Transcriptome-wide identification and expression analysis of the KT/HAK/KUP family in Salicornia europaea L. under varied NaCl and KCl treatments

Background The KT/HAK/KUP (KUP) transporters play important roles in potassium (K+) uptake and translocation, regulation of osmotic potential, salt tolerance, root morphogenesis and plant development. However, the KUP family has not been systematically studied in the typical halophyte Salicornia europaea L., and the specific expression patterns of SeKUPs under NaCl condition and K+ deficiency are unknown. Methods In this study, SeKUPs were screened from PacBio transcriptome data of Salicornia europaea L. using bioinformatics. The identification, phylogenetic analysis and prediction of conserved motifs of SeKUPs were extensively explored. Moreover, the expression levels of 24 selected SeKUPs were assayed by real-time quantitative polymerase chain reaction (RT-qPCR). Results In this study, a total of 24 putative SeKUPs were identified in S. europaea. Nineteen SeKUPs with the fixed domain EA[ML]FADL were used to construct the phylogenetic tree, and they were divided into four clusters (clusters I–IV). MEME analysis identified 10 motifs in S. europaea, and the motif analysis suggested that 19 of the identified SeKUPs had at least four K+ transporter motifs existed in all SeKUPs (with the exception of SeKUP-2). The RT-qPCR analysis showed that the expression levels of most SeKUPs were significantly up-regulated in S. europaea when they were exposed to K+ deficiency and high salinity, implying that these SeKUPs may play a key role in the absorption and transport of K+ and Na+ in S. europaea. Discussions Our results laid the foundation for revealing the salt tolerance mechanism of SeKUPs, and provided key candidate genes for further studies on the function of KUP family in S. europaea.


INTRODUCTION
Salt stress is one of the most important environmental factors affecting plant growth and development (Nabati et al., 2011). The excessive salt concentration in soil causes reduction in water potential, ions toxicity, osmotic stresses and induced secondary stress which even lead to plants' death (Munns & Tester, 2008). Halophytes are a special plant species: they can complete their life cycle in a saline environment of at least 200 mM NaCl condition (Flowers, Glenn & Volkov, 2019). Most halophytes are able to maintain the relative stability of potassium ion (K + ) content in the above-ground organs of plants in a high salt concentration environment (Flowers, Troke & Yeo, 1977), such as Lycium ruthenicum (Dai et al., 2019), Phragmites australis (Takahashi et al., 2007a) and Mesembryanthemum crystallinum (Su et al., 2002).
K + is an essential mineral for plant growth and development and is also the most abundant monovalent cation in plants, accounting for approximately 2% to 10% of plant dry weight (Clarkson & Hanson, 1980), and it plays a significant role in various physiological and biochemical processes, for instance, abiotic stress adaptation, stomatal movement, enzyme function and signal transduction (Véry & Sentenac, 2003). According to the transport characteristics of K + , K + transport families are divided into four types: Trk/HKT (tandem-pore K + channels) family, KT (K + transporter)/HAK (high-affinity K + )/KUP (K + uptake) family, CHX (cation/hydrogen exchanger) family and KEA (K + efflux antiporter) family (Gupta et al., 2008;Mäser et al., 2001). Among them, the KT/HAK/KUP (KUP) family belonging to the APC (amino acid polyamine organization) superfamily, is the largest and widely distributed in bacteria, fungi, and plants, but has not yet been identified in animal cells (Corratgé-Faillie et al., 2010). The KUP transporters were first identified in Arabidopsis thaliana (KUP1/KT1 and KUP2/KT2) and Hordeum vulgare (HAK1); thus, the composite name KUP, is widely used to refer to the whole family in plants (Véry et al., 2014;Epstein & Kim, 1971;Bañuelos et al., 1995). In the early stage, the KUP family was divided into four clusters (I-IV) (Rubio, Guillermo & Alonso, 2010). Recently, researchers discovered that this family has been re-divided into five clusters (clusters I-V) (Nieves-Cordones et al., 2016), and the main reason for this phenomenon is due to species diversity.
Firstly, the different cluster members have different physiological functions. The cluster I members can improve the absorption capacity of root system to K + under K + deficiency condition, such as AtHAK5 (A. thaliana), OsHAK1 (Oryza sativa) and SiHAK1 (Setaria italica) (Rubio, Guillermo & Alonso, 2010;Zhang et al., 2018). Members of cluster II have diverse functions in plant growth and development. For example, VvKUP2 (Vitis vinifera) can promote the expansion of berry epidermal cells (Davies et al., 2006;Elumalai, Nagpal & Reed, 2002). The members of cluster III can maintain K + /Na + homeostasis, like HcKUP12 (Halostachys capsica) (Yang & Wang, 2015) and PhaHAK5 (Phragmites australis) (Takahashi et al., 2007b). However, members in clusters IV and V have not yet been adequately studied (Bañuelos et al., 2002). Furthermore, some KUPs have been demonstrated to protect plants against salt stress. For instance, the constitutive overexpression of OsHAK5 in tobacco improved K + accumulation under salt stress (Elumalai, Nagpal & Reed, 2002). AtHAK11 and McHAK2 (Mesembryanthemum crystallinum) can promote the uptake of K + when plants are under salt stress (Su et al., 2002;Maathuis, 2006). These data indicate that members of the KUP family play critical roles in the uptake and transport of K + and in regulation of plant growth, development, and abiotic stress tolerance. Salicornia europaea L., a succulent halophyte, belongs to the family of Amaranthaceae, and it is a typical salt-resistant predominant species in the world (Nikalje et al., 2018). In the long-term evolutionary process, this special plant has gradually formed a strong salt tolerance mechanism in extremely saline environments. It can tolerate soil with more than 1,000 mM NaCl (Flowers & Colmer, 2008;Ozawa, Jianmei & Fujii, 2007), also accumulates large amounts of Na + than K + and compartmentalize Na + in the vacuole (Lv et al., 2012). Meanwhile, some research results show that S. europaea can still maintain a relatively stable K + /Na + even under increasing salt concentrations and longer treatment time (Wang et al., 2009;Fan et al., 2013), implying that S. europaea has a strong K + transport system under salt condition. Therefore, it is meaningful to elucidate the mechanism of K + uptake in S. europaea. However, the information about K + uptake family in S. europaea remains unknown.
In this study, we identified SeKUPs in S. europaea using PacBio sequencing system data (Tiika et al., 2021). We thoroughly performed multiple sequence alignment, presence of conserved motifs in the proteins, phylogenetic analysis, and real-time quantitative polymerase chain reaction (RT-qPCR) of SeKUPs in different tissues of S. europaea in response to salinity and K + deficiency. This study provides an important theoretical basis for the mechanism of K + uptake in S. europaea.
The four week old plantlets were subjected to NaCl and K + treatments. The seedlings were exposed to 1/2 Hoagland nutrient solution plus NaCl (0 mM, 50 mM and 200 mM) for a period of 0 h, 6 h, 24 h and 48 h, respectively. For K + treatment, the seedlings were exposed to modified 1/2 Hoagland nutrient solution (2 mM KNO 3 was substituted by 2 mM HNO 3 , 0.5 mM KH 2 PO 4 was substituted by 0.5 mM H 3 PO 4 ) plus 0.01 mM KCl (K + deficiency) or 2.5 mM KCl (normal K + ) for 0 h, 6 h, 24 h, and 48 h. Samples of shoots and roots were collected separately and quickly frozen in liquid nitrogen, and stored at −80 • C for RNA extraction.

Identification of the SeKUPs in S. europaea
We downloaded the transcriptome sequence of S. europaea from the NCBI database (https://www.ncbi.nlm.nih.gov/sra) (Accession number: PRJNA725943) (Tiika et al., 2021). Keywords related to potassium transport proteins were used to search candidate SeKUPs in S. europaea based on the transcriptome database. The amino acids of SeKUPs were predicted by finder searches for open reading frames (ORFs) (https: //www.ncbi.nlm.nih.gov/gorf/gorf.html), then were further identified (E-value <1e −5 ) by Blastp (protein-protein BLAST) search from NCBI. Finally, the sequences were subjected to conserved domains validation by InterProScan. We numbered the candidate SeKUPs by using the same overlapping prefix ''Se'' for S. europaea.
The conserved motifs of the deduced SeKUPs proteins were identified through MEME (Multiple Expectation Maximization for motif Elicitation) version 5.3.3 ( http://meme-suite.org/doc/cite.html) using the following parameters: the number of motifs searched was set to 10 and the range of the motif length was set to 5-50 aa (Bailey et al., 2015). All motifs were further annotated with InterProScan (http://www.ebi.ac.uk/interpro/) (Mulder & Apweiler, 2007).

RT-qPCR analysis
Total RNA was extracted from root and shoot tissues using TransZol Up Plus RNA Kit (Lot#M31018) referring to the manufacturer's instructions. The RNA quantity and quality were determined using a TGen Spectrophotometer (TianGen) based on the A260 nm/A280 nm and A260 nm/A230 nm ratio. Evo M-MLV RT Kit (AG11705, Accurate Biotechnology) was used to reverse transcribe the total RNA into cDNA and for removal of genomic DNA mixed in the cDNA before RT-qPCR analysis, following the manufacturer's protocol.
For RT-qPCR analysis, primers were designed based on mRNA sequences, using Primer 5.0 software and synthesized by TsingKe Biological Technology Co., Ltd. (Xi'an, China). The S. europaea Ubiquitin-conjugating (SeUBC) gene was used as the reference gene (Xiao et al., 2015). Three biological repeats were conducted and triplicate quantitative assays for each replicate were performed on 0.5 µL of each cDNA dilution using Heiff R qPCR SYBR R Green Master Mix kit (Yeasen Biotech Co., Ltd) per the manufacturer's protocol. The RT-qPCR analysis was performed using the QuantStudio TM 5 Real-Time PCR Instrument (ABI). Cycling parameters were: 95 • C for 5 min, 40 cycles at 95 • C for 10 s, and 60 • C for 30 s. The relative expression of the SeKUPs was calculated according to 2 − Ct (Livak & Schmittgen, 2001). The primer sequences for the SeKUPs and the housekeeping gene are listed in Table S1, and some SeKUPs share a pair of primers.

Data analysis
All values reported under gene expression levels are presented as means ± SE (n = 3). The significance level among means was analyzed by Duncan's multiple range tests (P ≤ 0.05) after performing a one-way ANOVA analysis using SPSS statistical software (Ver. 25.0, SPSS Inc., Chicago, IL, USA), and all histograms were generated using GraphPad Prism8.0.

Conserved motif analysis of SeKUPs
A total of 10 conserved motifs in putative SeKUP proteins were identified and designated as motifs (1-10) (Fig. 2). More detailed information on all conserved motifs can be found in Table S4. As revealed by our InterProScan search, most of the conserved motifs were found within the sequence of the K + transporters, with the exception of motif 10 (Table S4). As shown in Fig. 2, with the exception of SeKUP-2, all the identified SeKUPs contained at least four K + transporter motifs. In addition, motifs 1, 2, 6, 7, and 8 had the most amino acid sequences, whereas motif 10 contained the fewest protein sequences (Table S4). The majority of SeKUPs proteins contain the same types of motifs.

Expression patterns of SeKUPs under K + deficiency
Previous studies have demonstrated that the expression levels of KUPs are generally regulated by the concentration of K + (Zhou et al., 2020), such as OsHAK1 (Chen et al., 2015) in O. sativa and TaHAK1 in Triticum aestivum (Cheng et al., 2018). In addition, further studies have shown that members of the KUP family play an important role in the high affinity K + uptake process under K + deficiency condition (Rubio, Guillermo & Alonso, 2010;Santa-María, Oliferuk & Moriconi, 2018). To investigate the expression divergence of SeKUP s in response to different K + conditions in S. europaea, we analyzed the expression patterns of SeKUPs in the roots and in the shoots under 2.5 mM KCl (normal growth K + condition) and 0.01 mM KCl (K + deficiency) for different time periods (Table S5). Under the normal growth condition (2.5 mM KCl), the majority of SeKUPs were expressed in both shoots and roots, except for SeKUP-7/9, which was mainly expressed in the shoots. With the extension of treatment time under 2.5 mM KCl, most of the SeKUPs were induced more in the shoots than in the roots, and peaked at 24 h of treatment (Fig. 3). Under K + deficiency condition, some SeKUPs were induced significantly compared to the control (2.5 mM KCl treatment) both in the roots and shoots with time prolonged, for example, , and the relative expression h and SeKUP-7/9 in R-6 h were 8.7, 9.9, 9.6 and 8.7 times higher than their respective controls. Differently, SeKUP-6 showed induced expression in the roots and inhibited expression in the shoots. Besides, some SeKUPs like  exhibited reduced expression in the roots, but they were induced significantly in the shoots than control at short time treatment (6 h). Compared to the control, the expression of SeKUP-23 under K + deficiency were reduced at 6 h and 24 h treatment, then returned to the normal expression at 48 h treatment.

Expression patterns of SeKUPs under NaCl treatments
Studies have found that members of the KUP family are also involved in plant salt stress responses and regulate salt tolerance through a series of mechanisms (Chen et al., 2015;Horie et al., 2011). To further analyze the expression patterns of SeKUPs under salinity, we exposed the seedlings to 50 mM NaCl and 200 mM NaCl treatments for different times (0 h, 6 h, 24 h and 48 h) (Table S6, Fig. 4). With NaCl application, the majority of SeKUPs were induced in both shoots and roots, and the relative expression levels increased with time, and then peaked at 24 h, except for SeKUP-17 and 23 (expression was inhibited in both shoots and roots). Notably, the transcript abundance of SeKUP-2, -3, -6, -8/24, -15, -16 was 6.1 to 36.5 fold higher in the shoots under 50 mM NaCl condition at 24 h than at 0 h, and the values under 200 mM NaCl condition at 24 h were 9.3 to 45.7 fold higher compared to 0 h. Some SeKUPs like SeKUP-18/19 and SeKUP-20 were inhibited in the roots, while they were induced significantly in the shoots with increasing time under 50 mM and 200 mM NaCl treatments. Compared with 50 mM NaCl treatment, 200 mM NaCl significantly induced the expression of  in the shoots.
Conserved domains are the core of a protein family and have important functions in genes. At present, several typical conserved protein domains have been found in KUP family members, such as GVVYGDLGTSPLY (Rodríguez-Navarro, 2000) and LAYMGQAA, but the conserved domains vary among species. Although the conserved structure of KUP family is different, it has some relatively conserved amino acid domains, the highly conserved domains were searched by motif analysis to speculate whether these family members have functional differences during evolution (Wang et al., 2018). The results showed that 19 Values are means ± standard errors (SEs) (n = 3) and bars indicate SEs. Different letters (Duncan's test, p < 0.05) reflect the significant differences among different treatment times under the same NaCl concentration, respectively. The gene name is on the top left of each column graph. The seedlings of S. europaea are grown in the 1/2 Hoagland nutrient solution, and four weeks old seedlings are treated with 1/2 Hoagland nutrient solution plus different NaCl concentrations for varied times. The relative expression levels of all SeKUPs are calculated by 2 − Ct method, and 0 mM NaCl-0 h-root is used as the standard control. Some SeKUPs with ''/'' represent that they shared the same primers and the same expression patterns.
Full  (Ou et al., 2018), P. bretschneideri (Wang et al., 2018) P. persica (Song, Ma & Yu, 2015) and T. aestivum (Cheng et al., 2018). MEME revealed motifs that are conserved in the proteins originating from all four clusters. We searched for 10 motifs in S. europaea and 90% belonged to K + transporters. Motif analysis suggested that 19 of the identified SeKUPs had at least four typical motifs of K + transporters, with the exception of SeKUP2. A similar phenomenon also appeared in the motif analysis of O. sativa (Gupta et al., 2008). Although some homologous SeKUPs had different motifs structures (such as SeKUP-2/13), the majority of SeKUPs within the same subgroup shared similar motifs, and a similar number of motifs were present in SeKUPs proteins from each of the four clusters, indicating that the classification of SeKUPs was further supported by conserved motifs, with each subgroup sharing similar motifs. In S. europaea, with the exception of SeKUP-2, -13, -16, and -24, all SeKUPs contained 10 motifs, this phenomenon is similar to results in M. esculenta, where all MeKUPs contained 16 motifs with the exception of MeKUP-1, -7, -9, -10, -13, -15, -16, and -17 (Ou et al., 2018). These results support the high conservatism of sequences among KUP subgroup members. In our study, 19 SeKUPs were classified into four clusters based on their evolutionary relationships (Fig. 2). This is consistent with previous classifications of the KUP family in A. thaliana, O. sativa, V. vinifera and Z. mays (Zhang et al., 2012;Gupta et al., 2008). The results showed that most of the SeKUPs members were concentrated in cluster II. The number distribution of KUPs in the four clusters varied greatly, but this situation was consistent with previous studies that distributed KUPs unevenly in different clusters among angiosperms (Nieves-Cordones et al., 2016). Previous studies indicated that KUPs are widely expressed in different tissues of plants, such as roots, stems, leaves, flowers, and fruits (Corratgé-Faillie et al., 2010;Ahn, Shin & Schachtman, 2004). In the present study, we observed the consistent phenomenon that most SeKUPs were expressed in the shoots and roots, implying that they might play important roles in both shoots and roots. Besides, SeKUPs in the same cluster exhibited similar expression patterns. The representative members of cluster I, such as OsHAK1, OsHAK5 in O. sativa (Chen et al., 2015), and PbrHAK1 in P. bretschneideri (Wang et al., 2018) were reported to be induced by K + starvation, and they mainly mediate high affinity K + transport. SeKUP-6, -7, and -9 belonged to cluster I, and their expression abundance was significantly induced under K + deficiency than under normal K + condition, especially in the roots, implying that they might be mainly responsible for K + transport with high affinity. Meanwhile, the expression patterns of SeKUPs members from cluster II showed similar changes under normal K + condition and under K + deficiency (Gupta et al., 2008;Rubio, Guillermo & Alonso, 2010;Santa-María, Oliferuk & Moriconi, 2018). Previous reports have shown that members of the cluster II, for example, HvHAK2 in barley, AtKUP1, and AtKUP2 have different K + transport activities in dicotyledons (Véry et al., 2014). Our consistent results revealed that SeKUPs members from cluster II might be simultaneously involved in highaffinity and low-affinity K + absorption, and our speculation needs to be further validated.
Transcriptional regulation of K + transporter genes represents a major mechanism in plant responses to K + deficiency, and expression pattern analysis can provide insight into the potential functions of the SeKUPs in S. europaea.
The expression of SeKUPs was affected not only by the concentration of K + in the medium, but also by NaCl in the medium. Similar phenomenon also occurred in other plants with KUPs (Ou et al., 2018;Cheng et al., 2018), implying that these up-regulated KUPs may play a potential role under salt stress. In our study, we found that 22 SeKUPs were significantly up-regulated by salt stress, indicating that they could play a potential function under NaCl treatment, and further functional verification need to be explored among them. The up-regulation of SeKUP-16 was the most significant compared with the control, suggesting that SeKUP-16 might be a candidate gene for the adaptation of S. europaea to saline environment (Fig. 4). In addition, we found that the expression levels of some SeKUPs differed between shoots and roots, and that some SeKUPs were greatly suppressed under salt treatments. This is similar to Camellia sinensis (CsHAK17 ) , implying that they are not sensitive to high NaCl and K + deficiency.

CONCLUSIONS
In this study, we use Pac-Bio sequencing transcriptome data to discover 24 SeKUPs from S. europaea. Through conservative domain verification and motif analysis, we found that 19 SeKUPs have a fixed domain sequence (EA[ML]FADL) and were used to construct phylogenetic tree. Based on the phylogenetic relationships, the 19 SeKUPs could be divided into four clusters: I, II, III, and IV, in addition, clusters I to III were subdivided into subclusters a and b, rspectively. The RT-qPCR further validated the key role of 24 SeKUPs under abiotic stresses (salt and K + deficiency) in S. europaea.