Cloning and molecular characteristics of the ZmMATE6 sequence
The complete coding sequences of ZmMATE6 was amplified and sequenced from the Al-resistant inbred line 178 using gene-specific PCR primers (Fig. S1). The gene is 1596 bp long with 13 exons and 12 introns (Fig. 1a) and encodes a protein of 531 amino acids. Six residues were different between lines 178 and B73 (Fig. S2). The protein has an estimated molecular mass of 55.96 kDa and pI 9.59 and likely to contain 11 transmembrane spanning regions (Table 1). A multiple sequence alignment found that ZmMATE6 shared more than 30% identity at the amino acid level with ZmMATE1, SbMATE, HvAACT1 and OsFRDL4 (Fig. 1b). The 50 amino acid domain between A114 to I163 forms the citrate exuding motif (CEM) and the smaller region between P150 and I163 is a cytoplasmic loop (Fig. 1). Both of these domains have been linked with the subset of MATE proteins involved with citrate transport [27, 47].
Table 1
Predictions for the number of transmembrane regions (TMR) in ZmMATE6 and its subcellular localization.
Predictions for the transmembrane regions of ZmMATE6 |
Program | TMRs | Online linkage |
TMHMM | 9 | http://www.cbs.dtu.dk/cgi-bin/webface2.fcgi?jobid=5F686608000011CF37D23A6D&wait=20 |
TCDB | 11 | http://www.tcdb.org/progs/TMS.php |
TMpred | 11 | https://embnet.vital-it.ch/cgi-bin/TMPRED_form_parser |
SOSUI | 11 | http://harrier.nagahama-i-bio.ac.jp/sosui/cgi-bin/adv_sosui.cgi |
HMMTOP | 11 | http://www.enzim.hu/hmmtop/server/hmmtop.cgi |
Predictions for the subcellular location of ZmMATE6 |
Program | Location | Online linkage |
YLoc | Chlorop | https://abi-services.informatik.uni-tuebingen.de/yloc/webloc.cgi |
Cell-PLoc2.0 | PM | http://www.csbio.sjtu.edu.cn/cgi-bin/PlantmPLoc.cgi |
WoLFPSORT | Chlorop, PM | https://www.genscript.com/tools/wolf-psort/detail?file=2020/09/06/htdocs/results/159944442328764.detailed1.html#159944442328764 |
BUSCA | Mitochon | http://busca.biocomp.unibo.it/33ebe09c-e5fd-48e8-8bda-140d40b3d1cc/showresult/ |
A phylogenetic analysis was performed to compare ZmMATE6 with a range of other MATE genes including some that are known to transport citrate and linked with either aluminum resistance or iron nutrition (Table S2). Figure 2 shows that ZmMATE6 is included in the major clade that contains all members possessing the CEM domain, including ZmMATE1. Interestingly, within this larger clade, ZmMATE6 and another protein from rye, ScMATE3, separate early from the main cluster and become distinct lineages.
Distribution of pattern of ZmMATE6 expression in maize
A previous genome wide analysis showed that ZmMATE6 expression was induced in roots by Al treatment [46]. To confirm this result ZmMATE6 expression was first measured in the root tips of the Al-resistant inbred line 178 using quantitative RT-PCR. In the control treatment (-Al), ZmMATE6 expression was low but detectable and was increased by AlCl3 treatment (pH 4.2) (Fig. 3a). Expression was induced up to 20 µM Al but then saturated at higher concentrations at levels ~ 2.5-fold greater than controls.
We then investigated whether this response was specific for the Al3+ cation or whether different forms of Al or other cations can also induce ZmMATE6 expression in the same way. Al ions hydrolyse in solution and the molar fraction of several different soluble ions change as pH changes. Below pH 4.5, most Al exists as Al3+ which is largely responsible for the toxicity to plants in acidic soils. At values above pH 4.5, the molar fraction of Al3+ declines rapidly and the AlOH2+ and Al(OH)2+ species become more prevalent [48]. We measured ZmMATE6 expression in the root tips of plants treated with 60 µM AlCl3 at pH 4.2 and pH 5.6. At pH 5.6, no induction of ZmMATE6 expression was detected whereas at pH 4.2 treatment with 60 µM AlCl3 significantly induced expression by 50% and 60 µM AlCl3 induced expression by 350% (Fig. 3b). These results indicate that Al3+ could induce ZmMATE6 expression but AlOH2+ and Al(OH)2+ could not. We then tested whether a range of other divalent and trivalent cations including cadmium (Cd), lanthanum (La), zinc (Zn), copper (Cu), manganese (Mn) or iron (Fe) could induce expression. None of these cations were able to induce ZmMATE6 expression in the same way as Al3+.
The time-dependence of ZmMATE6 expression in Al-resistant and Al-sensitive genotype was measured through time in different plant tissues. The Al-resistant line 178 and the Al-sensitive line B73 were treated with 60 µM AlCl3 (pH 4.2) and expression was measured through time in the root tips (RT, apical 10 mm), the rest of roots (ROR) and the leaves (L). ZmMATE6 expression was detected in the roots and leaves of both genotypes and Al treatment steadily increased expression levels over approximately 12 h before declining again (Fig. 4a and 4b). While some variation in expression was detected between different tissues through time, we conclude that the expression in the Al-resistant and sensitive maize lines generally displayed similar patterns.
Examining ZmMATE6 expression with promoter–GUS fusions
To confirm these expression patterns, a 1.5 kb gDNA sequence upstream of the ZmMATE6 start codon was isolated and fused with the GUS reporter gene in an expression plasmid. The construct was transformed into Arabidopsis thaliana using the Agrobacterium-mediated method. Figures 5a–5c show that, in the absence of Al stress, GUS staining was low but detectable in the root tips, mature roots and leaves. After exposure to 60 µM AlCl3 (pH 4.5) for 9 h, GUS staining clearly increased in all tissues with stronger staining occurring in the mature root tissue than the root tips (Fig. 5d-5f). These results confirm the qRT-PCR results and suggest that the 1.5 kb region of sequence upstream of ZmMATE6 contains all the promoter information required to mimic the expression patterns observed in maize plants.
Subcellular localization of ZmMATE6
The ZmMATE6 protein is predicted to have multiple transmembrane regions and therefore is likely to localize to a membrane. Several predictive algorithms were utilized to determine the sub-cellular localization of the protein but the results were inconsistent. Some algorithms predicted localization of ZmMATE6 to the plasma membrane while others targeted the mitochondria or chloroplast (Table 1). In an attempt to determine the subcellular localization experimentally, we fused the GFP reporter gene to the 3’-terminus of the ZmMATE6 coding region and ligated it into the pCAMBIA2300 plasmid with the CaMV35S promoter to drive expression (35S:ZmMATE6::GFP). A control construct consisted of CaMV35S promoter driving expression of GFP alone (pCAMBIA2300-eGFP). Both constructs were transiently expressed in Nicotiana benthamiana leaves and protoplasts prepared from maize leaves. Fluorescence signals showed that the soluble GFP control localized to the cytoplasm and the nucleus in both cell types as expected (Fig. S3). Fluorescence from 35S:ZmMATE6:GFP constructs, however, was again inconclusive. Fluorescent signals were detected in the periphery of the tobacco cells and maize protoplasts and on some internal structures. These signals were not typical of the soluble GFP control and they could not be clearly linked with another membrane or organelle (Fig. S3). Neither transient expression system could confidently determine the sub-cellular localization of ZmMATE6 protein in these experiments.
Expression of ZmMATE6 in Arabidopsis increases citrate release and Al resistance
To investigate the function of ZmMATE6, the coding region was expressed in the Arabidopsis thaliana Columbia-0 (Col-0) using the CaMV35S promoter (Fig. S4). Seven independent T0 plants were selected and presence of the transgene was confirmed using PCR (Fig. 6a). The ZmMATE6 expression in leaf of these lines was four to 12-fold greater than the wild type control (Fig. 6b). Three T0 lines (#1, #4 and #6) were selected to generate homozygous T3 lines for further experiments.
MATE genes from other species whose expression is induced by Al often function as citrate transporters (see Introduction). We therefore measured the release of organic acids from the roots of 14 d old transgenic Arabidopsis seedlings with and without Al treatment. In the control treatment (0.5 mM CaCl2, pH 4.5) only malate and citrate were detected. Malate release was 0.02 pmol plant− 112h− 1 for all genotypes (data not shown) and citrate release was 0.3 nmol plant− 112h− 1 for all genotypes (Fig. 6c). When plants were treated with 60 µM AlCl3 (pH 4.5) malate release remained unchanged whereas citrate release was significantly increased in all genotypes but the increases were significantly larger in the transgenic lines than wild-type. Wild-type plants increased citrate release by four-fold to 1.3 nmol plant− 112 h− 1 whereas the two transgenic lines tested increased release by ten-fold to approximately 2.8 nmol plant− 112 h− 1 (Fig. 6c).
To determine whether the increase in citrate release affected Al resistance of the transgenic Arabidopsis, seedlings were transferred to plates containing 0 or 100 µM AlCl3 and grown for a further four days (Fig. 7a). Root growth of the wild-type and transgenic Arabidopsis lines was similar in the absence of Al. In the presence of 100 µM Al, net root growth was inhibited in all genotypes but the inhibition was significantly greater in the wild-type control than the transgenic lines (Fig. 7a, b). Relative root elongation (RRE) was less than 10% for wild-type plants compared to 46%, 58% and 86% for the three transgenic lines (Fig. 7c).
We tested whether the enhanced resistance to Al toxicity observed in the transgenic lines was associated with a reduction accumulation of Al in the roots and shoots. The first experiment estimated Al uptake by staining roots with hematoxylin. This compound turns a blue-purple colour when it chelates Al, so a darker stain indicates more Al is present in the tissue. Seedlings were treated with 0 or 60 µM AlCl3 for 12 h and the roots stained with hematoxylin (Fig. 8a). In control solution, the staining was very faint but it became darker in the root tips and mature roots after treatment with Al. The staining was less intense in the three transgenic lines than the wild-type plants, especially at the root tips (Fig. 8a). These results suggest that less Al accumulates in the roots of the transgenic lines than the wild-type controls. We quantified these differences by measuring the Al concentration in the roots before and after Al treatment using ICP-MS. In the control treatment, the transgenic lines and wild-type plants contained similar concentrations of Al of approximately 2.5 mg gDW− 1 (Fig. 8b). These concentrations increased in all lines after exposure to Al but the transgenic lines accumulated less than 30% of the Al that the wild-type plants accumulated. Al content in leaves was similar in all lines and did not change with Al treatment (Fig. S5). These results suggest that overexpression of ZmMATE6 increased Al resistance by reducing Al accumulation in the roots.