
==== Front
BMC Genomics
BMC Genomics
BMC Genomics
1471-2164
BioMed Central London

39237905
10712
10.1186/s12864-024-10712-5
Research
Genome-wide survey of KT/HAK/KUP genes in the genus Citrullus and analysis of their involvement in K+-deficiency and drought stress responses in between C. lanatus and C. amarus
Cheng Rui 123
Zhao Zhengxiang 12
Tang Yan 4
Gu Yan 3
Chen Guodong 4
Sun Yudong sunyudong@jaas.ac.cn

3
Wang Xuezheng wangxuezheng@neau.edu.cn

12
1 https://ror.org/0515nd386 grid.412243.2 0000 0004 1760 1136 College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin, Heilongjiang 150006 China
2 https://ror.org/05ckt8b96 grid.418524.e 0000 0004 0369 6250 Key Laboratory of Biology and Genetic Improvement of Horticulture Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, Harbin, Heilongjiang 150006 China
3 Huaiyin Institute of Agricultural Sciences of Xuhuai Region in Jiangsu, Huai’an, Jiangsu 223001 China
4 https://ror.org/0555ezg60 grid.417678.b 0000 0004 1800 1941 Huaiyin Institute of Technology, Huai’an, Jiangsu 223003 China
5 9 2024
5 9 2024
2024
25 83615 6 2024
14 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

The KT/HAK/KUP is the largest K+ transporter family in plants, playing crucial roles in K+ absorption, transport, and defense against environmental stress. Sweet watermelon is an economically significant horticultural crop belonging to the genus Citrullus, with a high demand for K+ during its growth process. However, a comprehensive analysis of the KT/HAK/KUP gene family in watermelon has not been reported.

Results

14 KT/HAK/KUP genes were identified in the genomes of each of seven Citrullus species. These KT/HAK/KUPs in watermelon were unevenly distributed across seven chromosomes. Segmental duplication is the primary driving force behind the expansion of the KT/HAK/KUP family, subjected to purifying selection during domestication (Ka/Ks < 1), and all KT/HAK/KUPs exhibit conserved motifs and could be phylogenetically classified into four groups. The promoters of KT/HAK/KUPs contain numerous cis-regulatory elements related to plant growth and development, phytohormone response, and stress response. Under K+ deficiency, the growth of watermelon seedlings was significantly inhibited, with cultivated watermelon experiencing greater impacts (canopy width, redox enzyme activity) compared to the wild type. All KT/HAK/KUPs in C. lanatus and C. amarus exhibit specific expression responses to K+-deficiency and drought stress by qRT-PCR. Notably, ClG42_07g0120700/CaPI482276_07g014010 were predominantly expressed in roots and were further induced by K+-deficiency and drought stress. Additionally, the K+ transport capacity of ClG42_07g0120700 under low K+ stress was confirmed by yeast functional complementation assay.

Conclusions

KT/HAK/KUP genes in watermelon were systematically identified and analyzed at the pangenome level and provide a foundation for understanding the classification and functions of the KT/HAK/KUPs in watermelon plants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-024-10712-5.

Keywords

Watermelon
Pangenome gene family
K+ transport
Gene expression
Huai’an Natural Science Research ProjectHABL202228 Seed Industry Vitalization Research Projects of Jiangsu ProvinceJBGS[2021]072 China Agriculture Research System of MOF and MARACARS-25 issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

K+ is an essential mineral for plant growth and development, and contributes to multiple physiological and metabolic processes, such as abiotic stress adaptation, stomatal movement, enzyme function, and signal transduction [1], and contributes up to 10% of the total plant biomass [2]. The plants absorbed and transported K+ primarily occurs through K+ channels or transporters [3, 4]. The KT/HAK/KUP family is the earliest identified and most abundant family of K+ transporters in plants [5], which functions in acquiring, transporting, and distributing K+, and maintaining internal ion homeostasis [6], they were also reported which play an essential role in enhancing plant tolerance to diverse abiotic stresses, including salt [5] and drought [7]. KT/HAK/KUP genes generally contain 10 to 15 transmembrane regions with a long loop in the second to third transmembrane region. and essential conserved ‘K+-trans’ domain (PF02705) [8, 9].

The KT/HAK/KUP gene family has been extensively investigated in many plants [10–15]. The number of KT/HAK/KUP genes varies among species, ranging from 13 to 56, with the lowest number in Arabidopsis (13) [10] and the highest number in wheat (56) [15]. Previous studies have shown that HAK/KUP/KT gene family divided into four groups: I, II, III, and IV [11–15], and each group has a conserved structure and function. The HAK/KUP/KTs in group I were mainly expressed in roots and are involved in the K+ uptake and transport from the environment by plant roots, and the HAK/KUP/KTs in group II were mainly involved in physiological processes such as plant growth regulation, while the functions of group III and IV were rarely reported [16, 17]. The expression patterns and functions of KT/HAK/KUP genes have also been reported in many plants. In Arabidopsis, most AtKT/HAK/KUPs are expressed in roots, siliques, leaves, and flowers [10]. The AtKUP4 is related to cell expansion of root hairs [18], AtHAK5 and AtKT1 are important transporter involved in K+ uptake in root system, and AtHAK5 is highly upregulated after K+ starvation (< 10 µM) [19], and AtKUP7 is also confirmed to be involved in K+ uptake and upregulated under K+ deficiency [20]. In rice, overexpressing OsHAK1 or OsHAK5 increases K+ uptake under K+ starvation, and they are all mainly expressed in roots, where they mediate K+ uptake and K+ translocation [21, 22], and a new study shows that OsHAK5 also involved in alters rice architecture via ATP-dependent transmembrane auxin fluxes [23]. In barley, HvHAK4 is involved in K+ uptake and translocation to leaves, where it ensures a high K+ concentration in chloroplasts [24, 25]. In addition, the KT/HAK/KUP genes in response to different abiotic stresses have also been investigated in many plant species [26–31]. For example, AtHAK5 plays a crucial role in enhancing salt tolerance in Arabidopsis by maintaining K+ homeostasis under high Na+ concentrations [32, 33], AtKUP4 is considered a critical link between root hair development and environmental signaling, knockout of AtKUP4 disrupts auxin distribution in root tips, highlighting its essential role in maintaining auxin balance during plant adaptation to environmental conditions [18, 34].

Sweet watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is one of the most popular fruit crops grown throughout the world [35, 36], which have a high demand for K+, especially during the fruit enlargement and maturation stage [37], while K+ insufficient is one of the major challenges to plant growth, and fruit yield and quality [6]. However, the functions of KT/HAK/KUPs in watermelon are largely unknown. Fortunately, the pangenome sequence of watermelon, which includes C.amarus (CA), C.ecirrhosus (CE), C.mucosospermus (CM), C.naudinianus (CN), C.rehmii (CR), and C.colocynthis (CC) alongside C.lanatus (CL), has been successfully assembled and made available (http://watermelondb.cn) [38]. This provides a foundation for the systematic analysis of the gene family functions of watermelons at the pangenome level. In this study, we identified KT/HAK/KUP family genes in seven Citrullus species and systematically analyzed their structures, conserved motifs, cis-regulatory elements, phylogenetic and collinear relationships. Moreover, the expression patterns of KT/HAK/KUPs under K+ starvation and drought stress during the vegetative growth of wild- and cultivated-type watermelon were also investigated. This work provides new insights for further functional studies of vegetative growth and stress-responsive of KT/HAK/KUPs in watermelon.

Materials and methods

Plant materials, growth conditions and treatments

The study was conducted in Huai’an (33.62°N, 119.02°E), China, using a cultivated watermelon breeding line G42 (C. lanatus var G42, developed by the Huaiyin Institute of Agricultural Sciences of Xuhuai Region in Jiangsu) and a wild watermelon accession PI 482276 (C. amarus var PI 482276, obtained from the USDA-ARS watermelon germplasm). G42 is a new germplasm of an excellent homozygous watermelon inbred line, with agronomic traits almost similar to cultivated varieties. It is the parent of popular watermelon varieties in China and represents a commercial variety to a certain extent. PI 482276 has excellent resistance to stress and disease and is an important raw material for cultivating disease-resistant varieties. The selected experimental materials have not only been widely used in watermelon breeding, but have also completed T2T gapless genome assembly, which is of great value for studying important agronomic traits and intermediate differences in watermelon. The seeds were sourced from the Huaiyin Institute of Agricultural Sciences of Xuhuai Region in Jiangsu. The watermelon seeds were disinfected and then wrapped in moist towels, and incubated at a constant temperature of 30 °C until germination. The germinated seeds were sowed in 72 cell trays filled with PINDSTRUP SUBSTRATE peat (Pindstrup Mosebrug A/S, Denmark) and covered with a layer of vermiculite, and the seedlings were irrigated with a 2‰ nutrient solution from the “Zhonghua Yangtian” series (Sinofert, China). The seedlings were grown in greenhouse with the environmental conditions until the seedlings reached the two-leaf stage.

To explore the effects of K+ starvation on the vegetative growth of watermelon and the expression of KT/HAK/KUPs in watermelon roots, 40 seedlings of each genotype were selected. The root substrate was carefully washed with water for hydroponic culture. Hydroponic growth containers were constructed from opaque blue plastic boxes (60 cm × 40 cm × 15 cm). Twenty holes, each 3 cm in diameter, were drilled in polystyrene foam plates used as lids. Each hole was intended to serve as a support for a single seedling surrounded by sponges. And then, each box was filled with 20 L of modified Hoagland’s solution (K+-Nor) with ample K+ supply. After 3 days, half of the seedlings (20) of each genotype were transferred to a nutrient solution without K+ (lacking KNO3 and adding 6 mM NaNO3 to maintain the concentration of N stabilization) for treatment of K+ deficiency (K+-Def), while the other half (20 seedlings) of each genotype remained in the normal K+ nutrient solution as control (K+-Nor). The nutrient solutions were aerated continuously by an air pump. After 15 days of K+ starvation treatment, roots, stems, and leaves of both control and treated seedlings were collected for growth index assessment, antioxidant enzyme activity measurement, and determination of K+ content. The analyses of enzyme activity and K+ content by Nanjing Ruiyuan Biotechnology Co., Ltd (Nanjing, China) using specialized assay kits. And roots collected and flash-frozen in liquid nitrogen to analysis KT/HAK/KUP gene expression in watermelon.

To explore the involvement of watermelon KT/HAK/KUPs response to drought stress in watermelons, 30 seedlings of each genotype were selected, and one seedling was transplanted per pot (16 cm × 13 cm × 18 cm, 1.5 L) filled with Profile Porous Ceramic substrate (Profile Products LLC, USA) in a semi-controlled greenhouse. All plants were irrigated with a 2‰ nutrient solution. After one week of cultivation, a single oversaturation irrigation treatment was performed. Subsequently, drought stress (Drought) was induced by withholding water. Normal irrigation was maintained as the control (CK), with each treatment involving 10 plants. Soil moisture sensors (Renke Control Technology Co., Ltd., Shandong, China) were inserted at a depth of 10 cm and connected to data loggers to record and monitor the percent volumetric water content (VWC) per pot at noon each day. The CK pots were regularly watered to maintain a VWC of 20 ± 5%. Conversely, for the drought treatment, after withholding water for 14 days, the plants reached the drought stress treatment level VWC below 10%, as indicated by VWC data collected daily from the data loggers. Roots collected at 17 days (when wilted phenotypes were apparent) after induction of drought stress were wrapped in aluminum foil and flash-frozen in liquid nitrogen to analyze watermelon KT/HAK/KUP gene expression.

Identification of KT/HAK/KUP genes in watermelon pangenome

In this study, all the sequences of watermelon genome (seven species) were downloaded from the Watermelon Database (http://watermelondb.cn/#/map). Candidate KT/HAK/KUP genes were searched against known AtKT/HAK/KUP protein sequences (downloaded from TAIR, https://www.arabidopsis.org/) using the BLASTP program (e-value ≤ 10 − 5). Hidden Markov Model (HMM) profiles of the K+ transporter domain (PF02705) of the KT/HAK/KUP gene family was downloaded from the PFAM database (http://pfam.xfam.org) for search by HMMER3.0. All candidate sequences were further examined using the NCBI-CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to confirm the presence of the K+ transporter domains.

Phylogenetic and conserved motifs analysis of watermelon KT/HAK/KUPs

The full-length amino acid sequences were extracted based on the identified KT/HAK/KUP genes ID from Watermelon Database (http://watermelondb.cn/#/map), and an evolutionary tree was constructed along with the 13 AtKT/HAK/KUPs from Arabidopsis and 27 OsKT/HAK/KUPs from rice (download from RGAP, http://rice.plantbiology.msu.edu/). All protein sequences were aligned by ClustalW software with the default parameters, and an unrooted neighbor-joining (NJ) phylogenetic tree was constructed by MEGA11 software with a bootstrap method of 1,000. The MEME online tool (http://meme-suite.org/tools/meme) was used to identify the motif sequence. And the phylogenetic tree and conserved motifs displayed by tvBOT software (https://www.chiplot.online/tvbot.html) [39].

Genome collinearity analysis and Ka/Ks, 4DTv calculated

The chromosomal localization information of watermelon KT/HAK/KUPs were obtained from genome gff3 files. All protein sequences and gff3 files of seven watermelon species were combined to analysis the genome collinearity by MCScanX software [40] with the default settings, and then displayed by Circos software [41]. For each pair of paralogs, KaKs_Calculator 2.0 [42] was employed to calculate the non-synonymous substitution rate (Ka), synonymous substitution rate (Ks), and corresponding ratio (Ka/Ks). The 4-fold synonymous third codon transversion (4DTv) value calculated by an in-house Perl script.

Physicochemical properties of KT/HAK/KUPs and cis-regulatory elements, subcellular localization predicted

The molecular weight, isoelectric point, and amino acid number of the candidate sequences were predicted by ProtParam (http://web.expasy.org/protparam/). The transmembrane structure was analyzed using the TMHMM online tool (http://www.cbs.dtu.dk/services/TMHMM/). The upstream 2,000 bp regions of the candidate genes were extracted, and PlantCARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to identify the cis-regulatory elements of the promoter region of all candidate genes and displayed by ggplot2. Furthermore, Cell-PLoc 2.0 (www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2) and WOLF PSORT ProtParam tool (https://wolfpsort.hgc.jp/) were used to predict the subcellular localization of the proteins encoded by KT/HAK/KUPs in watermelons.

Gene expression analysis using qRT-PCR

Total RNA was extracted from watermelon seedlings using the VeZol-Pure Total RNA Isolation Kit (Vazyme, Nanjing, China). This RNA was then used to synthesize cDNA with the HiScript II Q Select RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme, Nanjing, China), following the manufacturer’s instructions for all operations. The expression analysis of KT/HAK/KUPs was detected by qRT-PCR using the SYBR GREEN method with ClACTIN (ClG42_02g0007100). The fragments were amplified using specific primers that were designed by Primer Premier 6.0 (Supplemental Table S1). Three replicates were set up in each reaction. The relative expression levels of genes were calculated with 2−ΔΔCT methods [43].

Yeast complementation assay

The function of ClG42_07g0120700 was analyzed in the R5421 strain (Coolaber Technology Co., Ltd., Beijing, China), a K+-uptake-defective strain of Saccharomyces cerevisiae [44], following transformation with a pYES2.0 vector containing either the ClG42_07g0120700 gene or an empty vector following the manufacturer’s instructions. The pYES2.0-ClG42_07g0120700 vector was constructed by amplifying the ORF of the ClG42_07g0120700 gene using forward and reverse primers, which contain BamHI and XbaI restriction sites at their 5’ ends (Supplemental Table S1). Transformed R5421 strains were cultured on AP medium (Coolaber Technology Co., Ltd., Beijing, China) containing 50 mM K+ at 30 °C for 4–7 days. Positive clones were then selected and grown in liquid AP medium (with K+) until reaching OD600 = 1.0. The cultures were centrifuged at 600 rpm (× 41 g), the supernatants were removed, and washed with saline buffer [45]. The transformed yeast cells were serially diluted to 1:10, 1:100, and 1:1,000, and 4 µL aliquots were incubated in AP medium with varying K+ concentrations at 30 °C in the dark for 5–7 days.

Results

Identification and physicochemical analysis of KT/HAK/KUP gene family in Citrullus

The genomic information for seven watermelons, including C. amarus (CA), C. ecirrhosus (CE), C. mucosospermus (CM), C. naudinianus (CN), C. rehmii (CR), C. colocynthis (CC), and C. lanatus (CL), was obtained from the Watermelon Database (http://watermelondb.cn). For each species, a representative variety was selected, with genome sizes ranging from 369.32 to 420.61 Mb. Using BLASTP and HMM, along with NCBI-CDD and SMART, we conducted a comprehensive screening of the pangenome for KT/HAK/KUP candidate gene family members in watermelon (Citrullus). This analysis ultimately identified 14 KT/HAK/KUPs in each species (Table 1). The watermelon KT/HAK/KUP protein sequences lengths ranging from 720 (CE, CePI673135_09g005910) to 1801 (CN, CnPI596694_10g010300) residues, molecular weights ranging from 81.11 to 195.84 kDa, the number of transmembrane structures predicted from 8 (CE, CePI673135_10g009350) to 16 (CN, CnPI596694_10g010300), the isoelectric points ranging from 5.47 (CR, CrPI670011_10g010910) to 9.17 (CA, CaPI482276_07g014010), and all KT/HAK/KUPs of Citrullus exhibit hydrophilicity except CrPI670011_10g015820 in C.rehmii as predicted by ProtParam. Despite some variations in sequence length among individual KT/HAK/KUP genes in wild-type watermelons, all candidate KT/HAK/KUPs have complete conserved domains and conform to the characteristics of the KT/HAK/KUP family. Notably, subcellular localization analysis revealed that KT/HAK/KUPs of Citrullus were primarily located in the plasma membrane, endoplasmic reticulum, vacuoles (Supplemental Table S2).

Chromosomal localization and phylogenetic analysis of watermelon KT/HAK/KUPs

Based on the chromosomal localization information, the 14 KT/HAK/KUP genes in each watermelon species are predominantly distributed across specific chromosomes. The main concentrations are on chromosome 3 (3 genes), chromosome 5 (2 genes), chromosome 7 (2 genes), chromosome 9 (3 genes), chromosome 10 (3 genes), and chromosome 11 (1 gene). Notably, variations are observed in CR and CN. In CR, there is one KT/HAK/KUP gene on chromosome 1, accompanied by a reduction of one gene on chromosome 9 (2 KT/HAK/KUPs). In CN, there are only 2 KT/HAK/KUP genes on chromosome 3, with an additional KT/HAK/KUP gene on chromosome 9 (4 KT/HAK/KUPs) (Supplemental Table S2).

The phylogenetic tree was constructed using the full-length KT/HAK/KUP proteins obtained from watermelons, Arabidopsis (13 proteins), and rice (27 proteins). The analysis revealed four major groups, designated as I, II, III, and IV, based on the classification of KT/HAK/KUP genes in Arabidopsis [16] (Fig. 1). The distribution of KT/HAK/KUP genes across the phylogenetic tree of the seven watermelons showed remarkable consistency. Each species harbored 4 genes in group I, 6 in group II, 2 in group III, and 2 in group IV. Notably, group II and group I had the highest representation among watermelon KT/HAK/KUPs, accounting for 71.43%.

Fig. 1 Phylogenetic tree and motifs analysis of KT/HAK/KUP proteins sequences in Citrullus, Arabidopsis and rice. Classification of KT/HAK/KUP gene family based on phylogenetic tree. 98 watermelon KT/HAK/KUP genes, 13 AtKT/HAK/KUP genes, 27 OsKT/HAK/KUP genes are clustered into four groups (I-IV). KT/HAK/KUP genes from watermelon, Arabidopsis and rice are denoted by star, dot, square, respectively. The tree was generated with the MEGA11 software using the neighbor-joining (NJ) method

Interestingly, each group of the phylogenetic tree can be divided into multiple subgroups (2–6) based on the number of KT/HAK/KUPs per species (Fig. 1). We observed that the chromosomal locations of KT/HAK/KUPs from each species within the subgroups were generally consistent. However, in CN, the distribution of KT/HAK/KUPs on chromosomes 3 and 9 is distinct compared to others within the phylogenetic tree subgroups. For example, in subgroups IC and ID, where KT/HAK/KUP genes from other species are located on chromosome 9, the KT/HAK/KUPs in CN (CnPI596649_03g006210, CnPI596649_03g00622) are instead located on chromosome 3. Conversely, in subgroups IIIA and IIIB, where KT/HAK/KUP genes from other species are located on chromosome 3, the KT/HAK/KUPs in CN are located on chromosome 9.

To gain a deeper understanding of the classification and structural composition of watermelon KT/HAK/KUPs, the conserved motifs were analyzed. We identified ten conserved motifs in KT/HAK/KUPs protein sequences, and all members exhibited these motifs except for CePI673135_10g009350, which lacked some motifs due to the presence of a ‘ZntA’ domain (Fig. 1). Furthermore, we observed that all KT/HAK/KUPs in watermelons possess a motif known as the ‘K_trans’ superfamily.

Collinearity and expansion analysis of KT/HAK/KUPs in watermelons

Collinearity analysis revealed 132 collinear gene pairs among the 98 KT/HAK/KUP genes in Citrullus. CE, CN, CR, and CL exhibit extensive collinearity among each other, with an average of more than 16 collinear gene pairs between them. Additionally, there are 3–4 intragenomic collinear gene pairs of KT/HAK/KUPs among these four species. In contrast, CC has only four collinear gene pairs with CA and one collinear gene pair with CM. Similarly, CA and CM have only 1–4 collinear gene pairs with CE, CN, CR, and CL (Fig. 2).

Fig. 2 Chromosome localization and collinearity analysis of the KT/HAK/KUP genes in Citrullus. The different colors represent the different species in Citrullus: blue (C. amarus), green (C. colocynthis), red (C. ecirrhosus), orange (C. lanatus), purple (C. mucosospermus), light blue (C. naudinianus), and light green (C. rehmii). The lines represent collinear gene pairs of the KT/HAK/KUPs, where the blue line represents interspecific and the red line represents intraspecific

Among the KT/HAK/KUP family members in the Citrullus, many genes involved in collinear regions, indicating that numerous KT/HAK/KUP genes have undergone duplication events. The results from the MCScanX method suggested that segmental duplication may play a key role in amplifying the watermelon KT/HAK/KUP gene family (data not shown). Additionally, we calculated 4DTv and Ks value to evaluate the date of duplication events and further calculated Ka/Ks ratios to determine the pressures driving KT/HAK/KUP gene evolution. The results show that peaks in 4DTv values are primarily concentrated among syntenic gene pairs between CL and CE, CR, CA and CM at 0.0 to 0.025. In contrast, the peaks for CN and CC with other Citrullus species between 0.3 and 0.4 (Fig. 3A). The Ks peak values, ranging from 0 to 0.02, similarly are primarily concentrated among CL and CE, CR, CA and CM (Fig. 3B). These findings indicate that KT/HAK/KUP gene duplication events coincide with the divergence periods of these Citrullus species. Specifically, CL and CM diverged approximately 0.25 million years ago (Mya), CA diverged about 2.41 Mya, while CC diverged approximately 4.54 Mya, CN is speculated to be the ancestor of watermelons [36–38]. The Ka/Ks results, which predominantly range from 0.1 to 0.5, suggest that KT/HAK/KUP genes were subjected to evolutionary selection during their evolution (Fig. 3C).

Fig. 3 The evolution analysis of the KT/HAK/KUP gene family. The distribution of 4DTv (A) and Ks (B) values of homologous gene pairs among in Citrullus. (C) The distribution Ka/Ks values of homology gene pairs

Cis-regulatory elements analyzed of watermelon KT/HAK/KUPs

Promoter cis-regulatory elements play a key role in the gene regulation of gene expression patterns in response to plant growth and development and environmental stresses [46]. The cis-regulatory elements were analyzed with the upstream 2,000 bp sequence of watermelon KT/HAK/KUPs using the PlantCARE database. In total, 1,505 cis-regulatory elements were predicated in all KT/HAK/KUPs, which could be classified into five categories based on their functions (Fig. 4). The most abundant category was light response element (44.32%), followed by phytohormone response element (21.79%), promoter/enhancer element (16.28%), stress response element (14.22%), and plant growth and development element (3.39%). Within the promoter/enhancer element category, two prevalent elements responsible for transcriptional efficiency were identified: TATA-boxes (40.00%) and CAAT-boxes (40.00%); light responsive elements included 27 members, which was the largest subdivision including Box 4 (13.49%), GT1-motif (10.64) and G-box (9.45%) as representatives. Regulatory elements related to phytohormone-response comprised 11 components, including ABRE (22.26%), CGTCA/TGACG motifs (14.33%), TCA-element (12.5%), TGA-element (9.15%) and P-box (8.54%), which involved in abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA), auxin, and gibberellin (GA) responses, respectively. And the stress-related elements involved anoxic (ARE, 42.06.93%), low temperature (LTR, 15.42%) and drought (MBS, 13.08%) responsiveness were also found in watermelon KT/HAK/KUPs. Additionally, the related to plant growth and development elements were also found, which involved in meristem expression (CAT-box, 52.94%), endosperm expression (GCN4_motif, 31.37.9%) and differentiation of the palisade mesophyll cells (HD-Zip I, 9.8%). These results suggest that KT/HAK/KUP genes in watermelons are extensively involved in plants growth and developmental processes, phytohormone and stress responses.

Fig. 4 Cis-regulatory elements in watermelon KT/HAK/KUPs promoters. Left: The heat map employs the intensity of the red color to represent the number of promoters corresponding to each gene. Right: The colored histograms indicate the number of different cis-regulatory elements in five categories

Effects of K+ limited on vegetative growth and K+ distribution in watermelon seedlings

K+ is vital for plant growth and stress resistance [6]. In this study, we observed a noticeable inhibitory effect on the epigeal growth of watermelon under K+ limitation, while induced the taproot growth (Fig. 5A). Notably, the crown width of G42 is more inhibitory than PI 482276. To evaluate the impact of K+ deficiency on watermelon seedlings, we evaluated enzyme activities, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT), as well as the malondialdehyde (MDA) content in the leaves of both PI 482276 and G42 under conditions of both K+ starvation and sufficiency. The results demonstrated a significant increase in the activities of POD, SOD, CAT, and MDA content in watermelon seedlings subjected to K+ starvation. Importantly, PI 482276 exhibited significantly higher activities of POD, SOD, and CAT in its leaves compared to G42, while its MDA content was significantly lower than that of G42 (Fig. 5B). These findings suggest that K+ deficiency may lead to the accumulation of harmful substances, such as reactive oxygen species and MDA, in watermelon seedlings.

Fig. 5 The phenotype (A), oxidative stress-related markers (B) and K+ distribution (C) of watermelon seedlings were compared under K+-deficient (K+-Def) and -sufficient (K+-Nor) conditions

We also conducted a comprehensive analysis of K+ content in roots, stems, and leaves of watermelons. The results reveal variations in K+ absorption and distribution between the wild-type watermelon PI 482276 and the cultivated-type watermelon G42. PI 482276 exhibits a superior capacity for K+ absorption and transport, as evidenced by a higher K+ content in the stems compared to the roots. By contrast, G42 displays the highest K+ content in the roots, and its ability to transport K+ to the above-ground parts appears to be lower than PI 482276 (Fig. 5C). Under K+ limitation, the K+ content in roots, stems, and leaves of both PI 482276 and G42 decreased significantly, indicating that the K+ required for watermelon growth is mainly absorbed by the roots from external sources.

The qRT-PCR analysis of watermelon KT/HAK/KUPs in response to K +-deficiency and drought stress in CL and CA

To further validate the expression patterns of watermelon KT/HAK/KUP genes under the K+-deficiency and drought stress, we subjected two different watermelon genotypes, the cultivated watermelon G42 (CL) and the wild watermelon PI 482276 (CA), which are frequently utilized in breeding programs [47, 48], to simultaneous K+ deficiency and drought treatment. Sequence similarity often implies functional similarity. Based on sequence analysis and the proximal relationships in the phylogenetic tree, we conducted a comparative grouping analysis of the KT/HAK/KUPs expression profiles between the G42 and the PI 482276. The results indicate that the expression patterns of KT/HAK/KUPs in response to K+ deficiency are generally consistent between PI 482276 and G42 (Fig. 6). The expression levels of ClG42_07g0120700/CaPI482276_07g014010 and ClG42_09g0193000/CaPI482276_09g019970 were significantly upregulated under K+ deficiency in both PI 482276 and G42. In contrast, ClG42_03g0006000/CaPI482276_03g000580, ClG42_03g0042100/CaPI482276_03g004210, ClG42_03g0042300/CaPI482276_03g004230, ClG42_05g0139300/CaPI482276_05g014160, ClG42_10g0093200/CaPI482276_10g009570, ClG42_11g0018200/CaPI482276_11g000360 showed insignificant differences in expression levels under K+ deficiency in PI 482276 but exhibited a significant upregulation in G42. Moreover, specific K+ deficiency responsive genes were identified in PI 482276, such as CaPI482276_09g006110/ClG42_09g0060500 and CaPI482276_10g013140/ClG42_10g0143100. Notably, ClG42_09g0060500 is predominantly inactive in the roots of G42 but CaPI482276_09g006110 shows distinct expression in PI 482276, with significant upregulation under K+ starvation. These results indicate that the response mechanisms to K+ starvation stress may differ between the wild-type watermelon (PI 482276) and the cultivated-type watermelon (G42). However, one gene (ClG42_07g0120700/CaPI482276_07g014010) exhibited strong responses to K+ deficiency in both wild and cultivated types. It has a very low expression level under K+ sufficiency conditions but shows a significant upregulation under K+ deficiency. It may play a crucial functional role in the absorption of K+ by watermelons in K+ deficient environments.

The KT/HAK/KUP genes have been reported to play a crucial role in plant responses to abiotic stress [17–19]. In our study, we identified numerous cis-regulatory elements associated with drought (MBS) and low temperature (LTR) stress responses in their promoter regions (Fig. 4). Consequently, we investigated the expression patterns of KT/HAK/KUP genes in both CL and CA under drought treatment (Fig. 7). With the exception of CaPI482276_07g006110/ClG42_07g0060500, which was nearly undetectable in the roots of both PI 482276 and G42, the other KT/HAK/KUP genes generally responded to drought stress. CaPI482276_03g000580/ClG42_03g0006000, CaPI482276_03g004210/ClG42_03g0042100, CaPI482276_05g014160/ClG42_05g0139300, CaPI482276_07g014010/ClG42_07g0120700, and CaPI482276_09g019970/ClG42_09g0193000 exhibited significant upregulation in response to drought stress, whereas CaPI482276_10g010990/ClG42_10g0106000 showed downregulation under drought stress in both CL and CA. Notably, drought-responsive elements (MBS) were observed in the promoter regions of CaPI482276_03g000580/ClG42_03g0006000, CaPI482276_09g019970/ClG42_09g0193000, as well as CaPI482276_03g004210, CaPI482276_05g014160, and CaPI482276_07g014010 (Fig. 4).

Fig. 6 Expression profiles of KT/HAK/KUP genes in CL and CA in response to K+-deficiency by qRT-PCR. Mean values and standard deviations (SDs) were obtained from three replicates. The error bars indicate standard deviation. The least significant difference (LSD) with a significance level of * p < 0.05 and ** p < 0.01 by one-ANOVA

Fig. 7 Expression analysis of KT/HAK/KUP genes in CL and CA following drought treatment by qRT-PCR. Mean values and standard deviations (SDs) were obtained from three replicates. The error bars indicate standard deviation. The least significant difference (LSD) with a significance level of * p < 0.05 and ** p < 0.01 by one-ANOVA

Heterologous expression of ClG42_07g0120700 in yeast

The ClG42_07g0120700 is induced by K+-deficiency and shares homology with the Arabidopsis gene AtHAK5 (AT4G13420), which is known to play a crucial role in root responses to low K+ environments [19]. The qRT-PCR analysis confirmed that ClG42_07g0120700 is predominantly expressed in watermelon roots and shows minimal expression in leaves (Fig. 8A). Further validation using the yeast mutant R5421 demonstrated that both the empty vector (pYES2.0) and ClG42_07g0120700 transformed yeast strains grew well on AP medium with high K+ concentration (20 mM K+). However, under low K+ conditions (0.1 Mm K+), the growth of the yeast strain with the empty vector (pYES2.0) was suppressed, while the strain expressing ClG42_07g0120700 continued to grow (Fig. 8B). This confirms the functional role of ClG42_07g0120700 as a K+ transporter.

Fig. 8 Tissue-specific expression pattern and yeast complementation analysis of ClG42_07g0120700. A: expression pattern of ClG42_07g0120700 in indicated roots, stems, and leaves of watermelon seedlings. ClACTIN was an internal control. Data are mean ± SE (n = 3). B: yeast complementation assay of K+ acquisition by the K+ uptake-defective yeast mutant (R5421) complemented with ClG42_07g0120700. Growth status of R5421 cells expressing ClG42_07g0120700, empty vector (pYES2.0), on AP solid medium containing from 20 to 0.1 mM K+. The 1:10 serial dilutions of yeast cells were spotted on the AP solid medium and then incubated at 30 °C for 5–7 d

Discussion

K+ concentration in soil solution ranges from 0.025 to 5 mM, thus, K+ deficiency is definitely an abiotic stress to plants [49]. The growth and development of watermelon was hindered by K+ deficiency, leading to a reduction in both root and shoot dry weight, as well as a decrease in total root length and root surface area [50, 51]. The mechanisms of K+ uptake and transport in plants have been the focus of research, several studies have shown that KT/HAK/KUP gene family is associated with transmembrane transport of K+ and K+ supply in plants [28]. However, no study has investigated the structure and functional relationship of KT/HAK/KUP in watermelon. Sweet watermelon (C. lanatus) is a global fruit crop, which belongs to the genus Citrullus Schrad and has six known species including C. naudinianus (CN), C. colocynthis (CC), C. ecirrhosus (CE), C. rehmii (CR), C. amarus (CA), and C. mucosospermus (CM) that exist in the wild [52]. Wild relatives are important resources for watermelon breeding. Especially the extensive use of CA in watermelon breeding has significantly enhanced the genetic diversity of cultivated watermelons [38, 47]. With the advancement of sequencing technology and molecular biology, recent studies have reported the assembly results of the Citrullus genus pangenome, providing deeper insights into their origins, evolution and domestication [35, 36, 38]. However, studies on the individual gene families across different species are still rare. In this study, 98 KT/HAK/KUP genes were identified from seven species of Citrullus, with each species containing 14 KT/HAK/KUPs (Table 1). Although their genome sizes differ, our findings indicate that the number of KT/HAK/KUPs is not significantly correlated with genome size [53]. From the perspective of chromosome distribution, only CR and CN exhibit differences compared to the other five watermelon varieties. This suggests that CL, CA, CM, CE, and CC have relatively completely retained the chromosome regions of the KT/HAK/KUP gene family during the evolutionary process. Furthermore, phylogenetic tree analysis has identified CN as the most distant member in each subgroup, while CM is the closest relative to CL, suggesting that CM may be the direct ancestor of CL, while CN may be the original ancestor species of CL [36]. And phylogenetic analysis reveals that group II contains the highest number of KT/HAK/KUPs, which is consistent with previous classifications of the KT/HAK/KUP gene family in Arabidopsis [16], rice [11], maize [12], tomato [13] and bamboo [46]. However, a large number of KT/HAK/KUPs from watermelon are distributed in group I, whereas there is only one KT/HAK/KUP gene from Arabidopsis, similar to rice and bamboo [11, 16, 46].

Table 1 Summary of the KT/HAK/KUP gene family in watermelons (Citrullus)

Species	Variety	Genome size (bp)	Chromosome number (2n)	Whole gene number	KT/HAK/KUP gene number	
I	II	III	IV	
Citrullus amarus	PI 482276	387,890,241	22	24,011	4	6	2	2	
Citrullus colocynthis	PI 537300	386,341,915	22	24,081	4	6	2	2	
Citrullus ecirrhosus	PI 673135	409,055,546	22	23,964	4	6	2	2	
Citrullus lanatus	G42	369,321,917	22	24,205	4	6	2	2	
Citrullus mucosospermus	PI 595203	377,190,016	22	25,232	4	6	2	2	
Citrullus naudinianus	PI 596694	370,541,501	22	25,277	4	6	2	2	
Citrullus rehmii	PI 670011	420,611,209	22	26,952	4	6	2	2	

Gene duplication events play a crucial role in expanding and evolving gene families [54, 55]. Duplication events have caused the expansion of gene family members in plants, and mutations in upstream regulatory regions and coding regions cause changes in the expression pattern and function of new members [56, 57]. In this study, we identified segmental/WGD as the primary mechanisms potentially responsible for the evolution of KT/HAK/KUP genes in watermelon. Similar results of collinearity analysis have been found in other species such as pear and bamboo [14, 46]. In addition, the Ks and 4TDv values were consistent with previously reported the estimated times of divergence events of watermelon species [35]. The Ka/Ks ratios were less than 1, indicating that purifying selection is significant in the evolution of KT/HAK/KUP genes in watermelon species [58, 59].

The wild watermelon PI 482276 is widely used as a crucial source of resistance in watermelon breeding, with proven excellence in stress and disease resistance, such as resistance to gummy stem blight [47]. This study found that K+ deficiency stress has a smaller impact on the above-ground parts compared to the cultivated G42. Even under K+ deficiency, PI 482276 can supply more K+ to the above-ground parts. Moreover, the activities of oxidative enzymes such as POD, SOD, and CAT are higher under stress conditions (low K+) than in the cultivated type, indicating superior stress resistance (Fig. 5). Plant stress tolerance is closely associated with the expression of specific genes, such as KT/HAK/KUP genes, which are involved in plant response to abiotic stresses such as K+ deficiency, drought, and salt [28–31]. We discovered that the gene CaPI482276_09g006610 on chromosome 9 (corresponding to ClG42_09g0060500 in G42) is exclusively expressed in the roots of the wild type PI 482276 and significantly responds to K+ deficiency stress, suggesting that it may be a specific expression gene contributing to the stronger stress resistance of PI 482276. Additionally, in both wild and cultivated watermelons, the CaPI482276_07g014010/ClG42_07g0120700 gene significantly responds to K+ deficiency. Further analysis revealed that these genes belong to group IA in the phylogenetic tree, along with AtHAK5 (AT4G13420) which is involved in both K+ uptake and translocation, and AtHAK5 plays a crucial role in enhancing salt tolerance in Arabidopsis by maintaining K+ homeostasis at high Na+ concentrations [33, 60, 61]. Indeed, the rescue by ClG42_07g0120700 of the K+-transport-disabled yeast mutant in a K+-deficient medium suggests that ClG42_07g0120700 functions in high-affinity K+ uptake, similar to the demonstrated involvement of AtHAK5 in K+ transport in roots K+-limitation [60, 61]. Based on the relatively higher expression of ClG42_07g0120700 in roots (Fig. 8A), we speculate that ClG42_07g0120700 plays a critical role in K+ transfer under K+-deficiency conditions from outside to the root in the watermelon seedlings. Cis- regulatory elements are DNA sequences that interact with structural genes and serve as binding sites for transcription factors. These elements regulate gene expression in plants by binding to transcription factors and controlling the timing and efficiency of gene transcription [62]. In this study, the potential role of watermelon KT/HAK/KUPs in stress response was evaluated by examining KT/HAK/KUPs promoter regions and predicting the presence of various cis-regulatory elements associated with different abiotic stresses (Fig. 4). These elements include ARE elements, which are essential for anaerobic induction; LTR elements, which are involved in low-temperature responsiveness; and MBS elements, which are MYB binding sites involved in drought inducibility. The qRT-PCR analysis was performed under drought stress to determine KT/HAK/KUP expression patterns in wild and cultivated watermelon (Fig. 7). Most KT/HAK/KUP gene family members of the watermelon responded to drought stresses, indicating that KT/HAK/KUP genes regulate responses to environmental stress.

Conclusions

This is the first study related to pangenome-wide identification and comprehensive analysis of KT/HAK/KUP genes in the seven watermelon species of the genus Citrullus. In this study, we conducted a systematic comparative analysis of the KT/HAK/KUP gene family members in different species of the Citrullus, focusing on chromosome distribution, gene structure, evolution, and domestication. In addition, we investigated the responses of wild and cultivated watermelons to K+-deficiency and drought stress, revealing the expression patterns of KT/HAK/KUP genes in watermelons under these conditions. Therefore, this study provides new insights into the role of KT/HAK/KUPs in species differentiation and stress resistance in watermelons. KT/HAK/KUPs are promising candidate genes for transgenic plant breeding and warrant further exploration and innovation.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Acknowledgements

The authors express gratitude for the invaluable support received during the research process. We greatly appreciate the valuable technical, data, and platform support provided throughout the entire project by Professor Xingping Zhang and Yun Deng from Peking University Institute of Advanced Ag Sciences, and professor Feishi Luan, Peng Gao, and associate professor Shi Liu, Honglv Liu and Zicheng Zhu from our team. Their support has played a significant role in the success of this study.

Author contributions

C.R., W.X.Z. and S.Y.D. conceived and designed the research. C.R., T.Y., G.Y., and C.G.D. performed the experiments. C.R. and Z.Z.X analyzed the data and wrote the manuscript. W.X.Z. and S.Y.D. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by China Agriculture Research System of MOF and MARA (CARS-25), the Huai’an Natural Science Research Project (HABL202228), the Research and Development Fund Project of Huai’an Academy of Agricultural Sciences (HABL202228), and the Seed Industry Vitalization Research Projects of Jiangsu Province (JBGS[2021]072).

Data availability

The watermelon pangenome information and sequences used in this study can be obtained from the Watermelon Genome Database (http://watermelondb.cn). All other datasets supporting the conclusions of this article are included within the article and its additional files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Abbreviations

HAK/KUP/KT High-affinity K+ transporter / K+ uptake permease / K+ transporter

CL Citrullus lanatus

CA Citrullus amarus

CE Citrullus ecirrhosus

CM Citrullus mucosospermus

CN Citrullus naudinianus

CR Citrullus rehmii

CC Citrullus colocynthis

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Véry AA Hervé S Molecular mechanisms and regulation of K+ transport in higher plants Annu Rev Plant Biol 2003 54 575 603 10.1146/annurev.arplant.54.031902.134831 14503004
Véry AA, Hervé S. Molecular mechanisms and regulation of K+ transport in higher plants. Annu Rev Plant Biol. 2003;54:575–603.14503004 10.1146/annurev.arplant.54.031902.134831
2. Ahammed GJ Chen Y Liu C Yang Y Light regulation of potassium in plants Plant Physiol Bioch 2022 170 316 24 10.1016/j.plaphy.2021.12.019
Ahammed GJ, Chen Y, Liu C, Yang Y. Light regulation of potassium in plants. Plant Physiol Bioch. 2022;170:316–24.10.1016/j.plaphy.2021.12.019
3. Epstein E Rains DW Elzam OE Resolution of dual mechanisms of potassium absorption by barley roots PNAS 1963 49 5 684 92 10.1073/pnas.49.5.684 16591089
Epstein E, Rains DW, Elzam OE. Resolution of dual mechanisms of potassium absorption by barley roots. PNAS. 1963;49(5):684–92.16591089 10.1073/pnas.49.5.684
4. Isabelle C Cécile L Martin B Hervé S Molecular mechanisms involved in plant adaptation to low K+ availability J Exp Bot 2014 65 3 833 48 10.1093/jxb/ert402 24293613
Isabelle C, Cécile L, Martin B, Hervé S. Molecular mechanisms involved in plant adaptation to low K+ availability. J Exp Bot. 2014;65(3):833–48.24293613 10.1093/jxb/ert402
5. Song Z Yang S Zhu H Jin M Su Y Heterologous expression of an alligatorweed high-affinity potassium transporter gene enhances salinity tolerance in Arabidopsis thaliana Am J Bot 2014 101 840 50 10.3732/ajb.1400155 24824834
Song Z, Yang S, Zhu H, Jin M, Su Y. Heterologous expression of an alligatorweed high-affinity potassium transporter gene enhances salinity tolerance in Arabidopsis thaliana. Am J Bot. 2014;101:840–50.24824834 10.3732/ajb.1400155
6. Ashley MK Grant M Grabov A Plant responses to potassium deficiencies: a role for potassium transport proteins J Exp Bot 2006 57 2 425 36 10.1093/jxb/erj034 16364949
Ashley MK, Grant M, Grabov A. Plant responses to potassium deficiencies: a role for potassium transport proteins. J Exp Bot. 2006;57(2):425–36.16364949 10.1093/jxb/erj034
7. Chen G Liu C Gao Z Zhang Y Jiang H Zhu L Ren D Yu L Xu G Qian Q OsHAK1, a high-affinity potassium transporter, positively regulates responses to drought stress in rice Front Plant Sci 2017 8 1885 10.3389/fpls.2017.01885 29163608
Chen G, Liu C, Gao Z, Zhang Y, Jiang H, Zhu L, Ren D, Yu L, Xu G, Qian Q. OsHAK1, a high-affinity potassium transporter, positively regulates responses to drought stress in rice. Front Plant Sci. 2017;8:1885.29163608 10.3389/fpls.2017.01885
8. Liu S Wu B Xie Y Zheng S Xie J Wang W Xiang D Li C Genome-wide analysis of HAK/KUP/KT potassium transporter genes in banana (Musa Acuminata L.) and their tissue-specific expression profiles under potassium stress Plant Growth Regul 2022 97 51 60 10.1007/s10725-021-00793-7
Liu S, Wu B, Xie Y, Zheng S, Xie J, Wang W, Xiang D, Li C. Genome-wide analysis of HAK/KUP/KT potassium transporter genes in banana (Musa Acuminata L.) and their tissue-specific expression profiles under potassium stress. Plant Growth Regul. 2022;97:51–60.10.1007/s10725-021-00793-7
9. Yurtseven E Kesmez GD Ünlükara A The effects of water salinity and potassium levels on yield, fruit quality and water consumption of a native central anatolian tomato species (Lycopersicon Esculantum) Agr Water Manage 2005 78 1 28 35
Yurtseven E, Kesmez GD, Ünlükara A. The effects of water salinity and potassium levels on yield, fruit quality and water consumption of a native central anatolian tomato species (Lycopersicon Esculantum). Agr Water Manage. 2005;78(1):28–35.
10. Ahn SJ Shin R Schachtman DP Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+ uptake Plant Physiol 2004 134 3 1135 45 10.1104/pp.103.034660 14988478
Ahn SJ, Shin R, Schachtman DP. Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K+ uptake. Plant Physiol. 2004;134(3):1135–45.14988478 10.1104/pp.103.034660
11. Gupta M Qiu X Wang L Xie W Zhang C Xiong L Lian X Zhang Q KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice (Oryza Sativa) Mol Genet Genomics 2008 280 437 52 10.1007/s00438-008-0377-7 18810495
Gupta M, Qiu X, Wang L, Xie W, Zhang C, Xiong L, Lian X, Zhang Q. KT/HAK/KUP potassium transporters gene family and their whole-life cycle expression profile in rice (Oryza Sativa). Mol Genet Genomics. 2008;280:437–52.18810495 10.1007/s00438-008-0377-7
12. Zhang Z Zhang J Chen Y Li R Wang H Wei J Genome-wide analysis and identification of HAK potassium transporter gene family in maize (Zea Mays L) Mol Biol Rep 2012 39 8465 73 10.1007/s11033-012-1700-2 22711305
Zhang Z, Zhang J, Chen Y, Li R, Wang H, Wei J. Genome-wide analysis and identification of HAK potassium transporter gene family in maize (Zea Mays L). Mol Biol Rep. 2012;39:8465–73.22711305 10.1007/s11033-012-1700-2
13. Hyun TK Rim Y Kim E Kim JS Genome-wide and molecular evolution analyses of the KT/HAK/KUP family in tomato (Solanum Lycopersicum L) Genes Genom 2014 36 365 74 10.1007/s13258-014-0174-0
Hyun TK, Rim Y, Kim E, Kim JS. Genome-wide and molecular evolution analyses of the KT/HAK/KUP family in tomato (Solanum Lycopersicum L). Genes Genom. 2014;36:365–74.10.1007/s13258-014-0174-0
14. Wang Y Lü J Chen D Zhang J Qi K Cheng R Zhang H Zhang S Genome-wide identification, evolution, and expression analysis of the KT/HAK/KUP family in pear Genome 2018 61 755 65 10.1139/gen-2017-0254 30130425
Wang Y, Lü J, Chen D, Zhang J, Qi K, Cheng R, Zhang H, Zhang S. Genome-wide identification, evolution, and expression analysis of the KT/HAK/KUP family in pear. Genome. 2018;61:755–65.30130425 10.1139/gen-2017-0254
15. Cheng X Liu X Mao W Zhang X Chen S Zhan K Bi H Xu H Genome-wide identification and analysis of HAK/KUP/KT potassium transporters gene family in wheat (Triticum Aestivum L) Int J Mol Sci 2018 19 3969 10.3390/ijms19123969 30544665
Cheng X, Liu X, Mao W, Zhang X, Chen S, Zhan K, Bi H, Xu H. Genome-wide identification and analysis of HAK/KUP/KT potassium transporters gene family in wheat (Triticum Aestivum L). Int J Mol Sci. 2018;19:3969.30544665 10.3390/ijms19123969
16. Mäser P Thomine S Schroeder JI Ward JM Hirschi K Sze H Talke IN Amtmann A Maathuis FJ Sanders D Phylogenetic relationships within cation transporter families of Arabidopsis Plant Physiol 2001 126 4 1646 67 10.1104/pp.126.4.1646 11500563
Mäser P, Thomine S, Schroeder JI, Ward JM, Hirschi K, Sze H, Talke IN, Amtmann A, Maathuis FJ, Sanders D. Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol. 2001;126(4):1646–67.11500563 10.1104/pp.126.4.1646
17. Li P Luo T Pu X Zhou Y Yu J Liu L Plant transporters: roles in stress responses and effects on growth and development Plant Growth Regul 2021 93 253 66 10.1007/s10725-020-00684-3
Li P, Luo T, Pu X, Zhou Y, Yu J, Liu L. Plant transporters: roles in stress responses and effects on growth and development. Plant Growth Regul. 2021;93:253–66.10.1007/s10725-020-00684-3
18. Rigas S, Ditengou FA, Ljung K, Daras G, Tietz O, Palme K, Hatzopoulos P. Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex. New Phytol 2103; 197:1130–41.
19. Young JP Markus G Julian IS Myeon HC High-affinity K+ transport in Arabidopsis: ATHAK5 and AKT1 are vital for seedling establishment and post germination growth under low-potassium conditions Plant Physiol 2010 153 2 863 75 10.1104/pp.110.154369 20413648
Young JP, Markus G, Julian IS, Myeon HC. High-affinity K+ transport in Arabidopsis: ATHAK5 and AKT1 are vital for seedling establishment and post germination growth under low-potassium conditions. Plant Physiol. 2010;153(2):863–75.20413648 10.1104/pp.110.154369
20. Han M Wu W Wu WH Wang Y Potassium transporter KUP7 is involved in K+ acquisition and translocation in Arabidopsis root under K+-limited conditions Mol Plant 2016 9 3 437 46 10.1016/j.molp.2016.01.012 26851373
Han M, Wu W, Wu WH, Wang Y. Potassium transporter KUP7 is involved in K+ acquisition and translocation in Arabidopsis root under K+-limited conditions. Mol Plant. 2016;9(3):437–46.26851373 10.1016/j.molp.2016.01.012
21. Chen G Hu Q Luo L Yang T Zhang S Hu Y Yu L Xu G Rice potassium transporter OSHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges Plant Cell Environ 2015 38 12 2747 65 10.1111/pce.12585 26046301
Chen G, Hu Q, Luo L, Yang T, Zhang S, Hu Y, Yu L, Xu G. Rice potassium transporter OSHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges. Plant Cell Environ. 2015;38(12):2747–65.26046301 10.1111/pce.12585
22. Yang T Zhang S Hu Y Wu F Hu Q Chen G Cai J Wu T Moran N Yu L Xu G The role of a potassium transporter OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels Plant Physiol 2014 166 2 945 59 10.1104/pp.114.246520 25157029
Yang T, Zhang S, Hu Y, Wu F, Hu Q, Chen G, Cai J, Wu T, Moran N, Yu L, Xu G. The role of a potassium transporter OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels. Plant Physiol. 2014;166(2):945–59.25157029 10.1104/pp.114.246520
23. Yang T Feng H Zhang S Xiao H Hu Q Chen G Xuan W Moran N Murphy A Yu L Xu G The potassium transporter OsHAK5 alters rice architecture via ATP-dependent transmembrane auxin fluxes Plant Commun 2020 1 5 100052 10.1016/j.xplc.2020.100052 33367257
Yang T, Feng H, Zhang S, Xiao H, Hu Q, Chen G, Xuan W, Moran N, Murphy A, Yu L, Xu G. The potassium transporter OsHAK5 alters rice architecture via ATP-dependent transmembrane auxin fluxes. Plant Commun. 2020;1(5):100052.33367257 10.1016/j.xplc.2020.100052
24. Boscari A Clément M Volkov V Golldack D Hybiak J Miller AJ Amtmann A Fricke W Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development Plant Cell Environ 2009 32 12 1761 77 10.1111/j.1365-3040.2009.02033.x 19682291
Boscari A, Clément M, Volkov V, Golldack D, Hybiak J, Miller AJ, Amtmann A, Fricke W. Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development. Plant Cell Environ. 2009;32(12):1761–77.19682291 10.1111/j.1365-3040.2009.02033.x
25. Cuin TA Bose J Stefano G Jha D Tester M Mancuso S Shabala S Assessing the role of root plasma membrane and tonoplast Na+/H+ exchangers in salinity tolerance in wheat: in planta quantification methods Plant Cell Environ 2011 34 6 947 61 10.1111/j.1365-3040.2011.02296.x 21342209
Cuin TA, Bose J, Stefano G, Jha D, Tester M, Mancuso S, Shabala S. Assessing the role of root plasma membrane and tonoplast Na+/H+ exchangers in salinity tolerance in wheat: in planta quantification methods. Plant Cell Environ. 2011;34(6):947–61.21342209 10.1111/j.1365-3040.2011.02296.x
26. Liang M, Gao Y, Mao T, Zhang X, Zhang S, Zhang H, Song Z. Characterization and expression of KT/HAK/KUP transporter family genes in willow under potassium deficiency, drought, and salt stresses. BioMed Res Int. 2020; 2020:2690760.
27. Alexander G Plant KT/KUP/HAK potassium transporters: single family-multiple functions Ann Bot 2007 99 66 1035 41 17495982
Alexander G, Plant. KT/KUP/HAK potassium transporters: single family-multiple functions. Ann Bot. 2007;99(66):1035–41.17495982
28. Li W Xu G Alli A Yu L Plant HAK/KUP/KT K+ transporters: function and regulation Semin Cell Dev Biol 2018 74 133 41 10.1016/j.semcdb.2017.07.009 28711523
Li W, Xu G, Alli A, Yu L, Plant. HAK/KUP/KT K+ transporters: function and regulation. Semin Cell Dev Biol. 2018;74:133–41.28711523 10.1016/j.semcdb.2017.07.009
29. Huang Y Cao H Yang L Chen C Shabala L Xiong M Niu M Liu J Zheng Z Zhou L Peng Z Bie Z Shabala S Tissue-specific respiratory burst oxidase homolog-dependent H2O2 signaling to the plasma membrane H+-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae J Exp Bot 2019 70 20 5879 93 10.1093/jxb/erz328 31290978
Huang Y, Cao H, Yang L, Chen C, Shabala L, Xiong M, Niu M, Liu J, Zheng Z, Zhou L, Peng Z, Bie Z, Shabala S. Tissue-specific respiratory burst oxidase homolog-dependent H2O2 signaling to the plasma membrane H+-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae. J Exp Bot. 2019;70(20):5879–93.31290978 10.1093/jxb/erz328
30. Ankit A Kamali S Singh A Genomic & structural diversity and functional role of potassium (K+) transport proteins in plants Int J Biol Macromol 2022 208 844 57 10.1016/j.ijbiomac.2022.03.179 35367275
Ankit A, Kamali S, Singh A. Genomic & structural diversity and functional role of potassium (K+) transport proteins in plants. Int J Biol Macromol. 2022;208:844–57.35367275 10.1016/j.ijbiomac.2022.03.179
31. Yang T Lu X Wang Y Xie Y Ma J Cheng X Xia E Wan X Zhang Z HAK/KUP/KT family potassium transporter genes are involved in potassium deficiency and stress responses in tea plants (Camellia sinensis L.): expression and functional analysis BMC Genomics 2020 21 556 10.1186/s12864-020-06948-6 32791963
Yang T, Lu X, Wang Y, Xie Y, Ma J, Cheng X, Xia E, Wan X, Zhang Z. HAK/KUP/KT family potassium transporter genes are involved in potassium deficiency and stress responses in tea plants (Camellia sinensis L.): expression and functional analysis. BMC Genomics. 2020;21:556.32791963 10.1186/s12864-020-06948-6
32. Gobert A Park G Amtmann A Sanders D Maathuis FJM Arabidopsis thaliana cyclic nucleotide gated channel 3 forms a non-selective ion transporter involved in germination and cation transport J Exp Bot 2006 57 4 791 800 10.1093/jxb/erj064 16449377
Gobert A, Park G, Amtmann A, Sanders D, Maathuis FJM. Arabidopsis thaliana cyclic nucleotide gated channel 3 forms a non-selective ion transporter involved in germination and cation transport. J Exp Bot. 2006;57(4):791–800.16449377 10.1093/jxb/erj064
33. Rubio F Nieves-Cordones M Alemán F Martínez V Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity range of concentrations Physiol Plant 2008 134 4 598 608 10.1111/j.1399-3054.2008.01168.x 19000196
Rubio F, Nieves-Cordones M, Alemán F, Martínez V. Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity range of concentrations. Physiol Plant. 2008;134(4):598–608.19000196 10.1111/j.1399-3054.2008.01168.x
34. Daras G Rigas S Tsitsekian D Iacovides TA Hatzopoulos P Potassium transporter TRH1 subunits assemble regulating root-hair elongation autonomously from the cell fate determination pathway Plant Sci 2015 231 131 7 10.1016/j.plantsci.2014.11.017 25575998
Daras G, Rigas S, Tsitsekian D, Iacovides TA, Hatzopoulos P. Potassium transporter TRH1 subunits assemble regulating root-hair elongation autonomously from the cell fate determination pathway. Plant Sci. 2015;231:131–7.25575998 10.1016/j.plantsci.2014.11.017
35. Wu S Sun H Gao L Branham S McGregor C Renner SS Xu Y Kousik C Wechter WP Levi A Fei Z A Citrullus genus super-pangenome reveals extensive variations in wild and cultivated watermelons and sheds light on watermelon evolution and domestication Plant Biotechnol J 2023 21 10 1926 8 10.1111/pbi.14120 37490004
Wu S, Sun H, Gao L, Branham S, McGregor C, Renner SS, Xu Y, Kousik C, Wechter WP, Levi A, Fei Z. A Citrullus genus super-pangenome reveals extensive variations in wild and cultivated watermelons and sheds light on watermelon evolution and domestication. Plant Biotechnol J. 2023;21(10):1926–8.37490004 10.1111/pbi.14120
36. Sun Y Kou DR Li Y Ni JP Wang J Zhang YM Wang QN Jiang B Wang X Sun YX Xu XT Tan XJ Zhang YJ Kong XD Pan-genome of Citrullus genus highlights the extent of presence/absence variation during domestication and selection BMC Genomics 2023 24 332 10.1186/s12864-023-09443-w 37322453
Sun Y, Kou DR, Li Y, Ni JP, Wang J, Zhang YM, Wang QN, Jiang B, Wang X, Sun YX, Xu XT, Tan XJ, Zhang YJ, Kong XD. Pan-genome of Citrullus genus highlights the extent of presence/absence variation during domestication and selection. BMC Genomics. 2023;24:332.37322453 10.1186/s12864-023-09443-w
37. Menezes F de Andrade A Bergamin RA Moreira AC de Souza AG Productivity and quality of watermelon fruits as a function of doses potassium in the Western Amazon Sci Electron Archives 2021 14 11 1 6 10.36560/141120211458
Menezes F, de Andrade A, Bergamin RA, Moreira AC, de Souza AG. Productivity and quality of watermelon fruits as a function of doses potassium in the Western Amazon. Sci Electron Archives. 2021;14(11):1–6.10.36560/141120211458
38. Zhang Y Zhao M Tan J Huang M Chu X Li Y Han X Fang T Tian Y Jarret R Lu D Chen Y Xue L Li X Qin G Li B Sun Y Deng XW Deng Y Zhang X He H Telomere-to-telomere Citrullus super-pangenome provides direction for watermelon breeding Nat Genet 2024 10.1038/s41588-024-01823-6 39210046
Zhang Y, Zhao M, Tan J, Huang M, Chu X, Li Y, Han X, Fang T, Tian Y, Jarret R, Lu D, Chen Y, Xue L, Li X, Qin G, Li B, Sun Y, Deng XW, Deng Y, Zhang X, He H. Telomere-to-telomere Citrullus super-pangenome provides direction for watermelon breeding. Nat Genet. 2024. 10.1038/s41588-024-01823-6.39210046 10.1038/s41588-024-01823-6
39. Xie J Chen Y Cai G Cai R Hu Z Wang H Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees Nucleic Acids Res 2023 51 587 92 10.1093/nar/gkad359
Xie J, Chen Y, Cai G, Cai R, Hu Z, Wang H. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res. 2023;51:587–92.10.1093/nar/gkad359
40. Wang Y Tang H Debarry JD Tan X Li J Wang X Lee T Jin H Marler B Guo H Kissinger JC Paterson AH MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity Nucleic Acids Res 2012 40 7 e49 10.1093/nar/gkr1293 22217600
Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, Lee T, Jin H, Marler B, Guo H, Kissinger JC, Paterson AH. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.22217600 10.1093/nar/gkr1293
41. Krzywinski M Schein J Birol I Connors J Gascoyne R Horsman D Jones SJ Marra MA Circos: an information aesthetic for comparative genomics Genome Res 2009 19 9 1639 45 10.1101/gr.092759.109 19541911
Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19(9):1639–45.19541911 10.1101/gr.092759.109
42. Wang D Zhang Y Zhang Z Zhu J Yu J KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies Genom Proteom Bioinform 2010 8 1 77 80 10.1016/S1672-0229(10)60008-3
Wang D, Zhang Y, Zhang Z, Zhu J, Yu J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genom Proteom Bioinform. 2010;8(1):77–80.10.1016/S1672-0229(10)60008-3
43. Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method Methods 2001 25 4 402 8 10.1006/meth.2001.1262 11846609
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆CT method. Methods. 2001;25(4):402–8.11846609 10.1006/meth.2001.1262
44. Liu J, Liu J, Cui M, Chen X, Liu J, Chen J, Chen A, Xu G. Investigate the effect of potassium on nodule symbiosis and uncover an HAK/KUP/KT member, GmHAK5, strongly responsive to root nodulation in soybean. J Plant Biol. 2022; 65(6):459-471.
45. Cheng R, Cheng Y, Lü J, Chen J, Wang Y, Zhang S, Zhang H. The gene PbTMT4 from pear (Pyrus bretschneideri) mediates vacuolar sugar transport and strongly affects sugar accumulation in fruit. Physiol Plantarum. 2018; 164(3):307–319.
46. Guo H Tan J Jiao Y Huang B Ma R Ramakrishnan M Qi G Zhang Z Genome-wide identification and expression analysis of the HAK/KUP/KT gene family in Moso bamboo Front Plant Sci 2024 15 1331710 10.3389/fpls.2024.1331710 38595761
Guo H, Tan J, Jiao Y, Huang B, Ma R, Ramakrishnan M, Qi G, Zhang Z. Genome-wide identification and expression analysis of the HAK/KUP/KT gene family in Moso bamboo. Front Plant Sci. 2024;15:1331710.38595761 10.3389/fpls.2024.1331710
47. Gimode W Bao K Fei Z McGregor C QTL associated with gummy stem blight resistance in watermelon Theor Appl Genet 2021 134 2 573 84 10.1007/s00122-020-03715-9 33135096
Gimode W, Bao K, Fei Z, McGregor C. QTL associated with gummy stem blight resistance in watermelon. Theor Appl Genet. 2021;134(2):573–84.33135096 10.1007/s00122-020-03715-9
48. Deng Y Liu S Zhang Y Tan J Li X Chu X Xu B Tian Y Sun Y Li B Xu Y Deng XW He H Zhang X A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding Mol Plant 2022 15 8 1268 84 10.1016/j.molp.2022.06.010 35746868
Deng Y, Liu S, Zhang Y, Tan J, Li X, Chu X, Xu B, Tian Y, Sun Y, Li B, Xu Y, Deng XW, He H, Zhang X. A telomere-to-telomere gap-free reference genome of watermelon and its mutation library provide important resources for gene discovery and breeding. Mol Plant. 2022;15(8):1268–84.35746868 10.1016/j.molp.2022.06.010
49. Hasanuzzaman M Bhuyan MHMB Nahar K Hossain MS Mahmud JA Hossen MS Masud AAC Moumita, Fujita M. Potassium: a vital regulator of plant responses and tolerance to abiotic stresses Agronomy 2018 8 3 31 10.3390/agronomy8030031
Hasanuzzaman M, Bhuyan MHMB, Nahar K, Hossain MS, Mahmud JA, Hossen MS, Masud AAC. Moumita, Fujita M. Potassium: a vital regulator of plant responses and tolerance to abiotic stresses. Agronomy. 2018;8(3):31.10.3390/agronomy8030031
50. Fan M Huang Y Zhong Y Kong Q Xie J Niu M Xu Y Bie Z Comparative transcriptome profiling of potassium starvation responsiveness in two contrasting watermelon genotypes Planta 2014 239 397 410 10.1007/s00425-013-1976-z 24185372
Fan M, Huang Y, Zhong Y, Kong Q, Xie J, Niu M, Xu Y, Bie Z. Comparative transcriptome profiling of potassium starvation responsiveness in two contrasting watermelon genotypes. Planta. 2014;239:397–410.24185372 10.1007/s00425-013-1976-z
51. Ahmad I Maathuis FJ Cellular and tissue distribution of potassium: physiological relevance, mechanisms and regulation J Plant Physiol 2014 171 9 708 14 10.1016/j.jplph.2013.10.016 24810768
Ahmad I, Maathuis FJ. Cellular and tissue distribution of potassium: physiological relevance, mechanisms and regulation. J Plant Physiol. 2014;171(9):708–14.24810768 10.1016/j.jplph.2013.10.016
52. Nimmakayala P Natarajan P Lopez-Ortiz C Dutta SK Levi A Reddy UK Population Genomics of Sweet Watermelon Population Genomics 2022 Cham Springer 856 901
Nimmakayala P, Natarajan P, Lopez-Ortiz C, Dutta SK, Levi A, Reddy UK. Population Genomics of Sweet Watermelon. Population Genomics. Cham: Springer; 2022. pp. 856–901.
53. Santa-Marıa GE Oliferuk S Moriconi JI KT-HAK-KUP transporters in major terrestrial photosynthetic organisms: a twenty years tale J Plant Physiol 2018 226 77 90 10.1016/j.jplph.2018.04.008 29704646
Santa-Marıa GE, Oliferuk S, Moriconi JI. KT-HAK-KUP transporters in major terrestrial photosynthetic organisms: a twenty years tale. J Plant Physiol. 2018;226:77–90.29704646 10.1016/j.jplph.2018.04.008
54. Wei J Tiika RJ Cui G Ma Y Yang H Duan H Transcriptome-wide identification and expression analysis of the KT/HAK/KUP family in Salicornia europaea L. under varied NaCl and KCl treatments PeerJ 2022 10 e12989 10.7717/peerj.12989 35261820
Wei J, Tiika RJ, Cui G, Ma Y, Yang H, Duan H. Transcriptome-wide identification and expression analysis of the KT/HAK/KUP family in Salicornia europaea L. under varied NaCl and KCl treatments. PeerJ. 2022;10:e12989.35261820 10.7717/peerj.12989
55. Maher C Stein L Ware D Evolution of Arabidopsis microRNA families through duplication events Genome Res 2006 16 510 9 10.1101/gr.4680506 16520461
Maher C, Stein L, Ware D. Evolution of Arabidopsis microRNA families through duplication events. Genome Res. 2006;16:510–9.16520461 10.1101/gr.4680506
56. Hashemipetroudi SH Arab M Heidari P Kuhlmann M Genome-wide analysis of the laccase (LAC) gene family in Aeluropus littoralis: a focus on identification, evolution and expression patterns in response to abiotic stresses and ABA treatment Front Plant Sci 2023 14 1112354 10.3389/fpls.2023.1112354 36938021
Hashemipetroudi SH, Arab M, Heidari P, Kuhlmann M. Genome-wide analysis of the laccase (LAC) gene family in Aeluropus littoralis: a focus on identification, evolution and expression patterns in response to abiotic stresses and ABA treatment. Front Plant Sci. 2023;14:1112354.36938021 10.3389/fpls.2023.1112354
57. Yaghobi M Heidari P Genome-wide analysis of aquaporin gene family in Triticum turgidum and its expression profile in response to salt stress Genes (Basel) 2023 14 1 202 10.3390/genes14010202 36672943
Yaghobi M, Heidari P. Genome-wide analysis of aquaporin gene family in Triticum turgidum and its expression profile in response to salt stress. Genes (Basel). 2023;14(1):202.36672943 10.3390/genes14010202
58. Guo S Zhao S Sun H Wang X Wu S Lin T Ren Y Gao L Deng Y Zhang J Lu X Zhang H Shang J Gong G Wen C He N Tian S Li M Liu J Wang Y Zhu Y Jarret R Levi A Zhang X Huang S Fei Z Liu W Xu Y Resequencing of 414 cultivated and wild watermelon accessions identifies selection for fruit quality traits Nat Genet 2019 51 11 1616 23 10.1038/s41588-019-0518-4 31676863
Guo S, Zhao S, Sun H, Wang X, Wu S, Lin T, Ren Y, Gao L, Deng Y, Zhang J, Lu X, Zhang H, Shang J, Gong G, Wen C, He N, Tian S, Li M, Liu J, Wang Y, Zhu Y, Jarret R, Levi A, Zhang X, Huang S, Fei Z, Liu W, Xu Y. Resequencing of 414 cultivated and wild watermelon accessions identifies selection for fruit quality traits. Nat Genet. 2019;51(11):1616–23.31676863 10.1038/s41588-019-0518-4
59. Cvijovic I Good BH Desai MM The effect of strong purifying selection on genetic diversity Genetics 2018 209 4 1235 78 10.1534/genetics.118.301058 29844134
Cvijovic I, Good BH, Desai MM. The effect of strong purifying selection on genetic diversity. Genetics. 2018;209(4):1235–78.29844134 10.1534/genetics.118.301058
60. Gierth M Mäser P Schroeder JI The potassium transporter AtHAK5 functions in K+ deprivation-induced high-affinity K+ uptake and AKT1 K+ channel contribution to K+ uptake kinetics in Arabidopsis roots Plant Physiol 2005 137 3 1105 14 10.1104/pp.104.057216 15734909
Gierth M, Mäser P, Schroeder JI. The potassium transporter AtHAK5 functions in K+ deprivation-induced high-affinity K+ uptake and AKT1 K+ channel contribution to K+ uptake kinetics in Arabidopsis roots. Plant Physiol. 2005;137(3):1105–14.15734909 10.1104/pp.104.057216
61. Ragel P Ródenas R García-Martín E Andrés Z Villalta I Nieves-Cordones M Rivero RM Martínez V Pardo JM Quintero FJ Rubio F The CBL-interacting protein kinase CIPK23 regulates HAK5-mediated high-affinity K+ uptake in Arabidopsis roots Plant Physiol 2015 169 4 2863 73 26474642
Ragel P, Ródenas R, García-Martín E, Andrés Z, Villalta I, Nieves-Cordones M, Rivero RM, Martínez V, Pardo JM, Quintero FJ, Rubio F. The CBL-interacting protein kinase CIPK23 regulates HAK5-mediated high-affinity K+ uptake in Arabidopsis roots. Plant Physiol. 2015;169(4):2863–73.26474642
62. Hernandez-Garcia CM Finer JJ Identification and validation of promoters and cis-acting regulatory elements Plant Sci 2014 217 109 19 10.1016/j.plantsci.2013.12.007 24467902
Hernandez-Garcia CM, Finer JJ. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 2014;217:109–19.24467902 10.1016/j.plantsci.2013.12.007
