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

Plant materials and treatments

The wild seeds of S. europaea were collected from Liangcao Village, Jingtai County, Baiyin City, Gansu Province in China (37°21′2″N, 104°5′28″W). The seeds were disinfected with 2% NaClO solution for about 3 min and washed with distilled water and then germinated at 28 °C on filter paper in the dark for 72 h. The plantlets were transferred into containers with sterilized sand, and were irrigated with 1/2 Hoagland nutrient solution (pH = 5.7). The formulation of 1/2 Hoagland nutrient solution was: 2 mM KNO₃, 0.5 mM KH₂PO₄, 0.5 mM MgSO₄⋅7H₂O, 0.5 mM Ca(NO₃)₂⋅4H₂, 50 µM H₃BO₃, 10 µM MnCl₂⋅4H₂O, 1.6 µMZnSO₄⋅ 7H₂O, 0.6 µM CuSO₄, 0.05 µM Na₂MoO₄⋅ 2H₂O, 0.06 mM Fe-citrate ⋅2H₂O. All plantlets were grown in an artificial climate box with a temperature of 22 ± 2 °C, relative humidity of about 65% and a daily photoperiod of 16/8 h (day/night; the flux density was approximately 600 µmol/m² s). The nutrient solution was renewed every 3 days.

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 KNO₃ was substituted by 2 mM HNO₃, 0.5 mM KH₂PO₄ was substituted by 0.5 mM H₃PO₄) 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.

Sequence analyses of SeKUPs

The protein sequence of SeKUPs was translated by ORFs (Tian et al., 2019). The biochemical properties of the candidates SeKUPs were predicted using the ExPASy (Expert Protein Analysis System) tool (https://web.expasy.org/protparam/) (Feng et al., 2020), including the molecular weight (MW), isoelectric points (pI), extinction coefficients, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (GRAVY) (Gasteiger et al., 2003).

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).

Phylogenetic analysis of SeKUPs

The protein sequences for 13 AtKUPs, 27 OsHAKs, 17 ZmHAKs, and 27 VvKUPs were retrieved from NCBI (https://www.ncbi.nlm.nih.govorg), UniPort (https://www.uniprot.org) (Consortium, 2015) and maize website (https://maizesequence.org/), and the newly identified SeKUPs were selected to construct the phylogenetic tree by using MEGA 5.0 with 1,000 bootstrap replications of maximum likelihood. The phylogenetic tree was embellished by the online tool iTOL (http://itol.embl.de/).

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^® qPCR SYBR^® Green Master Mix kit (Yeasen Biotech Co., Ltd) per the manufacturer’s protocol. The RT-qPCR analysis was performed using the QuantStudio™ 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^−ΔΔCt (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.

Identification of SeKUPs in S. europaea

A total of 24 putative SeKUPs were obtained from S. europaea, which were designated as SeKUP1 - SeKUP24 based on the Blastp results of other plant KUP protein sequences as queries. Among the 24 SeKUPs, the predicted cDNA length varied from 1,641 bp (SeKUP-2) to 3,391 bp (SeKUP-24), and the predicted protein length varied from 101 bp (SeKUP-2) to 845 bp (SeKUP-6, -7, -8, and -9) (Table 1).

To further analyze the characteristics of SeKUPs with complete sequences, a total of 12 SeKUPs were predicted by ORF finder, including SeKUP-1, -3, -4, -6, -7, -8, -9, -10, -11, -12, -14 and -15 (Table S2). Then the MWs, pIs, estimated half-life, instability index, aliphatic index, and GRAVY of these 12 SeKUPs were also calculated. As shown in Table S2, the ORF protein length of the predicted 12 SeKUPs ranged from 772 bp (SeKUP-12) to 845 bp (SeKUP-6, -7, -8 and -9); the molecular weight (MW) varied from 86,940.07 kDa (SeKUP-1, -10, and -12) to 94,683.36 kDa (SeKUP-8); isoelectric point (pI) varied from 5.79 (SeKUP-6, -7, -9) to 8.08 (SeKUP-1, -10, -11, and -12), extinction coefficients varied from 107,565 (SeKUP-8) to 128,660 (SeKUP-3, -4), all estimated half-life >10 h, the instability index of 12 SeKUPs proteins was lower than 42 (with the exception of SeKUP-3), the aliphatic index varied from 105.5 (SeKUP-8) to 111.64 (SeKUP-15), and the GRAVY varied from 0.222 (SeKUP-11) to 0.367 (SeKUP-14, -15), respectively (Table S2). In summary, most SeKUPs of the same subfamily shared similar sequences characteristics (MW, pI estimated half-life, instability index, aliphatic index, and GRAVY).

Phylogenetic analysis of SeKUPs

For the KUP family, it is generally believed that the sequence containing the EA [ML] FADL motif is identified as a KUP member (Ou et al., 2018). Therefore, through analysis, 19 sequences in our data contain this motf. So we use these 19 sequences to construct the evolutionary tree. These SeKUPs were divided into four clusters (cluster I, II, III, and IV) by phylogenetic analysis (Fig. 1) through other KUP protein sequences from four model plants (Table S3). In addition, the SeKUPs members in clusters I, II, and III were further classified into sub-clusters Ia, Ib, IIa, IIb, and IIIa, IIIb, respectively. SeKUP -6, -7, -8, and -9 belonged to cluster I; SeKUP -1, -2, -3, -4, -5, -10, -11, -13, -17, -18, and -19 belonged to cluster II; SeKUP -14, -15, -16 belonged to cluster III and SeKUP-12 belonged to cluster IV (Fig. 1). In summary, cluster II is the most abundant and cluster IV is the least abundant in S. europaea.

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, SeKUP-1/10/12, -2, -3, -4, -7/9, -8/24, -17 and -18/19, and the relative expression levels of SeKUP-3 in the roots (R)-24 h, SeKUP-3 in the shoots (S)-48 h, SeKUP-4 in S-6 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 SeKUP-15, -16, -20, -21/22 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 SeKUP-2, -3, -6, -14, -18/19, and -21/22 in the shoots.