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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

BMC Genomics volume 25, Article number: 836 (2024) Cite this article

Abstract

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.

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Introduction

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 [10,11,12,13,14,15]. 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 [11,12,13,14,15], 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 [26,27,28,29,30,31]. 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
figure 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

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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
figure 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

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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 [36,37,38]. 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
figure 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

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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
figure 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

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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
figure 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

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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 [17,18,19]. 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
figure 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

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Fig. 7
figure 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

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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
figure 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

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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)
Full size table

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 [28,29,30,31]. 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.

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.

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

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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.

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).

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Authors and Affiliations

  1. College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin, Heilongjiang, 150006, China

    Rui Cheng, Zhengxiang Zhao & Xuezheng Wang

  2. Key Laboratory of Biology and Genetic Improvement of Horticulture Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, Harbin, Heilongjiang, 150006, China

    Rui Cheng, Zhengxiang Zhao & Xuezheng Wang

  3. Huaiyin Institute of Agricultural Sciences of Xuhuai Region in Jiangsu, Huai’an, Jiangsu, 223001, China

    Rui Cheng, Yan Gu & Yudong Sun

  4. Huaiyin Institute of Technology, Huai’an, Jiangsu, 223003, China

    Yan Tang & Guodong Chen

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  1. Rui Cheng
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  3. Yan Tang
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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.

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Correspondence to Yudong Sun or Xuezheng Wang.

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Cheng, R., Zhao, Z., Tang, Y. et al. 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. BMC Genomics 25, 836 (2024). https://doi.org/10.1186/s12864-024-10712-5

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Keywords

BMC Genomics

ISSN: 1471-2164

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