Genome-Wide Identification, Cloning and Functional Analysis of the Zinc/Iron-Regulated Transporter-Like Protein (ZIP) Gene Family in Trifoliate Orange (Poncirus trifoliata L. Raf.)

Abstract

Zinc (Zn) and iron (Fe) deficiency are widespread among citrus plants, but the molecular mechanisms regarding uptake and transport of these two essential metal ions in citrus are still unclear. In the present study, 12 members of the Zn/Fe-regulated transporter (ZRT/IRT)-related protein (ZIP) gene family were identified and isolated from a widely used citrus rootstock, trifoliate orange (Poncirus trifoliata L. Raf.), and the genes were correspondingly named as PtZIPs according to the sequence and functional similarity to Arabidopsis thaliana ZIPs. The 12 PtZIP genes were predicted to encode proteins of 334-419 amino acids, harboring 6-9 putative transmembrane (TM) domains. All of the PtZIP proteins contained the highly conserved ZIP signature sequences in TM-IV, and nine of them showed a variable region rich in histidine residues between TM-III and TM-IV. Phylogenetic analysis subdivided the PtZIPs into four groups, similar as found for the ZIP family of A. thaliana, with clustered PtZIPs sharing a similar gene structure. Expression analysis showed that the PtZIP genes were very differently induced in roots and leaves under conditions of Zn, Fe and Mn deficiency. Yeast complementation tests indicated that PtIRT1, PtZIP1, PtZIP2, PtZIP3, and PtZIP12 were able to complement the zrt1zrt2 mutant, which was deficient in Zn uptake; PtIRT1 and PtZIP7 were able to complement the fet3fet4 mutant, which was deficient in Fe uptake, and PtIRT1 was able to complement the smf1 mutant, which was deficient in Mn uptake, suggesting their respective functions in Zn, Fe, and Mn transport. The present study broadens our understanding of metal ion uptake and transport and functional divergence of the various PtZIP genes in citrus plants.

Keywords: ZIP gene; citrus; iron deficiency; yeast complementation; zinc deficiency.

Figure 1

Multiple sequence alignment of PtZIP and AtZIP proteins. Twelve PtZIP proteins isolated from trifoliate orange and seven representative AtZIP proteins from Arabidopsis were aligned using ClustalW. Similar amino acids are indicated by dark or light shading, while signal peptides in the N-terminal end are highlighted in red. Transmembrane (TM) domains were shown as lines above the sequences and numbered I to VIII. The variable region rich in histidine residues between TM-III and TM-IV is highlighted in blue. The 15 ZIP signature sequences in TM-IV domain are marked with asterisks.

Figure 2

Phylogenetic tree and gene structure of PtZIP and AtZIP proteins. Twelve of the PtZIP proteins (marked with red triangles) and 15 of the AtZIP proteins (marked with black spots) were used to construct the neighbor-joining tree. Bootstrap values above 50 and supporting a node used to define a cluster are indicated. The structures of each gene were analyzed online using the Gene Structure Display Server and displayed according to the order of the phylogenetic relationship. The black boxes, blue boxes and lines represent exons, UTRs and introns, respectively, and their lengths are shown proportionally. The proteins were divided into four groups (I to IV) according to their phylogenetic relationships.

Figure 3

Distribution of PtZIP genes on the chromosomes. The chromosomal positions of the PtZIP genes were mapped according to the information available in the sweet orange genome database. The arrows indicate transcription direction. The chromosome numbers are indicated at the top of each chromosome. The scale is in megabases (Mb).

Figure 4

Expression patterns of PtZIP genes in roots and leaves under normal conditions or Zn, Fe and Mn deficiency. (A) qPCR analysis of PtZIP gene expression in roots and leaves of 40 d-old trifoliate orange seedlings grown on normal nutrient solution. Data are the means ± SE of three technical replicates. (B) Heat map showing the expression patterns of PtZIP genes following treatment to induce Zn, Fe and Mn deficiency for 7, 12, and 20 d. Relative expression levels of PtZIP genes in roots and leaves were analyzed by qPCR and normalized to Actin (Cs1g05000.1). The relative expression values in samples those grown under normal conditions for 7, 12, and 20 d were used as controls. The fold change was then calculated as (PtZIP expression under treatment)/(PtZIP expression under control). The cluster 3.0 software was then used to generate heat maps based on the ${\text{log}}_2^{\text{fold }change}$ data. Green and red indicates lower and higher transcriptional levels of PtZIP genes, respectively.

Figure 5

Complementation of yeast metal uptake-defective mutants with PtZIP genes on selective medium. Yeast mutants defective in the uptake of Zn (zrt1zrt2), Fe (fet3fet4), and Mn (smf1) were transformed with any one of the 12 PtZIPs, the empty vector pFL613 (negative control), or the AtZIP4 (positive control for zrt1zrt2), AtIRT1 (positive control for fet3fet4) and AtZIP7 (positive control for smf1). To test the complementary ability of the genes, 5 μL of transformed yeast cells with OD₆₀₀ values of 1.0, 0.1, 0.01, and 0.001 were spotted on plates containing different metal limiting medium and grown for 3–5 d at 30°C. (A) Transformed zrt1zrt2 cells on synthetic complete and dropout mix (without Uracil) medium (SC-URA) plus 0.1 mM ZnSO₄ (control) or SC-URA plus 1.0 mM EDTA and 0.4/0.6 mM ZnSO₄ (Zn limited with EDTA). (B) Transformed fet3fet4 cells on SC-URA without BPDS (control) or SC-URA plus 0.01/0.02 mM BPDS (Fe limited with BPDS). (C) Transformed smf1 cells on SC-URA without EGTA (control) or SC-URA plus 10/20 mM EGTA (Mn limited with EGTA). (D) Transformed yeast cells (100 μL) with an OD₆₀₀ of 1.0 were initially inoculated into 15 mL liquid SC-URA medium supplemented with 1.0 mM EDTA and 0.4 mM ZnSO₄ (for testing zrt1zrt2), 0.01 mM BPDS (for testing fet3fet4), and 10 mM EGTA (for testing smf1). After 20, 32, 44, and 56 h of growth at 200 rpm and 30°C, the OD₆₀₀ of 2 mL from the 15 mL culture was measured. Data are the means ± SE of three independent repeats. + under the horizontal axis indicates the positive controls, AtZIP4, AtIRT1 and AtZIP7 for zrt1zrt2, fet3fet4, and smf1, respectively. ^* indicates the values are significantly different from those associated with pFL613 at P < 0.05 by LSD testing.