK23 and StCBL3 exhibited similar expression patterns under LN stress.

4. Discussion

Nitrogen is the key factor affecting tuber quality and yield [27]. Nitrate also serves as a signal regulating tissue development, seed germination, and multiple biological responses [28]. Some research showed red and blue light were both involved in nitrate sensing and nitrogen deficiency stress [29,30]. Plants must cope with complex and variable growth environments during their life cycle. Ca2+ plays a key role as a ubiquitous secondary messenger in regulating a wide range of stimuli or signals in plants [31]. In the majority of biological processes in eukaryotic cells, protein kinases play a crucial regulatory role. Moreover, CIPKs participate in the response to a wide range of signals in plants. The CIPK protein family is involved in different biotic and abiotic stresses by regulating phosphatases, transcription factors, or transporters/channels [32,33]. For example, CmCIPK23 was reported as a negative regulator in nitrate uptake and assimilation; more research suggested that CmTGA1 (a transcription factor involved in nitrate response) interacted with CmCIPK23 in Cucumis melo [34].
In the present investigation, first, equivalent findings for CIPK23 were observed across diverse plant species (Figure 1); this was determined by selecting CIPK23 protein sequences from 20 various plants and analyzing the phylogenetic correlation and conserved motifs. Similar structural features for CIPKs have been found in other plants [35,36]. The sequence and structure of the CIPK family were similar, sharing the same mode of action throughout the plant kingdom. We found that StCIPK23 is localized in the cell membrane, cytoplasm, and nucleus (Figure 2). This location is the same as that of StCIPK10 [20]. Kim et al. reported that AtCIPK24 was convened to the tonoplast by AtCBL10 to regulate Na+ balance [37]. AtCIPK1 was taken to the plasma membrane, guided by AtCBL9 and AtCBL1 together; meanwhile, AtCIPK1 was led on the tonoplast by AtCBL1 and AtCBL2 [38,39]. Similarly, Xu et al. also found AtCIPK23 to increase low potassium tolerance in the plasma membrane by AtCBL1 and AtCBL9 [40]. Therefore, we speculate that the interaction between StCIPK23 and CBL may alter StCIPK23 localization in cells. NO3− and NH4+ are two sources of nitrogen absorbed by plant roots from soil [41]. Mhamdi et al. reported that the expression levels of StNRT1.2, StNRT1.5, and StNRT2.1 were higher in N-efficient varieties [42], suggesting NRT plays a positive and important role.
Role in nitrate accumulation and uptake: In Arabidopsis, as a major hub, AtCIPK23 was involved in drought, salinity, potassium, nitrate, ammonium, magnesium, et al. processes, implying the vital role of AtCIPK23 in enhancing the activity of nutrient transporters [43]. In Arabidopsis, AtCIPK8 is upregulated by low-nitrate stress, and AtCIPK8 can phosphorylate NRT1.1 to accelerate primary root growth and obtain more nitrate [44]. CBL7 as a Ca2+ sensor: cbl7 mutants showed more sensitivity to LN stress by downregulating the expression of NRT2.4 and NRT2.5; thus, the CBL-CIPK signaling system might fulfill multiple functions in regulating plant nitrate homeostasis [45]. Meanwhile, AtCPK6 participated in drought stress by phosphorylating NRT1.1 at the Thr571 site to decrease its NONO3− transporting activity during drought stress in Arabidopsis [46].
In this study, we demonstrated that overexpression of StCIPK23 enhanced LN tolerance, nitrate, and ammonium nitrogen compared to the WT plants during LN stress (Figure 3). In particular, the expression of StAT, StNRT2.1, StNR, StGS1-3, and StGOGAT (Figure 4) was significantly upregulated in potato plant overexpression lines. Suggesting its function was similar to OsCIPK2, because OsCIPK2 was found to improve abiotic stress tolerance by increasing the activities of nitrate reductase and glutamine synthase [47]. We guessed that the overexpression of StCIPK23 enhanced tolerance by increasing the NRT expression level. More studies revealed that under different N conditions, overexpression of OsNRT1.1A and OsNRT2.3b significantly increased rice yield and NUE by 40% with a short maturity period [48,49]. Similarly, AtCIPK23 not only regulates the nitrate receptor NPF6.3/NRT1.1 activity but also inhibits ammonium uptake via phosphorylation of the C-terminal end of the ammonium transporter [50]. As we know, high levels of NH4+ are toxic, so under high concentration ammonium ion (NH4+) stress, the high-affinity ammonium ion transporter (AMT1) was phosphated by AtCBL1 and AtCIPK23 [51]. AMT1;1 and AMT1;2 were reported to be depressed by the CBL1-CIPK23 complex, which also activated SLAH2/3 protein activity [52]. Some research showed CPK10/30/32 could phosphoregulate transcription factor NLP7, which induced expression of NPF6.3 in adequate NH4+ conditions [53,54]. Suggesting Ca2+-CBL-CIPK pathway-regulated ion transporters/channels to maintain several nutritional balances under abiotic stress [38]. Sanyal et al. reported that CIPK can phosphorylate CBL, and the interaction between CBL and CIPK promotes CIPK’s phosphorylation of downstream target proteins [10]. The combination of CBL and CIPK leads to the release of N-terminal kinase domains from the C-terminal self-inhibitory domain of CIPK, thereby enhancing CIPK activity [55,56]. In this study, we evaluated the interaction between StCBL3 and StCIPK23 via Y2H and LUC experiments (Figure 5). Furthermore, qRT–PCR analysis of StCIPK23 and StCBL3 revealed that both genes exhibited the same expression pattern in potato roots, stems, and leaves under LN stress (Figure 6). The expression levels of StCIPK23 and StCBL3 were rapidly increased in roots, suggesting they played vital roles in roots, similar to how AtCBL1 and AtCBL2 function in Arabidopsis [57]. Speculating that StCIPK23 and StCBL3 form a complex to work together under LN stress, Ho et al. reported that AtCBL1/9-AtCIPK23 enhances its function by phosphorylating NRT1.1 to adapt to the absorption of nitrate at different concentrations under nitrate conditions [48]. Similarly, CBL1/9-CIPK23 interacts with SLAH2 and SLAH3 to participate in nitrate transport by absorbing or secreting NONO3− [58]. In Arabidopsis, NPF6.3 is phosphorylated by CBL1/9-CIPK23, and it forms a dimer that converts NO3− from a low-affinity mode to a high-affinity mode, adapting to LN stress [59]. Du et al. reported that transcription factor MYB59 regulates K+/NO3− translocation and is involved in low K+ stress in Arabidopsis [60]. Another C2H2 zinc finger protein, STOP1 (SENSITIVE TO PROTON RHIZOTOXICITY 1), increased AtCIPK23 expression levels to adapt to low-K+ and high-NH4+ stress [61] Therefore, our further work will focus on the study of substrate phosphorylation by StCIPK23 and its upstream regulatory protein, which is highly important for explaining the regulation of nitrogen nutrition by the CBL-CIPK signaling system in potato plants.

5. Conclusions

In this study, we found that StCIPK23 has the closest evolutionary relatives to CaCIPK23, SlCIPK23, and SpCIPK23. Later research suggested that StCIPK23 is localized in the plasma membrane, cytoplasm, and nucleus. Compared with WT plants, StCIPK23-overexpressing potato seedlings were taller and had significantly greater nitrate and ammonium nitrogen contents under LN stress. StCIPK23 overexpression can increase StAT, StNRT2.1, StNR, StGS1-3, and StGOGAT expression levels in StCIPK23 transgenic potato seedlings under LN stress. The results of the Y2H and LUC experiments suggested that StCIPK23 could interact with StCBL3. The qRT–PCR results of the potato roots showed that the StCIPK23 expression level peaked at 6 h and the StCBL3 expression level peaked at 9 h. The expression level of two genes decreased to the lowest level at 24 h under LN stress. StCIPK23 and StCBL3 exhibited similar expression patterns in potato stems and leaves, peaking at 9 h under LN stress. Our work provides supplemental theoretical knowledge to deepen our understanding of the regulatory function of the StCBL3-StCIPK23 complex in nitrogen metabolism. But it is just the beginning of revealing StCBL3-StCIPK23 complex function in potato plants; future interesting works are to investigate phosphorylation substrates of StCIPK23 in potato plant nitrate signaling.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10050526/s1, Table S1: All primer sequences involved in this study; Table S2: Information from 20 different plant CIPK23 proteins.

Author Contributions

L.L. (Liqin Li) and F.H. designed the experiments and wrote the first draft; Y.L., L.Z. and M.X. finished the experiments; B.R. and Z.L. handled the data; L.L. (Liming Lu) and C.Y. revised the draft. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Science and Technology Department of Sichuan Province (Program No. 2022ZHYZ0006).

Data Availability Statement

The data presented in this study are available within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic relationships of CIPK23 from 20 different plants: (A) phylogenetic trees of CIPK23; (B) the motifs of the CIPK23 proteins. The motifs of the CIPK23 proteins (1–12) are shown with colored boxes.
Figure 1. Phylogenetic relationships of CIPK23 from 20 different plants: (A) phylogenetic trees of CIPK23; (B) the motifs of the CIPK23 proteins. The motifs of the CIPK23 proteins (1–12) are shown with colored boxes.
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Figure 2. Localization analysis of the StCIPK23. GFP: EGFP fluorescence signal; auto: chlorophyll; bright: bright field. The scale bar represents 20 μm.
Figure 2. Localization analysis of the StCIPK23. GFP: EGFP fluorescence signal; auto: chlorophyll; bright: bright field. The scale bar represents 20 μm.
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Figure 3. StCIPK23 overexpression enhanced low-nitrogen tolerance in potato plants: (A) expression analysis of StCIPK23; (B) expression analysis of StCIPK23. WT: wild type, OE-1~OE-3: StCIPK23 overexpression lines; (C) phenotypic analysis. bar = 2 cm; (D) analysis of plant height. NN, nitrogen-sufficient conditions; LN, low-nitrogen conditions; (E) nitrate nitrogen content; (F) ammonium nitrogen content. The data represent the mean ± standard deviation (n = 3). Different letters show significant differences (p < 0.05) among samples.
Figure 3. StCIPK23 overexpression enhanced low-nitrogen tolerance in potato plants: (A) expression analysis of StCIPK23; (B) expression analysis of StCIPK23. WT: wild type, OE-1~OE-3: StCIPK23 overexpression lines; (C) phenotypic analysis. bar = 2 cm; (D) analysis of plant height. NN, nitrogen-sufficient conditions; LN, low-nitrogen conditions; (E) nitrate nitrogen content; (F) ammonium nitrogen content. The data represent the mean ± standard deviation (n = 3). Different letters show significant differences (p < 0.05) among samples.
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Figure 4. Expression levels of key genes in nitrogen metabolism. WT: wild-type OE-1~OE-3: StCIPK23 transgenic plants. The data represent the mean ± standard deviation (n = 3). Different letters show significant differences (p < 0.05) among samples.
Figure 4. Expression levels of key genes in nitrogen metabolism. WT: wild-type OE-1~OE-3: StCIPK23 transgenic plants. The data represent the mean ± standard deviation (n = 3). Different letters show significant differences (p < 0.05) among samples.
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Figure 5. StCIPK23 interacts with StCBL3: (A) yeast two-hybrid assays; (B) luciferase complementary imaging assays. In the experiment, nLUC + cLUC, StCIPK23-nLUC + cLUC, and StCBL3-cLUC + nLUC were used as negative controls. The data were obtained from three biological replicates.
Figure 5. StCIPK23 interacts with StCBL3: (A) yeast two-hybrid assays; (B) luciferase complementary imaging assays. In the experiment, nLUC + cLUC, StCIPK23-nLUC + cLUC, and StCBL3-cLUC + nLUC were used as negative controls. The data were obtained from three biological replicates.
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Figure 6. Expression pattern analysis of StCIPK23 and StCBL3: (A) StCIPK23 expression in the roots; (B) StCBL3 expression in the roots; (C) StCIPK23 expression in the stem; (D) StCBL3 expression in the stem; (E) StCIPK23 expression in the leaves; (F) StCBL3 expression in the leaves. The data represent the mean ± standard deviation (n = 3). Different letters show significant differences (p < 0.05) among samples.
Figure 6. Expression pattern analysis of StCIPK23 and StCBL3: (A) StCIPK23 expression in the roots; (B) StCBL3 expression in the roots; (C) StCIPK23 expression in the stem; (D) StCBL3 expression in the stem; (E) StCIPK23 expression in the leaves; (F) StCBL3 expression in the leaves. The data represent the mean ± standard deviation (n = 3). Different letters show significant differences (p < 0.05) among samples.
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Huang, F.; Lu, Y.; Li, Z.; Zhang, L.; Xie, M.; Ren, B.; Lu, L.; Li, L.; Yang, C. Overexpression of CBL-Interacting Protein Kinases 23 Improves Tolerance to Low-Nitrogen Stress in Potato Plants. Horticulturae 2024, 10, 526. https://doi.org/10.3390/horticulturae10050526

AMA Style

Huang F, Lu Y, Li Z, Zhang L, Xie M, Ren B, Lu L, Li L, Yang C. Overexpression of CBL-Interacting Protein Kinases 23 Improves Tolerance to Low-Nitrogen Stress in Potato Plants. Horticulturae. 2024; 10(5):526. https://doi.org/10.3390/horticulturae10050526

Chicago/Turabian Style

Huang, Feiyun, Yifei Lu, Zi Li, Lang Zhang, Minqiu Xie, Bi Ren, Liming Lu, Liqin Li, and Cuiqin Yang. 2024. "Overexpression of CBL-Interacting Protein Kinases 23 Improves Tolerance to Low-Nitrogen Stress in Potato Plants" Horticulturae 10, no. 5: 526. https://doi.org/10.3390/horticulturae10050526

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