Genome-wide identification and expression analysis of the CLC gene family in pomegranate (Punica granatum) reveals its role in salt resistance

doi:10.21203/rs.3.rs-54027/v1

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Genome-wide identification and expression analysis of the CLC gene family in pomegranate (Punica granatum) reveals its role in salt resistance

https://doi.org/10.21203/rs.3.rs-54027/v1

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published 11 Dec, 2020

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Backgrounds

Pomegranate (Punica granatum L.) is an important commercial fruit tree, with moderate tolerance to salinity. The balance of Cl⁻ and other anions in pomegranate tissues are affected by salinity, however, the accumulation patterns of anions are poorly understood. The chloride channel (CLC) gene family is involved in conducting Cl⁻, NO₃⁻, HCO₃⁻ and I⁻, but its characteristics have not been reported on pomegranate.

Results

In this study, we identified seven PgCLC genes, consisting of four antiporters and three channels, based on the presence of the gating glutamate (E) and the proton glutamate (E). Phylogenetic analysis revealed that seven PgCLCs were divided into two clades, with clade I containing the typical conserved regions GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III), whereas clade II not. Multiple sequence alignment revealed that PgCLC-B had a P [proline, Pro] residue in region I, which is suspected to be a NO₃^–/H⁺ exchanger, while PgCLC-C1, PgCLC-C2, PgCLC-D and PgCLC-G contained a S [serine, Ser] residue, with a high affinity to Cl⁻. We were determined the content of Cl⁻, NO₃⁻, H₂PO₄⁻, and SO₄²⁻ in pomegranate tissues after 18 days of salt treatments (0, 100, 200 and 300 mM NaCl). Compared with control, the Cl⁻ content increased sharply in tissues and was ranked as leaf > stem > root. The uptake of NO₃⁻ and SO₄²⁻was inhibited by high salinity, while that of H₂PO₄⁻ increased. The results of real-time reverse transcription PCR (qRT-PCR) revealed that PgCLC genes had tissue-specific expression patterns. The high expression levels of PgCLC-C1, PgCLC-C2 and PgCLC-D in leaves suggested they played roles in sequestrating Cl⁻into the vacuoles. However, the low expression levels of PgCLCs in roots might be contributed to the exclusion of Cl⁻from root cells. Also, the non-significantly changed concentration of NO₃⁻inleaves andthe up-regulated PgCLC-B indicated an acceleration of transporting NO₃⁻ into leaves to mitigate the nitrogen deficiency.

Conclusions

Our findings suggested that PgCLC genes played important roles in balance of Cl⁻ and NO₃⁻ in pomegranate tissues under salt stress. This study establishes a theoretical foundation for the further functional characterization of CLC genes in pomegranate.

Plant Molecular Biology and Genetics

Plant Physiology and Morphology

CLC gene family

phylogenetic analysis

anion content

expression pattern

NaCl stress

Chlorine is an essential micronutrient for plants, predominantly occurring in the form of the chloride anion (Cl⁻) [1, 2]. Cl⁻ is mainly involved in plant physiological activities, such as photosynthesis, regulation of stomatal opening and closing, stabilization of the membrane potential, regulation of intracellular pH gradients and electrical excitability [2]. Excess and/or deficiency of Cl⁻ leads to weak plant growth, low yield and poor quality [3, 4]. Under a hypersaline condition (a high concentration of NaCl), plants maintain a balance of Na⁺ and Cl⁻ at the cellular and the whole plant levels by excluding Na⁺ and Cl⁻ from the cell or sequestering them into the vacuoles and controlling Na⁺ and Cl⁻ transport from roots to the acrial parts [3, 5]. Previous studies on plant salt tolerance mostly focused on the effects of Na⁺, while few studies on Cl⁻ toxic effects [6].

Pomegranate (Punica granatum L.) is a salt-tolerant plant that is widely grown in arid and semiarid regions [7]. In our previous study, we found that the Cl⁻ content was two times more than Na⁺ content in pomegranate tissues, and the accumulation of other anions was also affected by various concentration of salinity [8]. CLC proteins are highly associated with uptake and transport of these anions, like Cl⁻, NO₃⁻, HCO₃⁻, I⁻, and Br⁻ [9–12]. However, the characteristics of CLC genes have not been reported on pomegranate. This study aimed to make a comprehensive, genome-wide inventory of the CLC gene family in pomegranate and to illuminate their roles in responses to NaCl stress.

The first chlorine channel (CLC) family gene (CLC-0) was identified from the electric organ of the marine ray (Torpedo marmorata) [13], and since then, some new members have been found in bacteria, yeast, mammals and plants [14]. In land plants, the first CLC gene, CLC-Nt1, was cloned in tobacco [15]. Subsequently, numerous CLC gene homologues were isolated from Arabidopsis [16], rice (Oryza sativa) [17], soybean and trifoliate orange (Poncirus trifoliata) [18], etc. All of the CLC proteins have a highly conserved voltage-gated chloride channel (Voltage-gate CLC) domain and two CBS (cystathionine beta synthase) domains of putative regulatory function [9]. CLC proteins may act as Cl⁻ channels or as Cl⁻/H⁺-exchangers (antiporters) [14]. The CLC gene family contains three highly conserved regions related to anion selectivity: GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III) [19]. If the x residue in the conserved region (I) is P [proline, Pro], NO₃⁻ is preferentially transported, whereas if it is substituted by S [serine, Ser], Cl⁻ is preferentially transported[20]. The first x residue in conserved region II and the next fourth residue of the conserved region III can both be E (Glu) residue, which are signatures for CLC antiporters [21, 22]. However, if any other amino acids are found at these positions, such as in AtCLCe, AtCLCf and AtCLCg, the CLC proteins may exert CLC channels activity [22]. The Cl⁻ channels mediate passive transport by dissipating pre-existing electrochemical gradients, while the antiporters mediate active transport by coupling with energy consumption to move the substrate against an electrochemical gradient [22]. In higher plants, CLC proteins are localized to the organelles membranes, including the plasma membrane, vacuole membrane, endoplasmic reticulum membrane, mitochondrial membrane, chloroplast membrane, etc[14]. These proteins play vital roles in the control of electrical excitability, turgor maintenance, stomatal movement, and ion homeostasis, as well as in responses to biotic and/or abiotic stress [23–25].

In Arabidopsis, there are seven reported CLCs: AtCLCa ~ AtCLCg, which play different roles in diverse cell organelles [26, 27]. Barbierbrygoo et al. [28] and Marmagne et al.[29] suggested that AtCLCa ~ AtCLCd and AtCLCg were clustered into a distinct branch, belonging to eukaryotic CLCs, while AtCLCe and AtCLCf are closely related to prokaryotic CLC channels. AtCLCa is an NO₃⁻/H⁺ exchangers localized in the vacuolar membrane, which is critically involved in this nitrate accumulation in the vacuole [16]. AtCLCb, a vacuolar antiporter that shares 80% identity with AtCLCa, is highly expressed in young roots, hypocotyl and cotyledons [27]. AtCLCc is essential for the detoxification of the cytosol by sequestrating Cl⁻ into the vacuoles under salt stress, and it is strongly expressed in guard cells, pollen and roots [23]. AtCLCd and AtCLCf, both localized in Golgi membranes, may play a role in the acidification of the trans-Golgi vesicles network [29, 30], while AtCLCe is targeted to the thylakoid membranes in chloroplasts [29]. AtCLCg, the closest homolog to AtCLCc (62% identity), plays a physiological role in the Cl⁻ homeostasis during NaCl stress [31]. In other plants, many CLC genes are involved in anions transport and in the response to salt stress. For instance, the expression level of OsCLC-1 is upregulated in rice under NaCl stress [17]; PtrCLC genes are profoundly induced in orange by salt stress[18]; GmCLC1 has been found to enhanced salt tolerance in transgenic Arabidopsis seedlings by reducing the Cl⁻ accumulation in shoots[6]; and GsCLC-c2 over-expression contributes to Cl⁻ and NO₃⁻ homeostasis, and therefore confers wild soybean the salt tolerance[32].

Identification of CLCs in pomegranate

A HMM profile was used to identify the putative CLC genes in the pomegranate genome. All seven putative CLC genes contained a highly conserved Volgate_CLC domain and two CBS domains, and they were named PgCLC-B to PgCLC-G according to the homologous AtCLCs (Table 1). The analysis of protein sequences showed that the PgCLCs contained 698 ~ 797 amino acids and had molecular weights of 75.7 ~ 87.9 kDa. The predicted isoelectric points (pI) of all the PgCLC proteins ranged from 5.86 to 8.44. The grand average of the hydrophobicity (GRAVY) values were all positive values, indicating that the PgCLCs were hydrophobic proteins. There were a number of transmembrane helices (TMs) in the PgCLCs, ranging from 9 to 11, which were associated with their location in the organelles membrane: six of PgCLCs were located on the plasma membrane and PgCLC-E was located on the chloroplast thylakoid membrane.

Table 1. Characteristics of the CLC genes in pomegranate

Gene ID	Name	AA	Mw(kDa)	pI	GRAVY	Orthologs	TMs	Subcellular location
CDL15_Pgr005627	PgCLC-B	797	87.9	6.49	0.259	AtCLC-B	9	Plasma membrane
CDL15_Pgr027626	PgCLC-C1	698	75.7	7.53	0.364	AtCLC-C	10	Plasma membrane
CDL15_Pgr013895	PgCLC-C2	717	78.1	5.92	0.325	AtCLC-C	10	Plasma membrane
CDL15_Pgr008552	PgCLC-D	788	86.9	8.57	0.175	AtCLC-D	11	Plasma membrane
CDL15_Pgr019810	PgCLC-E	764	81.3	5.86	0.188	AtCLC-E	10	Chloroplast thylakoid membrane
CDL15_Pgr012201	PgCLC-F	765	81.7	6.54	0.035	AtCLC-F	11	Plasma membrane
CDL15_Pgr015371	PgCLC-G	709	77.3	8.44	0.468	AtCLC-G	10	Plasma membrane

Phylogenetic analysis of the CLC gene family in pomegranate

To elucidate the evolutionary traits of the CLC gene family in land plants, we investigated fifteen interesting species that had available reference genome sequences. Our results showed two obvious clades of the CLC gene tree, clade I was the major group bearing a moderate support (BS=61%, Fig. S1) and clade II contained two subgroups (Fig. 1). The divergence of clades I and II might have occurred before the origin of land plants due to each clade consisting of taxa from embrophytes (Fig. 1). Phylogenetic analyses indicated multiple rounds of ancient gene expansion (Fig. 1). The diversity of gene copy number from different lineages (Fig.1A). The gene tree-species tree reconcilably identified a gene duplication (the red star in Fig.1B) with a strongly supported (BS=100, Fig.S1) topology of (core eudicots, core eudicots). A gene duplication (the purple star in Fig.1B) resulting in a topology of ((core eudicots, monocots), (core eudicots, monocots)) was identified as one duplicate shared by angiosperms. Our phylogenetic analyses also found gene expansion in seed plants, with a gene birth from an ancient gene duplication (the green star in Fig.1B) and a subsequent gene death. The tree topology [(angiosperms, gymnosperms) angiosperms] of the CLC-A/B/C/G genes (Fig.1) exhibited a gene loss event in gymnosperms.

Here, our phylogenetic results showed that seven putative CLC genes in the pomegranate genome originated before the divergence of land plants and were retained after experiencing six times of duplications, including at least one ancient core eudicots-specific duplication and one angiosperm-specific expansion (Fig. 1, Fig. S1).

Conserved motifs and residues of the CLC gene family

To further investigate the structural diversity of all CLCs in land plants, the conserved motifs and regions were analyzed. Here, a total of 10 motifs were selected, referring as motif 1-10, and five representative species of each taxa were shown (Fig. 2B, Fig. S1B). Different motif patterns were clearly observed in the two clades, as mentioned above (Fig. 1B). For clade I, most of the CLCs possessed 10 motifs (Fig. 2B, C; Fig. S2). For clade II, most of the CLC-E and CLC-F proteins possessed four motifs: 6, 1, 8 and 2, which were shared by all of the CLCs of clade I. Three conserved regions GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III) were included in motif 9, motif 6 and motif 1, respectively (Fig. 2B, C and D). Three highly conserved regions of the CLC gene family were shared by members of clade I, whereas they were not shared by members of clade II (Fig. 2B, C; Fig. S2).

Additionally, to meticulously analyze the conserved regions of CLC proteins, multiple sequence alignment was performed. Members of the CLC-A/B subfamily had a P [proline, Pro] residue in the conserved region GxGIPE (I), while other proteins of the CLC-C, CLC-G and CLC-D subfamilies in clade I had a S [serine, Ser] residue in the conserved region I (Fig. 3A). These critical residues were recognized to have a close relation with anion selectivity. The P [proline, Pro] preferentially transported NO₃^–, whereas the S [serine, Ser] preferentially transported Cl^–(Fig. 3A). The presence of the conserved gating glutamate (E) in conserved region (II) and the proton glutamate (E) residues in the next fourth residue of the conserved region (III) were signatures for CLC antiporters. Otherwise, the conserved gating glutamate (E) of CLC-G subfamily and the proton glutamate (E) residue of CLC-E and CLC-F subfamilies were substituted by other amino acids (Fig. 3A), which suggested that the members of these three subfamilies might be CLC ion channels. Based on these results, we assumed that four CLC proteins (PgCLC-B, PgCLC-C1, PgCLC-C2 and PgCLC-D) were CLC antiporters, while the other three PgCLCs (PgCLC-E, PgCLC-F and PgCLC-G) were likely CLC channels (Fig. 3A, B).

Gene strectures of PgCLC genes

The gene structure results showed that PgCLC-C2, PgCLC-C1, PgCLC-G and PgCLC-B contained five introns and six exons. PgCLC-E and PgCLC-F contained five exons and nine exons, respectively. A similar exon/intron structure profile, concerning intron number, intron phase, and exon length, was observed in PgCLC-C2, PgCLC-C1 and PgCLC-G. In general, the PgCLC genes had a distinct intron-exon arrangement style (Fig. 4).

Growth characteristics and anion content in pomegranate tissues

With the increasing concentration of salinity, the dry weights of roots and stems showed no significant changes among each treatment (Table S4, p < 0.05). The leaf weight and total dry weight first increased and then decreased, reaching a peak at the 100 mM salinity level.

As shown in Figure 5A, the contents of Cl⁻ in pomegranate roots, stems and leaves significantly increased with the increasing concentration of salinity (p < 0.05). Under 300 mM NaCl stress, the levels of Cl⁻ in roots, stems and leaves increased 6.19, 5.29 and 7.42 times, compared with the control, respectively. The contents of Cl⁻ in plant tissues was ranked as leaf > stem > root. The NO₃⁻ contents in roots first increased and then decreased, with the highest value at 100 mM salinity. However, the NO₃⁻ contents in stems and leaves had no obvious changes (except NO₃⁻ content in stem at 300 mM NaCl, p < 0.05) among the different treatments. The NO₃⁻contents in plant tissues was ranked as root > stem > leaf (Fig. 5B). By contrast, the H₂PO₄⁻ contents in roots increased along with the increaseing salinity, while no significant changes were observed in most leaf and stem samples (p < 0.05). Moreover, we found that H₂PO₄⁻was mainly accumulated in stems (Fig. 5C). First an increase and then a decrease of the SO₄²⁻ contents in roots and leaves were observed with peaks at 100 mM salinity. The leaf content of SO₄²⁻ fell sharply under higher salinity (> 200 mM NaCl). The results demonstrated that the accumulation of SO₄²⁻ mainly occurred in roots (Fig. 5D).

Expression patterns of PgCLC genes under NaCl stress

To further investigate the expression patterns of PgCLCs, we performed the qRT-PCR analysis in pomegranate roots and leaves. The results showed that all the PgCLC genes had tissue-specific expression patterns, with high expression levels in leaves and low expression levels in roots (Fig. 6). Notably, when plants were subjected to salinity, the expression levels of all the tested PgCLCs were upregulated in pomegranate leaves, but were down-regulated or not obviously changed in roots (p< 0.01). For instance, the relative expression levels of PgCLC-B, PgCLC-C1, PgCLC-C1 and PgCLC-D in leaves increased with the increasing salinity; meanwhile, those of PgCLC-E, PgCLC-F and PgCLC-G in leaves significantly increased at high salinity (200 mM). Also, the expression levels of PgCLC-B, PgCLC-F and PgCLC-G in roots decreased and those of PgCLC-C1, PgCLC-C2, PgCLC-D and PgCLC-E in roots first decreased at 100 mM salinity level and then recovered slightly at 200 mM and/or 300 mM salinity levels (Fig. 6). Under 300mM NaCl stress, the expression levels of PgCLC-C1, PgCLC-C2 and PgCLC-F in leaves increased by more than 16-fold relative to those of the controls.

Correlation between the anion contents and the expression levels of PgCLC genes

Correlation analysis showed that the PgCLC genes were positively correlated with each other (Fig. 7, p < 0.05). The Cl⁻ content had significantly positive correlations with PgCLC-B, PgCLC-C1, PgCLC-C2 and PgCLC-D, while the SO₄²⁻ content had significantly negative correlations with these genes. Meanwhile, the contents of Cl⁻ and SO₄²⁻ were negatively correlated with each other (p < 0.05). A significantly negative correlation between the NO₃⁻ content and the expression level of PgCLC-B, and a significantly positive correlation between the SO₄²⁻ were found. There was no significant relationship between the H₂PO₄⁻ content and the other indexes (Fig. 7). These findings suggested that accumulation of Cl⁻, SO₄²⁻ and NO₃⁻in pomegranate tissues was associated with the expression levels of PgCLC genes under salt stress.

Identification of the CLC gene family in pomegranate

The CLC gene family is an evolutionarily well-conserved family, which has been found in prokaryotes and eukaryotes [9, 14]. CLC channels form two-pore homodimers with two monomers, and each monomer has its own pore through which Cl⁻ and other anions (HCO₃⁻, I⁻, and NO₃⁻) may be conducted [9, 33]. CLC transporters and channels have regulatory functions when ATP, ADP, AMP, or adenosine are bound at the CBS domains [33]. In pomegranate, each CLCs contains one voltage-gated CLC domain near the N-terminus and two CBS domains at the C-terminus. The specific effect implies that individual CLC transporters and channels are sensitive to the cell’s metabolic state [9, 22]. We found that PgCLCs were all hydrophobic proteins with numerous transmembrane helices (TMs) that were associated with their functions of allowing ion transport through membranes [34]. Also, PgCLC-E was found to be located on the chloroplast thylakoid membrane and might be related to photosynthesis activity, which was consistent with previous studies on AtCLCe by Marmagne et al [29].

Phylogenic relationships of the CLC gene family

Numerous early whole-genome duplication (WGD) events in plants, including the gamma event shared by core-eudicots [35], the WGD event shared by angiosperms [36-38], and the seed-plant WGD event [36, 37], contribute to gene duplications. The diversity of gene copy numbers from different lineages (Fig.1A) might be related to the rounds of WGD events shared with the taxon [39]. Based on the phylogenic analysis of the CLC gene family in 15 land plants, seven PgCLCs were divided into two clades, with clade I belonging to a eukaryotic branch and the clade II belonging to a bacterial branch [28, 29]. The divergence of clade I and clade II might have occurred before the origin of land plants due to each clade consisting of taxa from embrophytes (Fig. 1). The CLCs topology was consistent with that of Arabidopsis [28, 29], tobacco [40], tea (Camellia sinensis) [19] and trifoliate orange [18]. Phylogenetic analysis also indicated multiple rounds of ancient gene expansion (Fig. 1). For example, the gene duplication between CLC-C1 and CLC-C2 (the red star in Fig.1B) was supported by the duplication burst shared by core eudicots [41]. The gene duplication between the CLC-C and CLC-G subfamilies was due to one duplicate shared with angiosperms (the purple star in Fig.1B) [41]. Our phylogenetic analyses also found a gene expansion in seed plants, with a gene birth from an ancient gene duplication (the green star in Fig.1B) and a subsequent gene death. The CLC-A/B/C/G genes (Fig.1) exhibited a gene loss event in gymnosperms after experiencing the seed-plant WGD event [36, 37] despite the fact that the absence of the gene might have resulted from the putative incompleteness of the genome assembly and annotation. Recent phylogenetic studies have also found land plant-scale gene birth and expansion, such as in the CYP75 gene family [39] and GH28 gene family [36].

Additionally, the distributions of conserved motifs and conserved regions were consistent with the phylogenetic relationships (Fig. 2). Three highly conserved regions of the CLC gene family, GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III) were shared by the members of clade I, whereas thesy were not shared by the members of clade II. This finding indicated that the divergence of clades I and clade II might be due to the presence of these conserved regions (Fig. 2C). Our study also found that the x residue in conserved region (I) of the CLC-A/B subfamily was P [proline, Pro] (Fig. 3; Fig. S2), which preferentially transports NO₃^–[20], while that of the CLC-C, CLC-D and CLC-G subfamily was S [serine, Ser] (Fig. 3; Fig. S2), which preferentially transports Cl^–[20]. Thus, PgCLC-B is likely a NO₃^–/H⁺ exchanger that mainly transports NO₃^–rather than Cl^– [16, 27], while PgCLC-C, PgCLC-D and PgCLC-G have high affinity for Cl^– [16, 27]. The presence of a gating glutamate (E) residue and proton glutamate (E) residue was a signature for CLC antiporters [21, 22]. However, if one or both of the Glu (E) residues were substituted by any other amino acids in the conserved region, the CLC proteins might exhibit CLC channels activity [22]. Therefore, we assumed that PgCLC-B, PgCLC-C1, PgCLC-C2 and PgCLC-D were CLC antiporters and the PgCLC-E, PgCLC-F and PgCLC-G were CLC channels. Our results were in line with the findings in Arabidopsis [21, 22].

PgCLCs played roles in the anion accumulation under NaCl stress

As an essential micronutrient for plants, chloride (Cl⁻) is beneficial for plants at low concentrations in media [1, 2]. However, high salinity (mainly NaCl) may cause a perturbation of Na⁺ and Cl⁻ at both the cellular and whole plant levels [42], which affects the uptake and transport of other mineral ions, such as K⁺, Ca²⁺, Mg²⁺, H₂PO₄⁻, NO₃⁻ and SO₄²⁻[43, 44]. In this study, we focused on the anion accumulation in pomegranate tissues. CLC proteins are expressed on the cell membrane and conduct Cl⁻ or other anions, such as HCO₃⁻, I⁻, and NO₃⁻[9-12]. Compared with the controls, the expression levels of the PgCLCs were upregulated in leaves and downregulated or not significantly changed in roots (p < 0.01). The tissue-specific expression of seven PgCLCs indicated different mechanisms of anions transport in pomegranate roots and leaves. Our study found that the Cl⁻ contents in pomegranate tissues sharply increased with an order of leaf > stem > root (Fig. 5A), indicating a relatively strong ability for the pomegranate to transport and accumulate toxic ions in the acrial parts [45]. In leaves, the high expression levels of PgCLCs suggested the inclusion of Cl⁻into leaf cells. Individually, the expression levels of three antiporters PgCLC-C1, PgCLC-C2 and PgCLC-D were significantly positive with the Cl⁻content, which might be contributed to the sequestration of Cl⁻into leaf vacuoles [23, 46]. However, the low expression levels of PgCLCs in roots suggested the exclusion of Cl⁻from root cells. The recovery of PgCLC-C1, PgCLC-C2 and PgCLC-D in roots were contributed to the sequestration of Cl⁻into the root vacuoles at high salinity levels [23, 46]. Therefore, PgCLCs are believed to alleviate the deleterious effects of Cl⁻via excluding the Cl⁻from root cells and sequestrating Cl⁻ into leaf vacuoles [23, 46]. Similarly, some halophytes prefer to transport and accumulate detrimental ions in the acrial parts under salt stress [47, 48]. On the other hand, the low expression levels of PgCLC-E, PgCLC-F and PgCLC-G coding Cl⁻channels in leaves at moderate salinity levels (≤ 200 mM NaCl Fig. 7), suggested the capacity for pomegranate to inhibit the Cl⁻influx into organelles, such as chloroplast and Golgi apparatus [8, 29].

Additionally, the NO₃⁻ contents of pomegranate roots first increased and then decreased, and that of leaves not changed under salt stress (Fig. 5B, D). The increases of the Cl⁻ contents were concomitant with the decreases of the NO₃⁻ contents in pomegranate tissues, which could be due to the antagonism between Cl⁻ and NO₃⁻ [49]. The expression level of PgCLC-B (a NO₃^–/H⁺ exchanger) [16, 27], was significantly positive correlated with the Cl⁻ contents, and significantly negative correlated with the NO₃⁻content (p < 0.05). These results suggested that the decreased uptake of NO₃⁻in roots might be due to the inhibition of PgCLC-B activity under salt stress [16, 27]. The inhibition of nitrogen uptake was also associated with nitrate transporter (NRTs) [50, 51]. Meanwhile, the increased expression level of PgCLC-B in leaves indicated an acceleration of transporting NO₃⁻ into leaves to mitigate the nitrogen deficiency [27]. Teakle et al. [3] reported that the increased concentration of NO₃⁻ in media reduced the Cl⁻ content in leaves and then mitigated the foliar salt damage, the NO₃⁻/Cl⁻ even equaled to the K⁺/Na⁺, which was confirmed as one of the critical determinants of plant salt resistance [52, 53]. In pomegranate, it was observed that a low ratio of NO₃⁻/Cl⁻ might cause a reduction in growth [54] (data not showed).

In a ward, these findings suggested that PgCLC genes played important roles in balance of Cl⁻ and NO₃⁻ in pomegranate tissues under salt stress [10-12, 23]. While the accumulation pattern of SO₄²⁻ was associated with the other genes, such as sulfate transporters [55]. Wei et al. [18] found that PtrCLC genes were dramatically induced in response to NaCl stress, and PtrCLC6 showed a leaf-specific expression pattern in trifoliate orange. Zhang et al. [40] observed that all of the expressed NtCLC genes had a low expression level in tobacco roots under salt stress. Our findings are consistent with these reports. In addition, the functional characterization of each PgCLC genes need to further study.

In this study, we identified and characterized seven CLC genes in pomegranate. Phylogenetic analysis indicated that the PgCLCs were divided into two distinct clades, with a similar distribution of motifs and conserved regions in members of each clade. In pomegranate, the PgCLC genes displayed a tissue-specific expression pattern, with the high expression levels in leaves and the low expression levels in roots under salt stress. PgCLC genes are believed to play important roles in balance of Cl⁻ and NO₃⁻ in pomegranate tissues under salt stress. Our study provides the basis for the further functional characterization of the PgCLC genes.

Identification of the CLC gene family in pomegranate

A Hidden Markov Model (HMM) profile of the voltage-gated chloride channel (Voltage-gate CLC) domain (Accession no. PF00654) was employed to identify the putative CLC proteins from genome sequences using the software HMMER v3.1b1 [56] accorrding to the methods of Zhang et al. [39] with a cut-off E-value of ≤1e^-10. To construct a representative phylogeny across land plants, ten angiosperms (eight core eudicots and two monocots), two gymnosperms and three bryophytes were selected, including Arabidopsis thaliana, Citrus sinensis, Eucalyptus grandis, Eutrema salsugineum, Glycine max, Populus euphratica, Punica granatum, and Vitis vinifera as the core eudicots; Oryza sativa and Zostera marina as monocots; Ginkgo biloba and Pinus taeda as gymnosperms; and Marchantia polymorpha, Selaginella moellendorffii and Sphagnum fallax as bryophytes. Seven CLC proteins from Arabidopsis thaliana were obtained from the Arabidopsis Information Resource (TAIR) (http://www.arabidopsis.org/). The genome sequences of 14 other species were downloaded from URLs (Table S1). Firstly, all the putative CLC proteins were identified from the genomes of the 14 species. Subsequently, the CLC candidates were manually curated, and the nonredundant CLC proteins were further analyzed using the NCBI Conserved Domain Database (CDD, http://www.ncbi.nlm.nih.gov/cdd/) and SMART programs (http://smart.embl-heidelberg.de/) to confirm the presence of the Voltage-gate CLC domain. The theoretical isoelectric point (pI), molecular weight (Mw) and grand average of hydrophobicity (GRAVY) of seven PgCLC proteins were predicted using the Prot-Param tool (http://web.expasy.org/protparam/). The number of transmembrane helices (TMs) was predicted using TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) and TMpred (https://embnet.vital-it.ch/software/TMPRED_form.html). Subcellular localization was inferred with WoLF PSORT II (https://www.genscript.com/wolf-psort.html?src=leftbar).

Phylogenetic analysis of CLC gene family

To estimate the origin and divergence of CLC genes, an maximum likelihood (ML) tree of these genes was reconstructed using iQ-TREE and used to map on a species tree of land-plants, which is a part of the tree of life as inferred in the OneKP project [36], by using the methods in Zhang et al [57]. All of the puative CLC proteins were aligned using MUSCLE v3.8.31 [58] with the ‘auto’ setting. To improve the valid phylogeny signals, the low-quality alignment regions and incorrect sequences with apparent splice variants were removed [59]. Finally, a total of 113 putative CLC candidates were retained, including seven PgCLCs (Table S2). The conserved blocks were retained by Gblocks v0.91b [60], and then, phylogenetic analysis was preformed using iQ-TREE v2 [61] with the LG+R6 model, 1000 bootstraps, and the Shimodaira-Hasegawa-like aLRT (SH-aLRT) test. Putative functional homologs were identified from a gene clade that contained the query gene from Arabidopsis and was likely derived from an ancestral gene from land plants [57].

Conserved motifs, residues and gene stuctures prediction of CLC proteins

The conserved motifs and regions of all CLC proteins were predicted by the MEME tool (http://meme-suite.org/tools/meme). The maximum number of motifs was set to 10, and the optimum motif width was ≥ 6 and ≤ 50. Three conserved regions (GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III)) of the CLC gene family were searched by the MAST tool (http://meme-suite.org/tools/mast) with a sequence threshold ≤ 30 and an E-value ≤1e^-10 for motifs. Multiple sequence alignment of CLCs was performed by Clustal X v2.0 [62] and visualized by Jalview v1.0 [63]. The gene structure information of each PgCLCs was retrieved from the pomegranate genome and visualized using GSDS software (http://gsds.cbi.pku.edu.cn/).

Plant materials and growth conditions

Pomegranate cv. ‘Taishanhong’ cuttings (one-year-old, collected from Pomegranate repository of Nanjing Forestry University. China) were planted in a phytotron for six months, with a 28/22 °C day/night temperature, 60% humidity and 14 h light/10 h dark photoperiod. Hoagland’s nutrient solution [64] was supplied at the beginning of the experiment. A total of 24 pots (one plant per plot) were arranged in a completely randomized 3 blocks, and 8 pots per block, and every 2 pots were designed as a biological replicate. All plants were fertigated with half-strength Hoagland’s solution containing 0 (control), 100, 200, or 300 mM NaCl every six days, respectively. A saucer was placed under the containers to keep the soil moist. After 18 days of treatments, we harvested all plants separately to collect roots, stems, and leaves.

Anion content measurement

The dry weights of pomegranate roots, stems and leaves were determined after drying in a heating oven at 75 °C for 48 h. Dry samples were finely milled to pass through a 40-mesh sieve. Then 0.4 g of samples were treated with 50 mL of deionized water for 1 h in an ultrasonic extractor at room temperature, and then the obtained extracts were used to determine the contents of Cl⁻, NO₃⁻, H₂PO₄⁻, and SO₄²⁻ using an ion chromatography (ICS900 ion chromatographic system; AS4A-SC ion-exchange column, CD M-II electrical conductivity detector, mobile phase: Na₂CO₃/NaHCO₃ = 1.7/1.8 mM; Dionex, Sunnyvale, USA) [65].

Expression levels of PgCLCs by quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from fresh root and leaf samples using the BioTeke plant total RNA extraction kit (BioTeke Corporation, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was prepared using a reverse transcription kit-PrimeScript^TMRT reagent Kit with gDNA Eraser (TaKaRa Bio Tech Co., Ltd., Beijing, China). The primers of seven PgCLCs were designed with NCBI primer-BLAST (Table S2). Real-time RT-PCR (qRT-PCR) was performed using a 7500 fast Real-Time PCR system (Applied Biosystems, CA, USA) with three biological and three technical replicates for each cDNA sample, and the results were quantitatively analyzed by the 2^−ΔΔCT method [66]. Each reaction was carried out in a final volume of 20 μL, containing 10 μL of TB Green Premix Ex Taq, 0.4 μL of ROX Reference Dye II, 0.8 μL of upstream/downstream primers, 1 μL of cDNA template and 7 μL of ddH₂O. The PCR thermal cycler was set as follows: pre-denaturation at 95°C for 30 s; 40 cycles of 95°C for 5 s and 60°C for 34 s; the dissociation stage was set as follows: 95°C for 15 s, 60°C for 60 s and 95°C for 15 s. Pomegranate PgActin was used as an internal reference gene.

Data analysis

All data of the anion contents and qRT-PCR were analyzed with one-way ANOVA, and multiple comparisons were evaluated with the Turkey test (p < 0.01) using the SPSS program (Version 19.0. Chicago, IL, USA) based on the values of three complete randomized blocks. The correlation among variables was analyzed based on the ion content and expressional level of PgCLCs and visualized by a ‘corrplot’ package in R [67].

CLC: Chloride channel; CBS:Cystathionine Beta Synthase; Glu:glutamate; pI:Theoretical Isoelectric Point, Mw:Molecular Weight; GRAVY:Grand Average of Hydrophobicity; TMs:Transmembrane Helices; ML:Maximum Likelihood; WGD:Whole-genome Duplication; OneKP preoject:One Thousand Plant Project; SH-aLRT:Shimodaira-Hasegawa-like aLRT; qRT-PCR:Quantitative real-time polymerase chain reaction.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article (and its additional files).

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Initiative Project for Talents of Nanjing Forestry University (GXL2014070, GXL2018032), the Doctorate Fellowship Foundation of Nanjing Forestry University, and the Priority Academic Program Development of Jiangsu High Education Institutions (PAPD), the National Natural Science Foundation of China (31901341), the Natural Science Foundation of Jiangsu Province (BK20180768). These funding bodies took part in the design of the study and collection, analysis, and interpretation of data, and the writing of the manuscript, as well as in the open access payment.

Authors' contributions

CL and TZ analyzed and interpreted the phylogenetic analysis. YZ and JD performed the expression level examination of the stressed plants. CL was a major contributor in determining the ion content, analyzing data and writing the manuscript, and XZ and ZY revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Marschner P. Marschner's Mineral Nutrition of Higher Plants. 3rd ed. Australia: Academic press; 2012.
White PJ, Broadley MR. Chloride in soils and its uptake and movement within the plant: A Review. Ann Bot. 2001;88(6):967–88.
Teakle NL, Tyerman SD. Mechanisms of Cl^– transport contributing to salt tolerance. Plant Cell Environ. 2010;33(4):566–89.
Karaivazoglou NA, Papakosta DK, Divanidis S. Effect of chloride in irrigation water and form of nitrogen fertilizer on Virginia (flue-cured) tobacco. Field Crops Res. 2005;92(1):61–74.
Tregeagle JM, Tisdall JM, Tester M, Walker RR, Barrettlennard EG, Setter TL. Cl^– uptake, transport and accumulation in grapevine rootstocks of differing capacity for Cl^– exclusion. Funct Plant Biol. 2010;37(7):665–73.
Wei P, Wang L, Liu A, Yu B, Lam HM. GmCLC1 confers enhanced salt tolerance through regulating chloride accumulation in soybean. Front Plant Sci. 2016;7(113):1082.
Holl D, Hatib K, Bar-Ya'Akov I. Pomegranate: botany, horticulture, breeding. Hortic Rev. 2009;35(2):127–91.
Liu C, Yan M, Huang X, Yuan Z. Effects of NaCl stress on growth and ion homeostasis in pomegranate tissues. Eur J Hortic Sci. 2020;85(1):42–50.
Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48(D1):D265–8.
Miller AJ, Fan X, Orsel M, Smith SJ, Wells DM. Nitrate transport and signalling. J Exp Bot. 2007;58(9):2297.
Amtmann A, Armengaud P, Salt DE, Williams L. Effects of N, P, K and S on metabolism: new knowledge gained from multi-level analysis. Curr Opin Plant Biol. 2009;12(3):275–83.
Gojon A, Nacry P, Davidian JC. Root uptake regulation: a central process for NPS homeostasis in plants. Curr Opin Plant Biol. 2009;12(3):328.
Jentsch TJ, Steinmeyer K, Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature. 1990;348(6301):510–4.
Jentsch TJ. CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit Rev Biochem Mol. 2008;43(1):3–36.
Lurin C, Geelen D, Barbierbrygoo H, Guern J, Maurel C. Cloning and functional expression of a plant voltage-dependent chloride channel. Plant Cell. 1996;8(4):701–11.
De Angeli A, Monachello D, Ephritikhine G, Frachisse J, Thomine S, Gambale F, et al. The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature. 2006;442(7105):939–42.
Nakamura A, Fukuda A, Sakai S, Tanaka Y. Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.). Plant Cell Physiol. 2006;47(1):32–42.
Wei Q, Gu QQ, Wang N, Yang C, Peng S. Molecular cloning and characterization of the chloride channel gene family in trifoliate orange. Biol Plantarum. 2015;59(4):645–53.
Xing A, Ma Y, Wu Z, Nong S, Zhu J, Sun H, et al. Genome-wide identification and expression analysis of the CLC superfamily genes in tea plants (Camellia sinensis). Funct Integr Genomic. 2020;20:497–508.
Zifarelli G, Pusch M. CLC transport proteins in plants. FEBS Lett. 2010;584(10):2122–7.
Lisal J, Maduke M. Proton-coupled gating in chloride channels. Philos T Roy Soc B. 2009;364(1514):181–7.
Accardi A, Picollo A. CLC channels and transporters: proteins with borderline personalities. Biochim Biophys Acta. 2010;1798(8):1457–64.
Jossier M, Kroniewicz L, Dalmas F, Le TD, Ephritikhine G, Thomine S, et al. The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance. Plant J. 2010;64(4):563–76.
Wei Q, Liu Y, Zhou G, Li Q, Yang C, Peng SA. Overexpression of CsCLCc, a chloride channel gene from Poncirus trifoliata, enhances salt tolerance in Arabidopsis. Plant Mol Biol Rep. 2013;31(6):1548–57.
Jentsch TJ, Michael P. CLC chloride channels and transporters: structure, function, physiology, and disease. Physiol Rev. 2018;98(3):1493–590.
Jossier M, Kroniewicz L, Dalmas F, Thiec DL, Ephritikhine G, Thomine S, et al. The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance. Plant J. 2010;64(4):563–76.
Fechtbartenbach JVD, Bogner M, Dynowski M, Ludewig U. CLC-b-mediated NO₃^–/H⁺ exchange across the tonoplast of Arabidopsis vacuoles. Plant Cell Physiol. 2010;51(6):960–8.
Barbierbrygoo HLN, Angeli AD, Filleur S, Frachisse JM, Gambale F, Thomine SB, et al. Anion channels/transporters in plants: from molecular bases to regulatory networks. Annu Rev Plant Biol. 2011;62(62):25–51.
Marmagne A, Vinaugerdouard M, Monachello D, De Longevialle AF, Charon C, Allot M, et al. Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively. J Exp Bot. 2007;58(12):3385–93.
Guo W, Zuo Z, Cheng X, Sun J, Li H, Li L, et al. The chloride channel family gene CLCd negatively regulates pathogen-associated molecular pattern (PAMP)-triggered immunity in Arabidopsis. J Exp Bot. 2014;65(4):1205–15.
Tam NC, Astrid A, Mathieu J, Sylvain D, Sébastien T, Sophie F. Characterization of the chloride channel-like, AtCLCg, involved in chloride tolerance in Arabidopsis thaliana. Plant Cell Physiol. 2016;57(4):764–75.
Wei P, Che B, Shen L, Cui Y, Wu S, Cheng C, et al. Identification and functional characterization of the chloride channel gene, GsCLC-c2 from wild soybean. BMC Plant Biol. 2019;19(1):1–15.
Elgebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019;47(D1):D427–32.
Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982;157(1):105–32.
Jaillon O, Aury J, Noel B, Policriti A, Clepet C, Casagrande A, et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007;449(7161):463–7.
Initiative OTPT. One thousand plant transcriptomes and the phylogenomics of green plants. Nature. 2019;574(7780):679–85.
Jiao Y, Wickett NJ, Ayyampalayam S, Chanderbali AS, Landherr L, Ralph PE, et al. Ancestral polyploidy in seed plants and angiosperms. Nature. 2011;473(7345):97–100.
Genome A. The Amborella Genome and the Evolution of Flowering Plants. Science. 2013;342(6165):1241089–9.
Zhang T, Liu C, Huang X, Zhang H, Yuan Z. Land-plant phylogenomic and pomegranate transcriptomic analyses reveal an evolutionary scenario of CYP75 genes subsequent to whole genome duplications. Journal of plant biology. 2019;62(1):48–60.
Hui Z, Jin J, Jin L, Li Z, Xu G, Ran W, et al. Identification and analysis of the chloride channel gene family members in tobacco (Nicotiana tabacum). Gene. 2018;676:56–64.
Ren R, Wang H, Guo C, Zhang N, Zeng L, Chen Y, et al. Wide-spread whole genome duplications contribute to genome complexity and species diversity in angiosperms. Mol Plant. 2018;11(3):414–28.
Teakle NL, Tyerman SD. Mechanisms of Cl^– transport contributing to salt tolerance. Plant Cell Environ. 2010;33(4):566–89.
Silva EN, Silveira JA, Rodrigues CR, Viégas RA. Physiological adjustment to salt stress in Jatropha curcas is associated with accumulation of salt ions, transport and selectivity of K⁺, osmotic adjustment and K⁺/Na⁺ homeostasis. Plant Biol. 2015;17(5):1023–9.
Ibrahim H. Tolerance of two pomegranates cultivars (Punica granatum L.) to salinity stress under hydroponic culture conditions. J Basic Appl Sci Res. 2016;6(4):38–46.
Munns R. Comparative physiology of salt and water stress. Plant Cell Environ. 2002;25(2):239–50.
Zhang H, Zhao F, Tang R, Yu Y, Song J, Wang Y, et al. Two tonoplast MATE proteins function as turgor-regulating chloride channels in Arabidopsis. P Natl Acad Sci USA. 2017;114(10):E2036–45.
Flowers TJ, Colmer TD. Salinity tolerance in halophytes. New Phytol. 2008;179(4):945–63.
Orsini F, Accorsi M, Gianquinto G, Dinelli G, Antognoni F, Carrasco KBR, et al. Beyond the ionic and osmotic response to salinity in Chenopodium quinoa: functional elements of successful halophytism. Funct Plant Biol. 2011;38(10):818–31.
Abdelgadir EM, Oka M, Fujiyama H. Nitrogen nutrition of rice plants under salinity. Biol Plantarum. 2005;49(1):99–104.
Wang H, Zhang M, Guo R, Shi D, Liu B, Lin X, et al. Effects of salt stress on ion balance and nitrogen metabolism of old and young leaves in rice (Oryza sativa L.). BMC Plant Biol. 2012;12(1):194–4.
Kiba T, Feria-Bourrellier A-B, Lafouge F, Lezhneva L, Boutet-Mercey S, Orsel M, et al. The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants. Plant Cell. 2012;24(1):245–58.
Apse MP, Blumwald E. Na⁺ transport in plants. FEBS Lett. 2007;581(12):2247–54.
Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59(1):651–81.
Grattan S, Grieve C. Salinity–mineral nutrient relations in horticultural crops. Sci Hortic-Amsterdam. 1998;78(1–4):127–57.
Rouached H, Wirtz M, Alary R, Hell R, Arpat AB, Davidian JC, et al. Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis. Plant Physiol. 2008;147:897–911.
Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39:29–37.
Zhang C, Zhang T, Luebert F, Xiang Y, Huang C-H, Hu Y, et al. Asterid phylogenomics/phylotranscriptomics uncover morphological evolutionary histories and support phylogenetic placement for numerous whole genome duplications. Mol Biol Evol. 2020.
Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–7.
Hartmann A, Tesch D, Nothwang HG, Binindaemonds ORP. Evolution of the cation chloride cotransporter family: ancient origins, gene-losses, and subfunctionalization through duplication. Mol Biol Evol. 2014;31(2):434–47.
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000;17(4):540–52.
Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, Von Haeseler A, et al. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020;37(5):1530–4.
Larkin MA, Blackshields G, Brown NP, Chenna RM, Higgins DG. Clustal W and Clustal X Version 2.0. Bioinformatics. 2007;23(21):2947–8.
Clamp M, Cuff J, Searle SM, Barton GJ. The Jalview Java alignment editor. Bioinformatics. 2004;20(3):426–7.
Feng ZT, Deng YQ, Fan H, Sun QJ, Sui N, Wang BS. Effects of NaCl stress on the growth and photosynthetic characteristics of Ulmus pumila L. seedlings in sand culture. Photosynthetica. 2014;52(2):313–20.
Rui G, Shi LX, Yang YF. Germination, growth, osmotic adjustment and ionic balance of wheat in response to saline and alkaline stresses. Soil Sci Plant Nutr. 2009;55(5):667–79.
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.
Wei T, Simko V, Levy M, Xie Y, Jin Y, Zemla J. Package ‘corrplot’. Statistician. 2017;56:316–24.

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Genome-wide identification and expression analysis of the CLC gene family in pomegranate (Punica granatum) reveals its role in salt resistance

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