Identification of CLCs in pomegranate
A HMM profile was used to identify the putative CLC genes in 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 (TMHs) in the PgCLCs, ranging from 9 to 11, which were associated with the ion transport.
Table 1. Characteristics of the CLC genes in pomegranate
Gene ID
|
Name
|
Length
|
Mw(kDa)
|
pI
|
GRAVY
|
Orthologs
|
TMHs
|
CDL15_Pgr005627
|
PgCLC-B
|
797
|
87.9
|
6.49
|
0.259
|
AtCLC-B
|
9
|
CDL15_Pgr027626
|
PgCLC-C1
|
698
|
75.7
|
7.53
|
0.364
|
AtCLC-C
|
10
|
CDL15_Pgr013895
|
PgCLC-C2
|
717
|
78.1
|
5.92
|
0.325
|
AtCLC-C
|
10
|
CDL15_Pgr008552
|
PgCLC-D
|
788
|
86.9
|
8.57
|
0.175
|
AtCLC-D
|
11
|
CDL15_Pgr019810
|
PgCLC-E
|
764
|
81.3
|
5.86
|
0.188
|
AtCLC-E
|
10
|
CDL15_Pgr012201
|
PgCLC-F
|
765
|
81.7
|
6.54
|
0.035
|
AtCLC-F
|
11
|
CDL15_Pgr015371
|
PgCLC-G
|
709
|
77.3
|
8.44
|
0.468
|
AtCLC-G
|
10
|
Note: molecular weight (Mw), isoelectric points (pI), grand average of the hydrophobicity (GRAVY), transmembrane helices (TMHs)
Phylogenetic analysis of the CLC gene family in pomegranate
To elucidate the evolutionary traits of the CLC gene family in land plants, we investigated 15 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). PgCLC-E and PgCLC-F were belonged to clade II and other PgCLCs were belonged to clade I. 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), which was contributed to the duplication between PgCLC-C1 and PgCLC-C2. 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, which was associated with the duplication between PgCLC-C and PgCLC-G. 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. There were two members from Arabidopsis and Eutrema in the CLC-A/B subfamily, while only one member PgCLC-B from pomegranate.
Here, our phylogenetic results showed that seven putative PgCLC genes 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 (PgCLC-C1 and PgCLC-C2) and one angiosperm-specific expansion (PgCLC-C1/C2 and PgCLC-G) (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 ten 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 ten 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). Thus, PgCLC-B was likely a NO3–/H+ exchanger that mainly transported NO3–, while PgCLC-C, PgCLC-D and PgCLC-G might preferentially transported Cl–. 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 the CLC-G subfamily and the proton glutamate (E) residue of the 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 PgCLC 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).
Growth characteristics and anion contents in pomegranate tissues
With the increasing concentration of salinity, dry weights of roots and stems showed no significant changes among each treatment (Table S4, p < 0.05). While leaf dry weight and total dry weight first increased and then decreased, reaching a peak at 100 mM salinity level.
As shown in Figure 4A, the contents of Cl− in pomegranate roots, stems and leaves significantly increased with the increasing concentration of NaCl (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 control, respectively. The contents of Cl− in plant tissues was ranked as leaf > stem > root. Compared to control, 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 salinity (p < 0.05). The NO3− contents in plant tissues was ranked as root > stem > leaf (Fig. 4B). By contrast, the H2PO4− contents in roots increased along with the increasing 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. 4C). For the SO42− contents, trends of first increasing and then decreasing in pomegranate roots and leaves were observed with peaks at 100 mM salinity. As Figure 4D shown, SO42− mainly accumulated in roots, and the content of SO42− in leaves fell sharply under higher salinity (> 200 mM NaCl).
Expression patterns of the PgCLC genes under NaCl stress
To further investigate the expression patterns of the PgCLC genes, 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. 5). Notably, when plants were subjected to salinity, the expression levels of all the tested PgCLCs were up-regulated 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. 5). 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 controls.
Correlation between the anion contents and expression levels of the PgCLC genes
Correlation analysis showed that the PgCLC genes were positively correlated with each other (Fig. 6, p < 0.05). The Cl− contents 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. 6). These findings suggested that accumulation of Cl−, SO42− and NO3− in pomegranate tissues was associated with the expression levels of the PgCLC genes under salt stress.