Structure, Function, Regulation and Phylogenetic Relationship of ZIP Family Transporters of Plants

Abstract

Zinc (Zn) is an essential micronutrient for plants and humans. Nearly 50% of the agriculture soils of world are Zn-deficient. The low availability of Zn reduces the yield and quality of the crops. The zinc-regulated, iron-regulated transporter-like proteins (ZIP) family and iron-regulated transporters (IRTs) are involved in cellular uptake of Zn, its intracellular trafficking and detoxification in plants. In addition to Zn, ZIP family transporters also transport other divalent metal cations (such as Cd²⁺, Fe²⁺, and Cu²⁺). ZIP transporters play a crucial role in biofortification of grains with Zn. Only a very limited information is available on structural features and mechanism of Zn transport of plant ZIP family transporters. In this article, we present a detailed account on structure, function, regulations and phylogenetic relationships of plant ZIP transporters. We give an insight to structure of plant ZIPs through homology modeling and multiple sequence alignment with Bordetella bronchiseptica ZIP (BbZIP) protein whose crystal structure has been solved recently. We also provide details on ZIP transporter genes identified and characterized in rice and other plants till date. Functional characterization of plant ZIP transporters will help for the better crop yield and human health in future.

Keywords: ZIP transporters; functional characterization; genetic modification; homology modeling; transcription factor.

FIGURE 1

Multiple sequence alignment of Bordetella bronchiseptica ZIP (BbZIP), Arabidopsis (AtZIP), rice (OsZIP), and maize (ZmZIP) transporters. The protein sequences were aligned by ClustalW alignment using molecular evolutionary genetics analysis, V 6.0 (MEGA6) tool (Tamura et al., 2013). The residues (BbZIP) involved in Cd/Zn binding and transport are highlighted in green and dark green. The His177 highlighted in dark green is involved in both metal recruiting and cytoplasmic transport of metal. The hydrophobic residues involved in blocking of metals at extracellular surface are highlighted in light blue. Metal recruiting residues are highlighted in pink. The residues involved in metal release into the cytoplasm are highlighted in red.

FIGURE 2

Homology modeling of plant ZIP transporters using Bordetella bronchiseptica ZIP (BbZIP) (PDB ID: 5TSA) as a template. The BbZIP structure was visualized using LiteMol viewer (https://www.litemol.org/Viewer/). Both whole model (A) and its Zn²⁺ binding site (B) is shown for each ZIP. The protein sequences of plant ZIPs were obtained from phytozome website (https://phytozome.jgi.doe.gov/pz/portal.html). The homology models of the plant ZIP proteins were generated using Modeler v9.22 (Eswar et al., 2007), which created 100 models for each target protein. The models were then ranked based on their modeler objective function value. The five best models were analyzed using MolProbity (http://molprobity.biochem.duke.edu/), an online tool. Ramachandran plots were generated for these five models and finally the best model was chosen based on the% residues in the favored and allowed regions and the number of outliers. The models were then visualized by LiteMol viewer.

FIGURE 3

Phylogenetic tree of ZIP transporter family proteins of plants. The ZIP phylogenetic tree was constructed from 113 ZIP transporter protein sequences collected from 14 plant species. These included 8 monocot (Blue) and 6 dicot plants (Black). Each major cluster (MC1-MC8) is highlighted with different color. The low-affinity and high-affinity ZIP transporters are highlighted in red and green, respectively. The protein sequences of ZIP transporter family members were collected from Phytozome (www.phytozome.net) website. The phylogenetic tree was constructed by MEGA version 6 software with the maximum likelihood method based on the Jones-Taylor-Thornton matrix-based model. The bootstrap values are from 1000 replicates. The phylogeny tree was visualized by iTOL (https://itol.embl.de/).

FIGURE 4

Localization of ZIP transporter family proteins in the plant cell. The ZIP transporter family proteins are actively involved in uptake, transport, detoxification and homeostasis of Zn within plant cells (Li et al., 2013). Under low Zn condition, many ZIP transporters (ZIP1-ZIP12) are expressed and are localized in plasma membrane of the cell in rice, (Ramesh et al., 2003; Ishimaru et al., 2005; Yang et al., 2009; Lee et al., 2010a, b) hardy orange (Fu et al., 2017), maize (Li et al., 2013; Mondal et al., 2013), Arabidopsis (Milner et al., 2013), and barley (Pedas et al., 2009; Tiong et al., 2015). Some of the ZIP transporters are also localized to the membranes of intracellular organelles such as chloroplast [ZmZIP5 and ZmZIP7 (Li et al., 2013)], vacuole [HvZIP5 (Tiong et al., 2015) and AtZIP1 (Milner et al., 2013)], and endoplasmic reticulum [ZmZIP1-ZmZIP8 (Li et al., 2013)].

FIGURE 5

Expression of ZIP transporter genes in different parts of rice (A), maize (B), and wheat (C). The ZIP transporters are expressed in root, nodes, clumps, shoot/stem, leaf, flag leaf, kernel, and spikelets under Zn deficient levels. Under low Zn condition, root tissues show the expression of ZIP genes in rice (Ramesh et al., 2003; Ishimaru et al., 2005; Chen et al., 2008; Yang et al., 2009; Suzuki et al., 2012), maize (Li et al., 2013; Mondal et al., 2013) and wheat (Durmaz et al., 2011; Evens et al., 2017; Deshpande et al., 2018), clumps in rice (Yang et al., 2009), shoot/stem in rice (Ramesh et al., 2003; Ishimaru et al., 2005; Chen et al., 2008; Yang et al., 2009; Suzuki et al., 2012), maize (Li et al., 2013; Mondal et al., 2013), and wheat (Durmaz et al., 2011; Evens et al., 2017). Leaf tissue also showed the over expression of ZIP genes under low Zn soil in rice (Ramesh et al., 2003; Ishimaru et al., 2005; Chen et al., 2008) and wheat (Evens et al., 2017; Deshpande et al., 2018), flag leaf tissues in maize (Mondal et al., 2013) and wheat (Deshpande et al., 2018), kernel tissue in maize (Li et al., 2013) and spikelets tissue in rice (Yang et al., 2009) and wheat (Deshpande et al., 2018).