Transmembrane transport plays a key role in controlling the distribution of metals between organs and compartments in cells. The transport proteins in plants mainly include heavy metal ATPase (HMA) (He et al. 2020), natural resistance-associated macrophage protein (NRAMP) (Lu et al. 2020), ATP-Bing Cassette (ABC) and the efflux proteins in the cationic diffusion promoter family, which is also called metal tolerance proteins. Metal cation binds to the cytoplasmic C-terminal domain (CTD) of the CDF protein causing the apolipoprotein to change from an open V-type dimer to a closed structure. The transmembrane domain then undergoes a conformational change allowing the transport of the metal cation (Barber-Zucker et al. 2019). Currently, the identification and functional analysis of MTP family members have been completed in grapes (Shirazi et al. 2019), rice (Farthing et al. 2017), tobacco (Liu et al. 2019) and other species but they are yet to be reported in potatoes.
Plant MTP genes are divided into 7 groups (Cotrim et al. 2019). The first branch contains MTP1-MTP4 (G1), MTP5 (G5) and MTP12 (G12) which belong to the Zn-MTP subfamily. The second branch is composed of MTP7 (G7) and MTP6 (G6) which are derived from Zn/Fe-MTP. The third branch consists of MTP8 (G8) and MTP9–MTP11 (G9) from the Mn-MTP subfamily. In the Zn-MTP subfamily, MTP1 is related to the transport of Zn2+, Cd2+, Ni2+and Co2+ (Chen et al. 2009; Kim et al. 2004). The MTP2 protein mediates the transport of Zn, Co and Ni (Papierniak-Wygladala et al. 2020) and MTP3 mediates Zn and Mn tolerance by chelating the metal ions into the vacuoles (Gu et al. 2020). The CsMTP5-CsMTP12 complex in cucumber is involved in the outflow of zinc from the cytoplasm of yeast cells (Migocka et al. 2018). The members of the Mn-MTP subfamily are mainly involved in the transport of manganese in plants (Chen et al. 2013; Ueno et al. 2015). There are currently few reports on the functional roles of members of the Zn/Fe-MTP subfamily. These reports indicate that the MTP gene is involved in the transport and tolerance to multiple heavy metals and plays an important role in maintaining metal homeostasis in plants.
In this study, 11 potato MTP genes were identified and screened. A phylogenetic tree was constructed and used to compare the identified potato MTP protein sequences with the phylogenetic distribution of the MTP sequences of known species such as rice and Arabidopsis thaliana. This may help to understand the function of potato MTP.
We found that the Zn/Fe-MTP subfamily did not contain any StMTPs and so we hypothesize that this subfamily had been lost during the evolution of the potato. Most potato and tomato genes were located in adjacent branches the species are closely related. The length of the proteins encoded by StMTPs ranged from 28.77–100.11 kDa. The predicted results of the subcellular localization indicated that all StMTPs are located in the vacuole which agreed with the results of Gao (2020). The predicted results of the TMHMM Server v. 2.0 analysis showed that StMTP4 has a transmembrane domain that is similar to OsMTP6 (without the domain) (Ram et al. 2019). StMTP2 has 12 transmembrane domains similar to 14 in Brassica rapa var. rapa MTP12 (BrrMTP12) (Li et al. 2018). Also Montanini (2007b) found that in two subfamilies of Zn-MTP and Zn/Fe-MTP, the highly conserved HXXXD (X = any amino acid) motifs of aspartic (D) and histidine (H) were identified near the TMDII and TMDV. The Mn-MTP sequence can be distinguished by the consensus sequence DxxxD (x = any amino acid) in the TMD V, where D is a highly conserved aspartic acid residue (Montanini et al. 2007).
Divalent d-block metal cations (DDMC) such as Zn2+, Mn2+, Fe2+ and Cu2+ can interact with negatively charged (Asp, Glu) and polar residue (such as Cys, His and Asn) containing protein to reduce their toxicity to plants (Montanini et al. 2007). In this study, aspartic acid-based DXXXD motifs were found on the TMDII or near the TMDV of Mn-MTP and the HXXXD motifs on Zn-MTP, Zn/Fe-MTP TMDII and TMDV. Studies have shown that the highly conserved histidine and aspartic acid residues may be related to the transport of metal ions (Russell and Soulimane 2012). Also, a histidine-rich region between the Zn-MTP subfamily TMDIV and TMDV was observed that is responsible for zinc-binding (Migeon et al. 2010).
A gene promoter is a DNA sequence located upstream of the coding region of a gene and contains multiple cis-acting elements that are specific binding sites for proteins involved in transcription initiation and regulation (Hernandez-Garcia and Finer 2014). Promoter prediction analysis found a large number of promoter core elements such as the CAAT-box, TATA-box and light-responsive elements. Each member contained cis-acting elements related to growth hormones such as abscisic acid, auxin, and salicylic acid. We hypothesize that these genes may be regulated by plant hormones.
We studied the relative expression of 11 potato MTP genes under heavy metal stress (Zn2+, Cd2+). Our results showed that these two metal ions can induce the expression of potato MTP genes indicating that potato MTP genes may be involved in the tolerance or transport of Zn2+ and Cd2+. Also, the overall expression levels of the MTP genes in leaves were higher than in roots and stems. Under cadmium stress, the relative expression levels of all genes in the leaves highest at 24 h. Of these genes, StMTP9 and StMTP10 were both upregulated around 24 fold at 24 h indicating that these genes are involved in response to cadmium stress. Following zinc treatment, except for StMTP1 and StMTP6 which reached the highest expression level at 24 h, the rest of StMTPs reached the highest expression levels at 12 h and all StMTPs were all down-regulated at 6 h. The expression of StMTP11 was highest after 6 hours of zinc stress reaching 13 times that of the control suggesting that StMTP11 is more sensitive to zinc stress.
Overall, this study identified and analyzed the MTP genes in potatoes to provide further understanding of the structure and function of these genes. However, further studies are needed to better understand the response mechanism of StMTPs to heavy metal stress.