Identification and sequence analysis of CsMGT gene family in C. sinensis
The HMM profiles of the conserved MIT CorA-like superfamily domain (PF01544) were used to carry out a BLAST search of the C. sinensis genome in order to systematically examine the CsMGT genes. Utilizing TBtools Simple HMM Search, we found twelve potential CsMGT genes. Motif analysis carried out through CDD and the SMART website identified seven CsMGT proteins with conserved, full-length aa sequences as putative Mg2+ transporters (Table 1). The seven CsMGT genes were mapped to 5 C. sinensis chromosomes. Two genes (CsMGT3 and CsMGT4) were located on chromosome 8, and CsMGT5, CsMGT7, CsMGT1, and CsMGT6 were found on chromosomes 3, 5, 7, and 9, respectively. The CsMGT2 gene did not correspond to any chromosome. The lengths of the ORFs ranged from 318 bp (CsMGT7) to 1473 bp (CsMGT4). The full-length CsMGT protein sequences ranged from 105–490 amino acids, and their molecular weights ranged between 11.90 kDa (CsMGT7) to 54.85 kDa (CsMGT4). The CsMGT protein pI values ranged between 4.83 (CsMGT4) to 6.24 (CsMGT7), and the GRAVY values ranged between − 0.381 (CsMGT4) to 0.490 (CsMGT7). All CsMGT proteins, except for CsMGT3 and CsMGT5, were determined to be localized at the plasma membrane. Furthermore, all CsMGTs contained 2 conserved TM domains.
Table 1
Detailed information of predicted CsMGT genes in Citrus sinensis. Note: Gene ID, it is annotated in Citrus sinensis genome; ORF, open reading frame; AA, amino acid; pI, isoelectric point; Mw, molecular weight; GRAVY, grand average of hydrophobicity; No. TMD, Numbers of transmembrane domains.
Gene ID | Predicted name | Genome position | Chr | ORF (bp) | AA (aa) | Mw (kDa) | pI | GRAVY | Subcellular localization | No. TMD |
Start End |
Cs7g11510.1 | CsMGT1 | 7567289 | 7572749 | 7 | 1248 | 415 | 46.32 | 5.46 | 0.016 | plasma membrane | 2 |
orange1.1t01328.1 | CsMGT2 | 22632181 | 22638094 | Un | 1239 | 412 | 45.83 | 4.85 | -0.294 | plasma membrane | 2 |
Cs8g03910.1 | CsMGT3 | 1984218 | 1987376 | 8 | 1458 | 485 | 54.78 | 5.04 | -0.374 | endoplasmic reticulum membrane | 2 |
Cs8g03920.1 | CsMGT4 | 1987807 | 1989823 | 8 | 1473 | 490 | 54.85 | 4.83 | -0.381 | plasma membrane | 2 |
Cs3g06130.1 | CsMGT5 | 8173325 | 8177635 | 3 | 1341 | 446 | 49.43 | 5.63 | -0.133 | mitochondrial inner membrane | 2 |
Cs9g08150.1 | CsMGT6 | 5286112 | 5291888 | 9 | 1185 | 394 | 43.86 | 4.97 | -0.107 | plasma membrane | 2 |
Cs5g30290.1 | CsMGT7 | 32204935 | 32205833 | 5 | 318 | 105 | 11.90 | 6.24 | 0.490 | plasma membrane | 2 |
Note: Gene ID, it is annotated in Citrus sinensis genome; ORF, open reading frame; AA, amino acid; pI, isoelectric point; Mw, molecular weight; GRAVY, grand average of hydrophobicity; No. TMD, Numbers of transmembrane domains.
Phylogenetic analysis of CsMGT proteins
To analyze the evolutionary relationships amongst the CsMGT family, a phylogenetic tree comprising 26 MGT members from A. thaliana (ten proteins), O. sativa (nine proteins), and C. sinensis (seven proteins) was constructed. The seven CsMGT proteins were divided into four clades (B, C, D, and E): CsMGT1 in clade B; CsMGT2, 3, and 4 in clade C; CsMGT5 in clade D; and CsMGT6 and 7 in clade E (Fig. 1a). Each clade also encompassed representatives from both A. thaliana O. sativa, suggesting that the MGT protein family is evolutionarily-conserved. The CsMGT proteins were found to share higher homology with AtMGT proteins than with OsMGT proteins. Sequence alignment revealed the presence of two putative TM domains near the C-termini of all CsMGT proteins. The conserved GMN motif was also found in all seven CsMGT proteins (Fig. 1b).
Sequence structural features of CsMGTs
The distribution of motifs in CsMGT proteins was explored. The MEME program discovered ten conserved motifs, ranging between 15 to 50 amino acids, across CsMGT proteins (Fig. 2a). Using Pfam (PF01544), four putative MIT CorA-like divalent cation transporter superfamily domains were identified (Table 2). Six of the CsMGTs were found to contain eight highly conserved motifs (motifs 1–8), with the exception of CsMGT7, which contained only three motifs (motifs 1, 6, and 10). Motifs 9 and 10 were present only in CsMGT3 and CsMGT4 belonged to clade C, while CsMGT6 and CsMGT7 belonged to clade E. All CsMGT proteins, except for CsMGT7, began at the N-terminus with motif 7 and ended with motif 6. Our phylogenetic classification was further supported by the discovery that CsMGT proteins belonging to the same clade also contained similar conserved motifs.
Table 2
Putative 10 motifs of CsMGT proteins in Citrus sinensis.
Motif | Width | Protein sequences | Pfam domain |
1 | 50 | REYIDDTEDYINIQLDBKQNQLJQLELMLTTATLVJSAYIVVAGIFGMNI | MIT_CorA-like superfamily |
2 | 50 | KHAIMRRTGLPARDLRILDPLLSYPSTILGREKAIVINLEHIKAIITAZE | MIT_CorA-like superfamily |
3 | 50 | YPALDELTSKISTLNLERVRQJKSRLVAJTGRVQKVRDEJEHLLDDDEDM | MIT_CorA-like superfamily |
4 | 29 | SBHNDVEELEMLLEAYFVQIDGTLNKLST | MIT_CorA-like superfamily |
5 | 37 | NRDGPKELPFEFRALEAALEAACSCLDAEVKELEQEA | — |
6 | 21 | GTAIGCILLYVIAIAYAKHKK | — |
7 | 25 | KKKAAGVRPWLLLDSTGQSZVVEVG | — |
8 | 21 | VLLLBSLDPSVVPFVEELQRR | — |
9 | 41 | ILCHYQATKAQEGDGEDSNWKNLYDLEAPZSRASSPPDFAG | — |
10 | 15 | PYTWKEGHGYMFKWV | — |
The structure and combination of exons and introns is reported to alter gene function (Shaul 2017). To analyze the structural diversity of CsMGTs, we analyzed the distribution of exons and introns across these genes. Overall, intron-exon structure tended to be similar between members of the same clade (Fig. 2b). For example, the three CsMGT genes (CsMGT2, CsMGT3, and CsMGT4) belonging to clade C contained six exons separated by five introns. Conversely, in clade E, CsMGT6 contained ten introns and eleven exons, while CsMGT7 contained four introns and five exons. The differences in gene structure between clades provided additional support for the phylogenetic analysis, and were suggestive of evolutionary and functional diversification within the magnesium transporter family.
CsMGT promoter cis-acting elements
Studies of gene promoters are essential for understanding the mechanism of transcriptional regulation. In order to identify any cis-elements present in CsMGTs, PlantCARE was utilized to analyze the 2000 bp sequences upstream of their translation start sites. Several essential cis-acting elements were discovered and classified into three biological categories: stress responsiveness, phytohormone responsiveness, and plant growth and development (Fig. 3a-b). In the plant growth and development category, the as-1 element was the most common (25.70%), suggesting that the seven CsMGT genes might be related to shoot development. The circadian element (Anderson et al. 1994) was identified only in CsMGT3, suggesting that this gene may be involved in photoperiod sensitivity. Several other plant grown-related promoter elements were discovered across the CsMGT genes, including an AAGAA-motif associated with endosperm development, an AT-rich element associated with flowering, a CAT-box associated with shoot and root meristem expression, and an O2-site associated with zein metabolism.
In the stress responsiveness category, several elements associated with wounding response (WRE3 and WUN-motif), drought-inducibility (MBS and MYC), defense and stress response (TC-rich repeats), low temperature (LTR), anaerobic induction (ARE), and dehydration (DRE) were identified. These cis-elements were extensively distributed across the promoter regions of different CsMGT genes, with MYC being the most common (43.06%). MYC, which regulates ABA homeostasis and drought stress response (Abe et al. 1997), was found in seven CsMGT genes, suggesting that these genes may be crucial for drought stress response in citrus.
In the phytohormone responsiveness category, the TGACG- and CGTCA-motif jasmonate (JA)-responsive elements (Wang et al. 2019) were identified in all seven CsMGT genes, with each gene containing the same number of these two elements. The abscisic acid (ABA) response element (ABRE) (Hobo et al. 1999) was found in five CsMGT genes, with the largest number of ABRE elements (9) found in CsMGT6. The ethylene (ET)-responsive element (ERE) (Fujimoto et al. 2000) was also found in five CsMGT genes, with CsMGT2 possessing the greatest amount of ERE elements (5). Several other phytohormone-responsive cis-acting elements were identified across the CsMGT genes, including the gibberellin (GA3)-responsive TATC-box (Washida et al. 1999), salicylic acid (SA)-responsive TCA-element (Zhang et al. 2017), P-box, auxin-responsive TGA-element (Ulmasov et al. 1997), and GARE-motif. The most common phytohormone response elements were linked to JA-responsiveness (CGTCA-motif and TGACG-motif, 31.04%), ABA-responsiveness (ABRE element, 25.86%), and ET-responsiveness (ERE element, 24.14%), indicating that JA, ABA, and ET may regulate CsMGT genes.
Analysis of CsMGT gene expression
CsMGT gene expression was quantified in the fruit pulp, peels, flowers, leaves, stems, and roots of untreated C. sinensis (Fig. 4). Across all tissues, the highest expression was exhibited by CsMGT3. In roots and stems, all seven CsMGT genes exhibited similar, low-level expression. In leaves, both CsMGT3 and CsMGT7 were highly expressed. In flowers, CsMGT1, CsMGT2, CsMGT3, CsMGT5, and CsMGT6 exhibited similarly high levels expression, with CsMGT2 exhibiting the highest expression of all. In fruit pulp and peels, CsMGT3 exhibited the highest expression, while the expression of CsMGT5 was almost undetectable. These results indicate that CsMGT gene expression is tissue-specific, suggesting that this set of genes may play diverse roles in the development and growth of citrus plants.
In order to explore the expression response of CsMGTs to Mg2+ deficiency, we analyzed their expression in leaves and roots under Mg2+ starvation (Fig. 5). In leaves, the expression of most CsMGTs, with the exception of CsMGT7, was upregulated by Mg2+ deficiency, reaching the highest levels of expression within 24 hr (Fig. 5a). The expression levels declined after the Mg2+-starved plants were transplanted into Mg2+-sufficient medium. The expression patterns of CsMGT1, CsMGT5, and CsMGT6 exhibited similar trends. On the contrary, the expression of CsMGT7 was down-regulated by Mg2+ deficiency. In roots, the expression of CsMGT1, CsMGT2, CsMGT3, CsMGT4, and CsMGT7 was up-regulated by Mg2+ deficiency, with CsMGT2 and CsMGT7 exhibiting the highest levels of expression (Fig. 5b). The expressions of CsMGT5 and CsMGT6 were down-regulated by Mg2+ starvation. Notably, CsMGT7 gene expression was markedly higher in roots than in leaves under Mg2+ starvation, suggesting that this gene may play a role in root-level Mg2+ uptake.
Functional complementation assay in mutant yeast and bacteria
To validate the Mg2+ transport ability of CsMGTs and to evaluate their functional complementation, seven CsMGTs were cloned and separately transformed into CM66 mutant yeast cells, which are deficient in the plasma membrane-localized Mg2+ transporter genes ALR2 and ALR1. The wild-type yeast strain CM52 was employed as a growth control. The CM52 + EV yeast transformant was able to grow effectively on all media with varying concentrations of Mg2+ (Fig. 6a). However, the CM66 + EV yeast transformant was unable grow when the medium contained either 0.01 or 0.1 mM Mg2+. When CsMGT2, CsMGT3, CsMGT4, CsMGT5, CsMGT6, and CsMGT7 were expressed individually in CM66, the yeast transformants outperformed the CM66 + EV yeast transformant on solid medium containing either 0.01 or 0.1 mM Mg2+. The CsMGT7 yeast transformant consistently demonstrated a clear growth advantage on the solid medium supplemented with 0.01 mM Mg2+. CsMGT1 did not display any evidence of complementation ability (Fig. 6a).
A functional complementation assay was also carried out in the mutant S. typhimurium strain MM281, which is deficient in the MgtB, MgtA, and CorA Mg2+ transporters (Maguire 1992). The wild-type strain MM1927 was employed as a growth control. The MM1927 + EV control grew well on each medium, the MM281 + AtMGT10 positive control grew best on medium supplemented with 0.001 mM Mg2+, and the MM281 + EV negative control was unable to grow on medium supplemented with less than 0.01 mM Mg2+ (Fig. 6b). Among the seven CsMGT genes, only the growth of MM281 cells transformed with either CsMGT7, CsMGT4, or CsMGT5 were restored on medium containing 0.001 mM Mg2+. However, the growth of MM281 cells transformed with CsMGT7 was superior, demonstrating that CsMGT7 mediates Mg2+ uptake, albeit with lower affinity than AtMGT10.
Because yeast and bacterial transformants harboring CsMGT7 exhibited superior performance under low Mg2+ conditions, we further carried out a liquid culture experiment. The patterns of growth observed in the liquid medium was largely in agreement with the growth patterns observed on the solid medium (Fig. 6c-d). CsMGT7 gene expression similarly increased the growth of CM66 and MM281 in the liquid medium supplemented with 0.1 mM Mg2+, indicating that CsMGT7 can mediate Mg2+ uptake in both yeast and bacterial cells.