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Article

Genome-Wide Identification, Characterization, and Expression of the HAK/KUP/KT Potassium Transporter Gene Family in Poncirus trifoliata and Functional Analysis of PtKUP10 under Salt Stress

by
Longfei Jin
1,
Xinxing Yin
2,
Mingxia Wen
1,
Bei Huang
1,
Feng Liu
1,
Xinguo Li
3 and
Peng Wang
1,*
1
Institute of Citrus Research, Zhejiang Academy of Agricultural Sciences, Taizhou 318026, China
2
Taizhou Vocational College of Science & Technology, Taizhou 318020, China
3
Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Department of Horticulture, School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(6), 628; https://doi.org/10.3390/horticulturae10060628
Submission received: 8 May 2024 / Revised: 6 June 2024 / Accepted: 7 June 2024 / Published: 12 June 2024
(This article belongs to the Topic Tolerance to Drought and Salt Stress in Plants, 2nd volume)
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Abstract

:
Potassium is an essential mineral nutrient for citrus growth and stress response. In this study, the HAK/KUP/KT gene family was identified from the genome of trifoliate orange (Poncirus trifoliata). The physical and chemical properties, chromosomal location, gene structure, evolutionary relationship, conserved motifs, and tissue expression characteristics were analyzed. The expression characteristics under low potassium and salt stress were analyzed by fluorescence quantitative PCR. The function of PtKUP10 was investigated by heterologous expression in Arabidopsis thaliana. The results showed that at least 18 PtKUPs were distributed in seven chromosomes. Phylogenetic analysis showed that four PtKUPs clustered in clade I, which mediated the high-affinity potassium absorption. Gene expression analysis showed that four PtKUPs were highly expressed in root, seven PtKUPs were up-regulated by low potassium stress, and nine PtKUPs were up-regulated by salt stress. The cis-acting elements on the promoter of PtKUPs were predominantly involved in stress and hormone responses. Overexpression of PtKUP10 in Arabidopsis thaliana could enhance salt tolerance by accumulating more potassium in the shoot and reducing sodium content in the shoots and roots. These results indicated that PtKUPs play important roles in potassium absorption and salt stress response, and PtKUP10 might enhance salt tolerance by maintaining potassium and sodium homeostasis.

1. Introduction

Potassium (K+), the most abundant cation in plants (K2O content: 0.3% to 5% of dry weight), is involved in many biological processes, such as photosynthesis, assimilation products transportation, protein synthesis, osmotic regulation, stomatal movement, enzyme activation, organic acid metabolism, and stress resistance [1]. An adequate supply of K+ is a guarantee of yield and quality. K+ deficiency (below 250 mM dry weight) causes severe growth defects and yield decline in many crops [2]. In addition, K+ application can enhance the crops’ tolerance to various abiotic and biotic stresses [3,4,5,6]. Due to the similar physical and chemical properties of K+ and Na+, salinity significantly impairs K+ availability to plants. Maintaining Na+ and K+ homeostasis can effectively alleviate the inhibition of salt stress on plants [7]. In addition, the application of K+ helps crops to overcome oxidative cell damage [8].
Salt stress is a main obstacle to the productivity and quality of crops. More than 20% of irrigated soils worldwide are affected by salt stress, which seriously affects the sustainability and profitability of agriculture [9]. The economic loss caused by agricultural salination is as high as USD 27.30 billion annually [10]. Excess sodium chloride in the soil restrains water and nutrient absorption of crops. Moreover, excess sodium (Na+) and chloride ions are also toxic to crop growth.
Plants’ K+ acquisition and transportation mainly depend on K+ channels and K+ transporters [1]. Under high external K+ concentrations (>0.5 mM), plants acquire K+ through channels, recognized as a low-affinity K+ transport system [1]. In contrast, under low external K+ concentration (<0.2 mM), plants absorb mainly by transporters, which is a high-affinity K+ absorption system [1]. According to current research, K+ transporters can be divided into four major categories: Trk/HKT-type, KT/HAK/KUP (K+ transporter/high-affinity K+ transporter/K+ uptake permease), CHX (cation/hydrogen exchanger), and KEA (K+ efflux antiporter) [8,11]. The KT/HAK/KUP is reported to be the largest K+ transporter gene family found in plants. Evolutionary analysis divides the KUP gene family into four subgroups, of which clade I mediates highaffinity K+ absorption, and clade II mediates low-affinity K+ absorption [11].
In Arabidopsis, AtHAK5 and AtAKT1 are the two major transporters for K+ uptake [12,13]. The expression of AtHAK5 was markedly up-regulated at 48 h, 96 h, and 7 d after K+ deprivation [14]. In addition, AtKUP7 also mediated K+ acquisition and translocation in Arabidopsis root under low K+ conditions [15]. In rice, OsHAK1 and OsAKT1 played key regulatory roles in K+ absorption and transportation [16]. Under low K+ treatment, OsHAK1 was observably induced in the root epidermises and steles, and shoot vascular bundles [16]. Overexpression of OsHAK1 in rice increased K+ uptake, while knockout of OsHAK1 reduced the K+ absorption rate and stunted root and shoot growth [16]. Numerous studies revealed that HAK/KUP/KT transporters could protect plants against salt stress. For example, overexpression of OsHAK1 enhanced the salt tolerance of transgenic rice by increasing K+ accumulation and maintaining a higher K+/Na+ ratio [16]. Ectopic expression of Pumpkin’s CmHKT1;1 in cucumber increased salt tolerance of shoots [17]. Overexpression of PeHKT1;1 improved salt tolerance of transgenic Populus by regulation of antioxidant systems [18].
Citrus is one of the most important fruit crops widely grown worldwide. K+ is pivotal in citrus growth and quality improvement [19]. In recent years, soil salinization has become more serious due to overfertilization and unreasonable irrigation in citrus cultivation [19]. Exploring K+ absorption genes and studying the molecular mechanism of absorption is the basis for improving citrus potassium utilization efficiency and alleviating adverse effects of salinity. The citrus HAK/KUP/KT genes structures and functions have scarcely been researched. Trifoliate orange (Poncirus trifoliata) is the most widely used citrus rootstock, possessing important traits for citrus production including early bearing, high yield, desirable fruit quality, dwarfing, and disease resistance. In this study, the HAK/KUP/KT gene family was identified from the trifoliate orange genome, and the phylogeny, conserved motifs, structures, chromosome distribution, and expression characteristic of HAK/KUP/KT genes in low K+ and salt stress treatments were analyzed. In addition, PtKUP10 was functionally characterized by heterogeneous expression in Arabidopsis.

2. Materials and Methods

2.1. Materials and Treatments

The trifoliate orange seeds were collected from the germplasm nursery of the Zhejiang Citrus Research Institute. 15-week-old seedlings were grown in a greenhouse using hydroponics. After 2 weeks of adaptive growth in a hydroponic environment (the solution contains NH4NO3 3.0 mM, Na2HPO4·12H2O 0.3mM, NaH2PO4·H2O 0.6 mM, KCl 1.0 mM, CaCl2·2H2O 2 mM, MgSO4·7H2O 2 mM, Fe-EDTA 50 μM, MnCl2·4H2O 9 μM, CuSO4·5H2O 0.3 μM, ZnSO4·7H2O 0.8 μM, H3BO3 4 μM, H2MoO4 0.01 μM, pH = 6.0, and the solution was ventilated for 20 min every 3 h). Low K+ and salt stress treatments were performed with slight adjustments based on previous research [20,21]. The normal K+ concentration was 1.0 mM, and the low K+ concentration was 0.1 mM. Salt stress: NaCl (60 mM) was added to the normal total nutrient solution. One treatment contains six seedlings, and each treatment was repeated three times. The 2 cm root tips were collected at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h after treatment, frozen in liquid nitrogen immediately, and stored at −80 °C for later use.

2.2. Bioinformatics Analysis

The known Arabidopsis and rice KUP/HAK/KT protein sequences were downloaded from TAIR (https://www.arabidopsis.org/, accessed on 29 August 2022) and the rice genome website (http://rice.uga.edu/, accessed on 29 August 2022), respectively. The annotation files, protein sequences, gene sequence, and gene RPKM value of trifoliate orange were downloaded from the citrus pan-genome to the breeding database (http://citrus.hzau.edu.cn/index.php, accessed on 14 May 2022). The KUP/HAK/KT protein sequences of Arabidopsis and rice were used as reference sequences for trifoliate orange KUP/HAK/KT gene identification. The candidate PtKUPs were identified based on a BLASTp search, based on the thresholds (score value ≥ 100 and e value ≤ 1 × 10−10) of previous studies [22]. The molecular weight (Mw), isoelectric point (pI), number of amino acids, and total average hydrophilicity of candidate PtKUPs were predicted by utilizing ExPASy (https://web.expasy.org/protparam/, accessed on 1 September 2022). The subcellular localization of PtKUPs was predicted using Cell-PLoc [23]. The exon number and chromosome localization of genes were analyzed using TBtool [24], and heat maps of tissue expression were drawn. The phylogenetic tree of KUP gene families among different plants was constructed using MEGA X software via a neighbor-joining method with 1000 bootstrap replicates [25]. The conservative motifs of PtKUPs were analyzed using the online tool MEME (http://alternate.memesuite.org/tools/meme, accessed on 1 September 2022) [26]. A 2.0 Kb fragment upstream region of the PtKUP gene was identified from the trifoliate orange genome sequences. Further, the cis-acting elements in the promoter regions of PtKUPs were predicted using Plantcare [27].

2.3. RNA Isolation and Fluorescence Quantitative Real-Time PCR Analysis

The total RNA was isolated from root tips using a Plant total RNA extraction kit (Takara, Dalian, China). The RNA quantity and quality were checked with spectrophotometry (Thermo Scientific NANODROP ONEC, Waltham, MA, USA) and agarose gel electrophoresis. The cDNA library was constructed using a PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara, Dalian, China). Fluorescence quantitative real-time PCR (qRT-PCR) reactions were performed in the CFX96 PCR PCR Detection System (Bio-rad, Hercules, CA, USA) using the SYBR Green PCR Master Mix (Takara, Dalian, China) as described previously [20]. The qRT-PCR primers of 18 PtKUP genes were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 26 July 2022) and are given in Supplementary Table S1. The citrus β-actin gene reported in previous research was used as an internal control [28]. The relative expression level was calculated by the 2−ΔΔCT method [29], and three biological experimental repeats were performed.

2.4. Transformation of Arabidopsis and Phenotypic Analysis

The coding region of PtKUP10 was amplified with gene-specific primers (Supplementary Table S1). The plasmid was digested with restriction endonuclease (Kpn I and Mlu) and ligated into pCAMBIA1300-35S-E9 vector with T4-ligase. Then the overexpressing vector was mobilized into Agrobacterium tumefaciens (strain EHA105) and transformed into Arabidopsis according to the method previously reported [30]. Positive transformants were obtained by screening with 0.1 mM hygromycin and PCR amplification. Two T3 generation homozygous plants and wild-type (WT) Arabidopsis were treated with or without 200 mM NaCl. After 30 days of treatment, the plants were harvested, and dry weight was measured. The shoots and root Na+ and K+ contents were measured by using atomic absorption spectrometry (Thermo Electron Corporation, Waltham, MA, USA) [31], and three biological experimental repeats were performed. The leaf’s chlorophyll content was determined by a SPAD-502 chlorophyll meter (Konica Minolta, Tokyo, Japan) [32], and six biological experimental repeats were performed. The leaf’s malondialdehyde (MDA) content was determined by using malondialdehyde assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions, and three biological experimental repeats were performed.

2.5. Data Analysis

All data are shown as mean ± standard error (SE). GraphPad Prism 8 software was used for plotting. The tests for normality and homogeneity of variances were analyzed using SPSS 13.0, and the results were given in Supplementary Table S2. The significant differences were analyzed using SPSS 13.0 with the Duncan test at the 0.05 level.

3. Results

3.1. Genome-Wide Identification of PtKUP Gene Family

A total of 18 KUP genes were identified and named PtKUP1 to PtKUP18. The PtKUP gene sequence information is given in Supplementary Table S3. The amino acid length of PtKUP proteins ranged from 467 to 1102. The predicted MW and pI values ranged from 52.66 to 110.51 kDa and 5.37 to 9.41 range, respectively. The total mean hydrophilicity ranged from −0.166 to 0.645, of which 2 were negative and 16 were positive. Chromosome localization analysis showed that PtKUPs were mainly located on chromosomes 2, 3, 4, 7, and 8 (Table 1). The number of exons ranges from 6 to 16, among which PtKUP8 and PtKUP16 have the largest number of exons (16) (Table 1). Subcellular localization analysis found that all PtKUPs were located on the plasma membrane (Table 1).

3.2. Phylogenetic Analyses of PtKUP Gene Family

To further clarify the phylogenetic relationship and function of PtKUP gene family members, a phylogenetic tree was constructed based on the alignment protein sequences of 13 Arabidopsis AtKUP/HAK/KTs, 27 rice OsHAKs, and 18 trifoliate orange PtKUPs. The KUP gene family was divided into four clades (I~IV). Clade I contained eight OsHAKs, four PtKUPs (PtKUP7, PtKUP10, PtKUP11 and PtKUP16) and AtHAK5; Clade II contained three AtKUPs, three OsHAKs, and four PtKUPs (PtKUP8, PtKUP9, PtKUP13 and PtKUP15). Clade III contained nine OsHAKs, four AtKUPs, and seven PtKUPs (PtKUP1, PtKUP2, PtKUP5, PtKUP6, PtKUP12, PtKUP14 and PtKUP17); Clade IV contained four OsHAKs and two PtKUPs (PtKUP3 and PtKUP18) (Figure 1).

3.3. Conserved Motifs Analysis of PtKUP Gene Family

A total of 10 conserved motifs were identified from the protein sequences of PtKUPs and named motif1~motif10. The detailed motif information is given in Supplementary Table S4. Among them, PtKUP2, PtKUP4, PtKUP6, PtKUP7, PtKUP10, PtKUP13, PtKUP14, PtKUP15, PtKUP16 and PtKUP17 contain 10 conserved motifs. All PtKUP gene family members contained motif1 and motif3 (Figure 2).

3.4. Expression Analysis of PtKUP Gene Family in Different Tissues

The expression levels of PtKUP gene family members varied greatly in different tissues of trifoliate orange. Among them, PtKUP1 and PtKUP17 were mainly highly expressed in leaves, PtKUP7, PtKUP9, PtKUP10, and PtKUP16 were mainly expressed in roots, and PtKUP3 and PtKUP12 were mainly expressed in flowers. PtKUP8 was mainly expressed in young fruits, PtKUP18 was mainly expressed in mature fruits, and PtKUP6 and PtKUP15 were mainly expressed in seeds (Figure 3).

3.5. Expression Analysis of PtKUP Gene Family under Low K+ and Salt Stress

The expression characteristics of 18 PtKUPs were analyzed by qRT-PCR at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h after low K+ treatment. It was found that seven PtKUPs were induced by low K+ stress. The expression level of PtKUP1 gradually increased under low K+ stress, peaked at 6 h after treatment, and then gradually decreased. The expressions of PtKUP3, PtKUP8, PtKUP10, PtKUP14, and PtKUP16 gradually increased under low potassium stress, peaked at 3 h after treatment, and then gradually decreased. The expression of PtKUP11 increased immediately after 1 h treatment with low K+ stress, and then gradually decreased (Figure 4).
The expression characteristics of 18 PtKUPs were analyzed by qRT-PCR after 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h of salt treatment. A total of nine genes were induced by salt stress. The expression levels of PtKUP1, PtKUP3, PtKUP7, PtKUP10, PtKUP14, and PtKUP17 peaked after 3 h of salt treatment and then gradually decreased. The expression level of PtKUP8 peaked after 24 h of salt treatment. The expression level of PtKUP9 peaked after 12 h of salt treatment and then decreased gradually. The expression level of PtKUP16 peaked after 6 h of salt treatment, and then its expression decreased gradually (Figure 5).

3.6. Promoter Component Analysis of PtKUP Gene Family

To predict the transcription features and functions of the PtKUP gene family, cis-acting elements in the 20 kb promoter of each gene were analyzed. A large number of cis-acting elements was identified in promoters of the PtKUP gene family, including 70 light-responsive elements, 9 auxin response elements, 31 abscisic acid response elements, 23 gibberellin response elements, 27 methyl jasmonate response elements, 12 salicylic acid response elements, 23 drought induction elements, 13 low-temperature response elements, 10 defense and stress response elements, and 46 anaerobic induction elements (Figure 6). These results suggest that PtKUPs are involved in plant development and stress response.

3.7. Functional Analysis of PtKUP10 under Salt Stress

To investigate the function of PtKUP10 under salt stress, the growth and physiological parameters of WT and two transgenic lines (PtKUP10-OE2 and PtKUP10-OE5) treated with or without 200 mM NaCl were measured. The phenotype of PtKUP10 transgenic Arabidopsis plants was not significantly different from that of the WT under control conditions. However, under salt stress, PtKUP10 transgenic Arabidopsis showed better growth compared to WT (Figure 7). Under salt stress, the dry weights of PtKUP10 transgenic plants were significantly higher than WT (Figure 8). Under control conditions, the Na+ and K+ contents, and the Na+/K+ ratio of shoots and roots among WT and two transgenic lines were not significantly different (Figure 8). Under salt stress, the Na+ content of shoots and roots in both WT and transgenic Arabidopsis was significantly increased, while the Na+ content of shoots and roots in transgenic Arabidopsis was significantly lower than in WT (Figure 8). Under salt stress, the K+ content of shoots and roots in WT and transgenic plants was significantly decreased, while the K+ content of shoots in transgenic plants was significantly lower than in WT (Figure 8). Under control conditions, the shoot and root Na+/K+ ratio between WT and PtKUP10 transgenic plants was not significantly different. However, the shoot and root Na+/K+ ratio in PtKUP10 transgenic plants was significantly lower than in WT, under salt stress (Figure 8). Furthermore, the chlorophyll content of leaves in PtKUP10 transgenic Arabidopsis was significantly higher than in WT, while the MDA content was significantly lower than in WT, under salt stress (Figure 8).

4. Discussion

K+ is an important macroelement for the growth and quality of citrus. Increasing K+ fertilizer can significantly increase the soluble solid content of fruit and reduce fruit cracking [19,33]. Overuse of K+ fertilizer caused magnesium absorption disorders in citrus [34]. Exploring the essential genes for K+ absorption and studying the molecular mechanism of K+ transport are crucial for enhancing K+ utilization in citrus. Moreover, maintaining the balance of K+ and Na+ is an important strategy for enhancing the salt tolerance of crops. Citrus is one of the most salt-sensitive species, and excess soil salinity leads to a sharp decline in citrus yield [20,35]. Thus far, the HAK/KUP/KT family has been identified in many plants, including Arabidopsis [36], rice [8], maize [37], wheat [21], cassava [3], bamboo [38], tea [39], pear [40]. The gene and protein structures, phylogenetic features, gene expression characteristics, and functions of some KUP gene families have already been well characterized.
In this study, 18 members of the PtKUP gene family were excavated from the genome of trifoliate orange. Evolutionary analysis revealed that the KUP genes of plants were categorized into four evolutionary clusters. Increasing evidence suggests that KUP gene members in evolutionary cluster I were involved in high-affinity absorption of K+, and evolutionary cluster II mediated low-affinity absorption of K+ [8,9,41]. In this research, 18 gene family members were categorized into four evolutionary clusters, and four PtKUPs (PtKUP7, PtKUP10, PtKUP11, and PtKUP16) were in cluster I, indicating that these four genes might be involved in the high-affinity K+ absorption of trifoliate orange. Gene structure analysis showed that PtKUPs contained 6–16 exons, which was consistent with studies in Arabidopsis, rice, tea tree, and cassava [8,39], indicating that the KUP gene family is highly conserved in plants. The “K_trans” superfamily (cl15781) is the conserved domain model of K+ transporter across phyla. In this research, ten motifs in PtKUPs belonging to the “K_trans” superfamily were identified in trifoliate orange. Although the conserved motifs of different gene families were not completely consistent, they all contained motifs 1 and 3, indicating that these two motifs might be the core motifs for maintaining the K+ transport function in citrus. A large number of studies have found that the KUP gene is expressed in various tissues and organs of plants, and plays an important role in the absorption, transport, and utilization of K+. For example, five HAK genes (HAK2, HAK10, HAK15, HAK23, and HAK25) were expressed in all tissues of the three rice varieties [8], and CsHAK3 was consistently highly expressed in eight tissues of the tea tree [39]. In this study, PtKUP7, PtKUP9, PtKUP10, and PtKUP16 were highly expressed in the roots, which indicated that these four PtKUPs were involved in K+ absorption from soil to roots in trifoliate orange.
Transcriptional regulation plays a key role in helping plants cope with low K+ stress. For example, AtHAK5 was significantly induced under low K+ stress in Arabidopsis [16,42]. Studies in rice also showed that the expression levels of OsHAK1 and OsHAK5 are induced by low K+ stress, which mediates the absorption and transport of K+ in both low and high K+ environments [16,43]. The expression levels of PbHAK2 and PbHAK12 were rapidly up-regulated under K+ deficiency in pear [40]. In this study, seven PtKUP genes were induced by low K+ stress. Among these, PtKUP10 and PtKUP16 were predominantly expressed in the roots, indicating their crucial roles in responding to low K+ stress. The expression levels of PtKUP1 and PtKUP17 in leaves were significantly higher than those in other tissues, suggesting that these two genes may play important regulatory roles in leaf K+ absorption. A large number of studies have found that HAK/KUP/KT genes are involved in plant responses to abiotic stress [3]. For example, MeKUPs were up-regulated by low temperature, drought, salt, and oxidative stress in cassava [3], and the expression level of CsHAKs in tea tree was induced by drought and salt stress [39]. Studies in pear showed that PbHAK expression was induced by low temperature, salt, and drought stress [40]. Studies in rice found that OsHAK16 maintained K+ and Na+ balance and enhanced tolerance to salt stress by transporting K+ to the shoots [44]. In this study, the expression of nine PtKUPs was induced by salt stress, suggesting that PtKUPs may also be involved in the response of trifoliate orange to salt by mediating K+ transport. Meanwhile, a large number of cis-acting elements related to drought, low temperature, and ABA response were also identified on the promoter of PtKUPs, which may be the main reason for the salt stress-induced expression of PtKUPs.
In the long process of evolution, plants have also obtained a series of mechanisms for salt adaptation, including morphological [45], physiological [46], and molecular biological mechanisms [47]. Under salt stress, the plant root structure can be modified, such as increasing periderm cell diameter which excludes Na+ from the stele and compartmentalizing Na+ into a vacuole or cell wall [45,48,49]. Plants can also enhance salt tolerance by balancing homeostasis between different ions. High soil salinity usually inhibits the K+ uptake of crops, leading to K+ deficiency [50]. Thus, it is essential to promote plants’ K+ uptake under salt stress to maintain Na+/K+ homeostasis and improve salt tolerance. One effective strategy for enhancing K+ acquisition under high-salinity conditions is to increase the expression of K+ transporter genes [51]. The shoot Na+/K+ ratio is commonly used to evaluate a plant’s salt tolerance, with a lower shoot Na+/K+ ratio normally indicating a better salinity tolerance [52,53]. Compared with the WT, transgenic Arabidopsis plants which overexpress PtKUP10 accumulated more K+ and less Na+ under salt stress. Consequently, in our study, the Na+/K+ ratio in the shoots and roots of transgenic plants was also significantly lower than in WT. Similar results were observed in transgenic Arabidopsis plants overexpressing the rice OsHAK1 and cassava MeHAK5 [9,16]. Moreover, the higher chlorophyll contents in PtKUP10 transgenic Arabidopsis plants indicated that the transgenic plants had higher photosynthetic efficiency than WT. The lower MDA content in PtKUP10 transgenic Arabidopsis suggested that the membranes were more effectively protected from salt stress than WT plants. According to the expression characteristic of 18 PtKUPs and the functional analysis of PtKUP10, a schematic model was proposed (Figure 9). PtKUP1 and PtKUP17 might play important roles in the transportation of K+ from roots to leaves. PtKUP8 and PtKUP18 might play important roles in the transportation of K+ from roots to fruits. Under low K+ conditions, PtKUP10 and PtKUP16 were induced in roots to increase K+ uptake, and under salt stress, PtKUP9, PtKUP10, and PtKUP16 were induced in roots to increase K+ uptake and inhibit Na+ transport to the shoot. Overall, these results indicate that PtKUP10 can enhance salt tolerance by maintaining K+/Na+ homeostasis, which might be used as a candidate gene for salt-tolerant citrus breeding in the future. In citrus production, increased application of potassium fertilizer might alleviate the harm of soil salinization.

5. Conclusions

In this study, 18 members of the PtKUP gene family were identified from the trifoliate orange genome using bioinformatics methods. Phylogenetic analysis showed that PtKUP7, PtKUP10, PtKUP11, and PtKUP16 clustered in clade I, which mediated high-affinity K+ absorption. Gene expression analysis showed that seven members of the PtKUP gene family were induced by low K+ stress, and nine members of the PtKUP gene family were induced by salt stress. These results indicated that the PtKUP gene family was involved in the absorption of K+ and response to salt stress. Furthermore, overexpression of PtKUP10 in transgenic Arabidopsis improved the salt tolerance of transgenic plants by decreasing the Na+/K+ ratio. Therefore, PtKUP10 could be a candidate gene for salt-tolerant citrus breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10060628/s1, Table S1: List of primers used in this study. Table S2: The tests for normality and homogeneity of variances. Table S3: The cDNA and protein sequence of the PtKUP gene family. Table S4: Conserved motifs identified from the PtKUP gene family.

Author Contributions

Conceptualization, L.J., methodology, L.J. and X.Y., investigation, M.W., B.H. and F.L., data curation, L.J. and X.Y., writing—original draft preparation, L.J.; writing—review and editing, P.W. and X.L.; supervision, P.W. and X.L.; funding acquisition, P.W. and L.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the “San Nong Jiu Fang” Science and Technology Cooperation Plan of Zhejiang Province (2023SNJF014) and the Open Project of Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province (HNZDSYS (23)-4).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of KUP/HAK/KT family proteins among trifoliate orange, rice, and Arabidopsis. The red circles represent trifoliate orange. The blue squares represent Arabidopsis. The green triangle represents rice. Different color lines represent four clades (I, II, III, IV) of the KUP/HAK/KT family.
Figure 1. Phylogenetic tree of KUP/HAK/KT family proteins among trifoliate orange, rice, and Arabidopsis. The red circles represent trifoliate orange. The blue squares represent Arabidopsis. The green triangle represents rice. Different color lines represent four clades (I, II, III, IV) of the KUP/HAK/KT family.
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Figure 2. Conserved protein motifs of PtKUPs. Ten motifs were identified in PtKUP proteins and marked by different colors.
Figure 2. Conserved protein motifs of PtKUPs. Ten motifs were identified in PtKUP proteins and marked by different colors.
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Figure 3. The heatmap of the PtKUP gene family in different tissues.
Figure 3. The heatmap of the PtKUP gene family in different tissues.
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Figure 4. The expression characteristics of the PtKUP gene family under K+ deficiency. The bars represent SE (n = 3). The bars of different colors represent different hours after low K+ treatment. Different lowercase letters on the bar indicate significant differences (p < 0.05) among treatments.
Figure 4. The expression characteristics of the PtKUP gene family under K+ deficiency. The bars represent SE (n = 3). The bars of different colors represent different hours after low K+ treatment. Different lowercase letters on the bar indicate significant differences (p < 0.05) among treatments.
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Figure 5. The expression characteristics of the PtKUP gene family under salt stress. The bars represent SE (n = 3). The bars of different colors represent different hours after salt treatment. Different lowercase letters on the bar indicate significant differences (p < 0.05) among treatments.
Figure 5. The expression characteristics of the PtKUP gene family under salt stress. The bars represent SE (n = 3). The bars of different colors represent different hours after salt treatment. Different lowercase letters on the bar indicate significant differences (p < 0.05) among treatments.
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Figure 6. Analysis of the cis-acting elements present in the promoter regions of PtKUPs.
Figure 6. Analysis of the cis-acting elements present in the promoter regions of PtKUPs.
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Figure 7. Performance of WT and PtKUP10 transgenic Arabidopsis under control and salt stress.
Figure 7. Performance of WT and PtKUP10 transgenic Arabidopsis under control and salt stress.
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Figure 8. Growth and physiological parameters of WT and PtKUP10 transgenic Arabidopsis under control and salt stress. The black bars represent control, and the gray bars represent salt treatment (200 mM NaCl). The bars represent SE (Chl contents, n = 6, the others, n = 3). Different lowercase letters on the bar indicate significant differences (p < 0.05) among treatments.
Figure 8. Growth and physiological parameters of WT and PtKUP10 transgenic Arabidopsis under control and salt stress. The black bars represent control, and the gray bars represent salt treatment (200 mM NaCl). The bars represent SE (Chl contents, n = 6, the others, n = 3). Different lowercase letters on the bar indicate significant differences (p < 0.05) among treatments.
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Figure 9. A Schematic model for the proposed roles of PtKUPs based on gene expression profiles in citrus. In the roots, PtKUP7, PtKUP9, PtKUP10, and PtKUP16 were the main expressed genes. In the leaves, PtKUP1 and PtKUP17 were the main expressed genes. In the fruit, PtKUP8 and PtKUP18 were the main expressed genes. Under low potassium stress, PtKUP10 and PtKUP16 were up-regulated in roots to increase potassium acquisition. Under salt stress, PtKUP9, PtKUP10, and PtKUP16 were up-regulated in roots to enhance salt tolerance by maintaining K+/Na+ homeostasis. The heatmap showed gene expression level at 0 h, 1 h, 3 h, 6 h,12 h and 24 h after salt or low K+ treatment.
Figure 9. A Schematic model for the proposed roles of PtKUPs based on gene expression profiles in citrus. In the roots, PtKUP7, PtKUP9, PtKUP10, and PtKUP16 were the main expressed genes. In the leaves, PtKUP1 and PtKUP17 were the main expressed genes. In the fruit, PtKUP8 and PtKUP18 were the main expressed genes. Under low potassium stress, PtKUP10 and PtKUP16 were up-regulated in roots to increase potassium acquisition. Under salt stress, PtKUP9, PtKUP10, and PtKUP16 were up-regulated in roots to enhance salt tolerance by maintaining K+/Na+ homeostasis. The heatmap showed gene expression level at 0 h, 1 h, 3 h, 6 h,12 h and 24 h after salt or low K+ treatment.
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Table 1. Physicochemical properties of PtKUP gene family.
Table 1. Physicochemical properties of PtKUP gene family.
GeneAccessionAA No.MW (kD)pIGravyChromosome LocationExons No.Subcellular Localization
PtKUP1Pt2g007790.1975109.667.81−0.147211plasmalemma
PtKUP2Pt2g009280.178387.368.410.43229plasmalemma
PtKUP3Pt2g017510.152059.268.260.36926plasmalemma
PtKUP4Pt2g021520.179288.488.380.36928plasmalemma
PtKUP5Pt2g022230.158064.199.000.645210plasmalemma
PtKUP6Pt3g002490.177687.289.350.31838plasmalemma
PtKUP7Pt3g033530.177887.266.880.24938plasmalemma
PtKUP8Pt3g033540.182593.328.90−0.166316plasmalemma
PtKUP9Pt3g037370.146752.669.410.38337plasmalemma
PtKUP10Pt3g037380.177887.206.670.25438plasmalemma
PtKUP11Pt4g001420.163069.656.680.25449plasmalemma
PtKUP12Pt4g004240.161668.818.780.45847plasmalemma
PtKUP13Pt4g004250.183993.015.990.38549plasmalemma
PtKUP14Pt4g020610.179388.497.590.36649plasmalemma
PtKUP15Pt7g001340.184593.855.370.305710plasmalemma
PtKUP16Pt7g012630.1962107.358.850.195710plasmalemma
PtKUP17Pt8g012100.11002110.516.460.230816plasmalemma
PtKUP18PtUn023130.173181.778.690.363Un8plasmalemma
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Jin, L.; Yin, X.; Wen, M.; Huang, B.; Liu, F.; Li, X.; Wang, P. Genome-Wide Identification, Characterization, and Expression of the HAK/KUP/KT Potassium Transporter Gene Family in Poncirus trifoliata and Functional Analysis of PtKUP10 under Salt Stress. Horticulturae 2024, 10, 628. https://doi.org/10.3390/horticulturae10060628

AMA Style

Jin L, Yin X, Wen M, Huang B, Liu F, Li X, Wang P. Genome-Wide Identification, Characterization, and Expression of the HAK/KUP/KT Potassium Transporter Gene Family in Poncirus trifoliata and Functional Analysis of PtKUP10 under Salt Stress. Horticulturae. 2024; 10(6):628. https://doi.org/10.3390/horticulturae10060628

Chicago/Turabian Style

Jin, Longfei, Xinxing Yin, Mingxia Wen, Bei Huang, Feng Liu, Xinguo Li, and Peng Wang. 2024. "Genome-Wide Identification, Characterization, and Expression of the HAK/KUP/KT Potassium Transporter Gene Family in Poncirus trifoliata and Functional Analysis of PtKUP10 under Salt Stress" Horticulturae 10, no. 6: 628. https://doi.org/10.3390/horticulturae10060628

APA Style

Jin, L., Yin, X., Wen, M., Huang, B., Liu, F., Li, X., & Wang, P. (2024). Genome-Wide Identification, Characterization, and Expression of the HAK/KUP/KT Potassium Transporter Gene Family in Poncirus trifoliata and Functional Analysis of PtKUP10 under Salt Stress. Horticulturae, 10(6), 628. https://doi.org/10.3390/horticulturae10060628

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