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BRIEF COMMUNICATION
Genome-wide Analysis of Zinc Transporter Genes
of Maize (Zea mays )
Tapan Kumar Mondal & Showkat Ahmad Ganie &
Mukesh Kumar Rana & Tilak Raj Sharma
#
Springer Science+Business Media New York 2013
Abstract Zinc (Zn) is an essential micronutrient for plants
and animals. Zinc-regulated transporters and iron-regulated
transporter-like proteins (ZIP) are important zinc transporters
in plants with the characteristic ZIP domain (Pfam:PF02535).
Although individual genes belonging to the ZIP family had
been discovered in various plants, genome-wide analysis of
the paralouges (ZmZIP ) in maize and their relationships with
other related genera has so far not been conducted. We per-
formed a genome-wide analysis and identified 12 members of
the ZIP gene family in maize. Chromosomal locations, motif
organization, and biochemical characterizations of proteins, as
well as exon–intron, trans-membrane domains of these
ZmZIP genes were determined, which indicated the structural
diversity of ZmZIP. Additionally, apart from the identification
of the canonical form of the metal binding signature in ZIP
domains of the ZmZIP proteins, we also identified a new
conserved plant ZIP signature. Further, tissue-specific expres-
sions of those genes were determined by real-time PCR in the
flag leaf as well as in 10-day-old-baby kernel among the high
and low kernel zinc-containing maize inbreds. We found that
overall transcript abundance was higher in the flag leaf than
the kernel in both the inbreds for all the members except two,
namely ZmZIP5 and ZmZIP11 were expressed more in flag
leaf of a high-kernel zinc-containing inbreds than a low-
kernel zinc-containing inbreds. Therefore, these results
provide a basis for further functional characterization of spe-
cific ZmZIP genes in the future.
Keywords Abiotic stress
.
Biofortification
.
Zinc transporter
.
Zea mays
.
Zinc use efficiency
Introduction
Zinc (Zn), as an essential micronutrient, is a component of
more than 300 enzymes involved in plant biological process-
es. It plays important roles in gene expression and cellular
development. However, a pre-requisite to developing zinc
biofortified foods is the understanding of the molecular mech-
anism of zinc uptake by root, transport through stem, and
loading to the specified organs such as seed and fruit.
Maize is an important cereal in the world with high pro-
duction and productivity. Maize is grown in more than 166
countries for various uses, and in diverse seasons and agro-
ecological conditions due to its high yield potential. In India,
while 25 % of the maize produce is consumed as food, the
remaining 75 % is used as livestock and poultry feed. Thus,
micronutrient-deficient maize not only affects human beings
but also livestock and poultry. Therefore, biofortified maize
will be immensely helpful for the eradication of malnutrition
as it is sustainable and affordable for the poorest of the poor. It
is well documented that several genes control the Zn homeo-
stasis in strategy II model plant rice (Ramesh et al. 2003;
Ishimaru et al. 2006, 2007; Lee et al. 2010a, 2010b;Bashir
et al. 2012). However, genes responsive to zinc uptake as well
as loading in the kernel of maize have not so far been studied,
which is a prerequisite for understanding the molecular mech-
anism of Zn uptake. Members of the ZRT-IRT-like protein
(ZIP) family were the first metal transporters to be identified in
plants (Eide et al. 1996). Along with other factors, the expres-
sions of ZIP genes are regulated by the tissue metal status of
Electronic supplementary material The online version of this article
(doi:10.1007/s11105-013-0664-2) contains supplementary material,
which is available to authorized users.
T. K. Mondal (*)
:
S. A. Ganie
:
M. K. Rana
Division of Genomic Resource, National Bureau of Plant Genetic
Resource, IARI, Pusa Campus, New Delhi 110012, India
e-mail: mondaltk@yahoo.com
T. R. Sharma
National Research Centre on Plant Biotechnology, IARI, Pusa
Campus, New Delhi 110012, India
Plant Mol Biol Rep
DOI 10.1007/s11105-013-0664-2
the plant (Grotz et al. 1998). Genes with the ZIP domain have
also been identified and characterized from other plant species
including soybean, Medicago truncatula, Noccaea
caerulescens and Thlaspi japonica (Pence et al. 2000;
Assunção et al. 2001; Burleigh et al. 2003;Mizunoetal.
2005;Moreauetal.2002; Plaza et al. 2007). For instance,
NcZNT1 from Noccaea caerulescens was shown to mediate
high-affinity Zn uptake and low-affinity Cd uptake in yeast
(Pence et al. 2000). Homologous genes TjZNT1 and TjZNT2
isolated from the Ni hyper-accumulator Thlaspi japonica
were shown to enhance the transport of Zn, Cd, and Mn
(Mizuno et al. 2005).
The complete genome sequences derived from large-scale
sequencing projects are important for comparative and func-
tional genomics research, providing the opportunity to scan
various gene families. Like any other sequencing project, the
complete maize genome sequence also provides a valuable
resource for comparative analysis of the gene families. With
decoding of the maize genome sequence, several gene fami-
lies have already been characterized in maize (Liu et al. 2013a,
b; Zhang et al. 2013). Although individual zinc-regulated
transporters (ZRT) and iron-regulated transporter-like (IRT)
protein (ZIP) genes have been extensively studied in yeast
(Wu et al. 2011), as well as in higher plants such as rice (Lee
et al. 2010b; Lan et al. 2013), genome-wide analysis of the
members of this family has yet to be studied in maize.
Therefore, the objectives of present studies were, (1) iden-
tification of non-redundant members of maize ZIP family
genes, (2) characterization of their biochemical properties,
genomic organization, motif analysis, and phylogenetic rela-
tionship, and (3) expression analysis of these genes in differ-
ent genotypes of maize. The results of this work provide a
foundation to better understand functional and evolutionary
history of the ZIP gene family in angiosperms. In the present
study, we identified 12 putative members of maize ZIP genes
(ZmZIPs) on the basis of ZIP domain and validated their
expression among the two inbred lines which differ in Zn
use efficiency. This is the first attempt to describe the ZIP
gene family along with their expression in maize.
Materials and Methods
Identification, Characterization and Mapping of ZmZIP
Genes
The complete genome sequence of maize was downloaded
from the public database (www.maizesequence.org) and used
in this study. AtZIP1 was the first zinc transporter with ZIP
domain (Pfam:PF02535), characterized from Arabidopsis
(Grotz et al. 1998). Thus, to identify the ZmZIP family genes,
the AtZIP1 gene (AT3G12750 ) was used as query against the
maize genome sequence using basic local alignment search
tool (blastn). The blast search identified the
GRMZM2G045849 gene of maize with the same ZIP domain
(Pfam:PF02535). Subsequently, the Hidden Markov Model
(HMM) profile of the ZIP domain from the Pfam database
(pfam.janelia.org) was then used to search for maize ZIP
genes using the blastp program (E-value=0.001). The Pfam
database was used to ensure that each predicted ZmZIP gene
encoded the ZIP domain. All confirmed ZmZIP were aligned
using Clustal W (Thompson et al. 1994)inMEGA5.05
software (Tamura et al. 2011) to exclude overlapping ZmZIP
genes. Various biochemical parameters such as length of the
protein sequence, isoelectric point (pI), post-translational
modification, signal peptide, transmembrane domain (TM),
and grand average of hydropathicity (GRAVY) values (Kyte
and Doolittle 1982) of the 12 numbers of ZmZIP genes were
determined using various proteomics tools of ExPySy server
(www.expasy.org). Cellular/subcellular targeting sites, were
assessed using WoLF PSORT (www .wolfpsort.seq.cbrc.jp).
Each non-overlapping ZmZIP gene sequence was then
used as a query against the whole maize genome sequence
(maizesequence.org), by using the tblastn program and phys-
ically positioned on different maize chromosomes. The names
of ZmZIP genes were given according to their position from
the top to the bottom on the maize chromosomes 1 to 10. Thus
physical locations of all ZmZIP genes were generated (www.
maizesequence.org) against a search of the ZIP domain
(PF02535).
Synteny analysis between maize and sorghum was con-
ducted locally using the similar method developed for the
Plant Genome Duplica tion Database (Tang et al. 2008).
First, blastp was conducted using all ZmZIP proteins to search
for potential anchors (E<1e
−5
,top5matches)inthesorghum
genome. Afterwards, MCscan was employed to identify ho-
mologous regions. Finally, syntenic blocks were evaluated by
ColinearScan. Alignments with an E value <1 e
−10
were
considered as significant matches.
Phylogenetic and Syntenic Analyses
We identified and retrieved non-redundant protein sequences
of ZIP family members of four monocots such as
Brachypodium distachyon , Oryza sativa, Setaria italic,
Sorghum bicolor from the Phytozyme database (www.
phytozome.net) that had a ZIP domain (Pfam:PF02535)
(Supplementary Table 1). After that, conserved sequences of
those proteins were aligned using the ClustalW program in
BioEdit software (www .mbio.ncsu.edu/bioedit/bioedit)with
default parameters (Hall 1999). Based on the conserved se-
quences alignment of the proteins, the rooted phylogenetic
tree was constructed using MEGA 5.05 software (Tamura
et al. 2011), by both the neighbor-joining method (Saitou
and Nei 1987) and the minimum evolution method. The
reliability of the phylogenetic tree was estimated using
Plant Mol Biol Rep
bootstrap values with 1,000 replicates. Using these methods,
evolutionary relationships of ZIP family members were
established between maize and the four other monocots men-
tioned above. In contrast, the phylogenetic relationships of
ZmZIP genes were established on the basis of the ZIP domain
only, keeping the rest of the parameters unchanged.
Determination of Exon-intron and Cis -elements
in the Promoter
To determine the exon–intron organization, genomic and cod-
ing sequences (predicted, cDNA when available) of ZmZIPs
were aligned. To identify TM (trans-membrane) domains of
ZmZIP proteins, we used Conpred II (http://bioinfo.si.
hirosaki-u.ac.jp/~ConPred2/), a consensus prediction method
for obtaining transmembrane topology models. To identify the
cis-elements, promoter sequences from +1 to −1,000 bp of
each of the ZmZIPs were extracted from www.
maizesequence.org and analyzed for stress-responsive cis-
elements in the PLACE database (Higo et al. 1999)
Motif Analysis
To further analyze the structure of the ZIP domains, w e
identified their protein sequence throu gh SMART (www.
smart.embl-heidelberg.de) and aligned them using
CLUSTALW software. Conserved motifs were identified
from the 12 ZmZIPs using the MEME 4.6.1/MAST motif
search software (Bailey a nd Elkan 199 4; Bailey and
Gribskov, 1998)(www .meme.sdsc.edu/meme/cgi-bin/meme.
cgi) with the following parameters: (1) distribution of motif
occurrences with any number of repetitions, (2) 6 and 60
amino acids as minimum and maximum width of motifs, (3)
only motifs with expected value lower than 1×10
−20
,and(4)a
maximum 10 number of motifs per peptide sequence. The
functional annotations of these motifs were analyzed by
InterProScan (www.ebi.ac.uk/Tools/pfa/iprscan), SMART
and the MOTIF search database (genome.jp/tools/motif).
Sequence logos of conserved motifs were also generate d
with WebLogo (Crooks et al. 2004).
In silico Expression Patterns of the ZmZIPs
To id en t if y the expression patterns, sequ ence tags of all
ZmZIPs were investigated at the available transcriptional level
in public domain. Maize ESTs were obtained through blastn
searches against the database (www.maizesequence.org). The
ZmZIP genes were analyzed by using the tblastn program with
the following parameters: (1) maximum identity of 95 % and
(2) minimum length of 400 bp with E value>10
−10
.Inaddition
to the maize EST database, maize expression data of ZmZIPs
were also extracted from the Maize Assembled Genomic
Island (MAGI) (www.magi.plantgenomics.iastate.edu), the
Plant Genomic Database (Plant GDB) (www.plantgdb.org)
including EST, cDNA and PUTs (Plant GDB unique
transcripts) and from the MPSS database (www.mpss.udel.
edu/maize).
Expression Analysis of ZmZIPs by qPCR
We analyzed the expression levels of ZmZIP transcripts by
real-time PCR in VQL-2 and CM-145 which were the con-
trasting high and low kernel zinc-containing maize inbreds,
respectively, the former one being an isogenic line of the latter
(Chakraborti et al. 2009;Prasannaetal.2008). The tissue from
flag leaf at the age of 10-day-old kernel and 10-day-old baby
kernel were both sampled in liquid nitrogen from the plant of
those two inbreds, namely VQL-2 and CM-145. Total RNA
was extracted from 100 mg of tissue using Trizol reagent
(Invitrogen, Carlsbad, CA, USA) following the manufacturer’s
instructions. The yield and quality of DNAase (Promega Life
Sciences)-treated RNA were determined by Nanodrop 1000
(M/S; Thermo Scientific, USA) and 2 % agarose gel electro-
phoresis in MOPS [3-(N-morpholino) propanesulfonic acid]
buffer, respectively. The cDNA was synthesized using 1 μgof
RNA with 200 U l
−1
reverse transcriptase Superscript TM III
(Invitrogen), 10 mM dNTPs and 250 ng oligo (dT). The
resulting cDNA samples were diluted 20 times (1:20) in
RNase-free water, and 2 μl of the diluted cDNA was used in
a total reaction volume of 25 μl for determining the relative
expression of ZmZIPs using QuantiFast SYBR Green PCR
Master Mix (Qiagen, India). The primers used to amplify
ZmZIPs are listed in Supplementary Table 2. Real-time PCR
analysis was performed in a 96-well plate using Roche 454
qPCR system (Roche, USA). The thermal cycling conditions
of 95 °C for 5 min followed by 45 cycles of 95 °C for 15 s,
60 °C for 30 s, and 72 °C for 30 s were used. The expression of
each ZmZIP gene in various samples was normalized with
actin 1 as reference gene (GRMZM2G126010) as an internal
control (Zhao et al. 2011). The experiment was performed with
at least three independent biological replicates and two techni-
cal replicates for each biological replicate. The specificity of
the PCR reactions was confirmed by melting curve analysis of
the amplicons. The comparative 2
-Δct
[Δ C
T
=C
T
,geneof
interest - C
T
, actin 1] method was used to calculate the relative
quantities of each transcript in the samples (Schmittgen and
Livak 2008). Statistical analyses were conducted using the
SAS software of JMP Genomics (SAS Institute, NC, USA).
Results and Discussion
Zinc is an essential micronutrient for plant metabolism and
growth. The deficiency of Zn decreases plant growth and
affects cereal production and grain quality (Ishimaru et al.
2011), but excess Zn may cause significant toxi city to
Plant Mol Biol Rep
biological systems (Ishimaru et al. 2007). Therefore, plants
have established a tightly controlled system to balance the
uptake, utilization, and storage of these metal ions. The ZRT
and IRT-like protein (ZIP) family has been characterized
ubiquitously in organisms, including archaea, bacteria, fungi,
plants, and mammals, and has been demonstrated to be in-
volved in metal uptake and transport (Ishimaru et al. 2011).
ZIP proteins generally contribute to metal ion homeostasis by
transporting cations into the cytoplasm (Nozoye et al. 2013).
Functional complementation in yeast indicated that ZIP pro-
teins are able to transport various divalent cations, including
Fe
2+
,Zn
2+
,Mn
2+
,andCd
2+
(Guerinot, 2000). Although ZIP
genes have been characterized from several plants, their infor-
mation on maize is as yet very scanty. With the completion of
the maize genome, several gene families have been character-
ized (Liu et al. 2013a, b; Zhang et al. 2013). Therefore, in the
present study, we have identified, through genome-wide in
silico analysis, and characterized the ZmZIP family genes of
maize.
Identification, Characterization and Mapping of ZmZIPs
As key Zn transporter, the ZmZIPs family plays an important
role in Zn homeostasis in maize which affects plant growth
and development. We used Arabidopsis AtZIP1
(AT3G12750) gene for blastn search against the maize ge-
nome (maizesequence.org) for the identification of ZIP genes.
We identified a maize gene GRMZM2G045849 which had
54 % similarity with AT3G12750 with a characteristic ZIP
domain (Pfam:PF02535). This ZIP domain is responsible for
the transport of Zn metal ions in plants (Grotz et al. 1998). A
total of 12 non-redundant putative ZIP family genes were
finally identified by a genome-wide survey of maize that
significantly had ZIP domains (Table 1). In contrast, Sharma
and Chauhan (2008) identified 13 ZmZIPs . Although the
basis of the identification of ZmZIPs was not mentioned in
their report, it clearly differed from ours. This may be due to
the fact that, at that time, the maize genome had not been fully
annotated which led them to identify one redundant se-
quence as we noticed in that analysis. Such an observation
about redundancy of the gene has also been reported else-
where in identifying the members of the gene family (Jami
et al. 2011). Additionally, we also found two proteins, namely
GRMZM2G379348 and GRMZM2G045531, in the
Phytozome database that were initially identified by a cross-
check with the key word ‘zinc transporter’ in the search;
however, these two proteins were finally excluded as either
they did not have a ZIP domain or were found to be a
truncated protein sequence. Thus, deduced polypeptides of
the corresponding 12 ZmZIP genes were analyzed for the
number of amino acids (length), molecular weight, and iso-
electric point (pI). The amino acids number varied from a
minimum of 279 (ZmZIP6) to maximum of 573 (ZmZIP12).
Similar amino acid lengths for ZIP genes have also been
reported earlier in rice (Chen et al. 2008). The molecular
weight of the ZmZIP proteins varied from a minimum of
29.6 kDa (ZmZIP6) to 59.5 kDa (ZmZIP12). Only two pro-
teins, namely ZmZIP1 and ZmZIP12, were found to be large,
with a molecular weight of 51.8 and 59.5 KDa, respectively
(Table 1). Lengths of the ZmZIP genes were found to be
minimum of 1,340 bp (ZmZIP3) to maximum of 2,430 bp
(ZmZIP12). It has also been observed that all the genes begin
with an initiation codon and end with a stop codon, indicating
that they were functional in nature. In addition, the pI value is
also considered to be an important biochemical property for
ZmZIPs because these genes having different acidic or basic
features that might respond differentially to various environ-
mental factors (Allagulova et al. 2003). Theoretical pI values
Table 1 Properties of ZmZIP genes and their proteins
Gene name Sequence ID Chr Genomic sequence (bp) ORF length (bp) Deduced peptides
Length (aa) MW (kDa) PI GRAVY
ZmZIP1 GRMZM2G001803 1 6,610 1,899 483 51.8 6.01 -0.207
ZmZIP2 GRMZM2G118821 1 2,205 1,504 381 40.7 9.28 0.425
ZmZIP3 GRMZM2G115190 1 1,419 1,340 361 37.6 8.65 0.600
ZmZIP4 GRMZM2G045849 2 2,490 1,490 367 38.5 7.87 0.553
ZmZIP5 GRMZM2G111300 4 2,687 1,668 386 38.6 7.22 0.642
ZmZIP6 GRMZM2G050484 4 5,598 1,701 279 29.6 6.65 0.719
ZmZIP7 GRMZM2G064382 6 3,170 1,714 402 41.8 6.02 0.436
ZmZIP8 GRMZM2G015955 6 2,579 1,749 387 40.4 6.97 0.528
ZmZIP9 GRMZM2G047762 6 2,240 1,525 341 34.7 5.30 0.707
ZmZIP10 GRMZM2G093276 7 2,010 1,430 397 40.6 6.33 0.551
ZmZIP11 GRMZM2G034551 8 2,898 1,634 396 41.5 6.15 0.449
ZmZIP12 GRMZM5G813470 10 4,270 2,430 573 59.5 8.44 0.793
Plant Mol Biol Rep
of ZmZIPs varied from a minimum of 5.30 (ZmZIP9)toa
maximum of 9.28 (ZmZIP2)(Table1). Further analysis of the
amino acid compositions of all ZmZIP proteins indicated that
they shared the common features; just one exceptional exam-
ple was ZmZIP1 which had comparatively low alanin (9 %)
and a negative GRAVY (−0.207) value. All the other ZmZIPs
were found to have a positive GRAVY value (0.425–0.793)
(Table 1) indicating the presence of the very hydrophobic
nature of peptides which is a common feature for
membrane-spanning proteins (Grotz et al. 1998). It has also
been found that, except for three genes, namely, ZmZIP6,
ZmZIP7 ,andZmZIP8, all the other genes had signal peptides
indicating that they might be involved for the movement from
one organelle to other within the cytoplasm. Based on
PROSITE analysis, most of the ZmZIP proteins were predict-
ed to be located in the plasma membrane, except for ZmZIP4 ,
ZmZIP9 ,andZmZIP7, ZmZIP5 proteins which were predict-
ed to be located in cytoplasm and chloroplast, respectively.
Similar results were also obtained with rice OsZIP genes,
where OsZIP4, OsZIP5, and OsZIP8 were located in the
plasma membrane while others are located in cytoplasm
(Chen et al. 2008; Ishimaru et al. 2006, 2007).
The deduced protein sequences when scanned through
PROSITE (www.expasy.org/tools/ scanprosite) showed the
presence of sites for various post-translational modifications,
and other sequence-specific features (Table 2). There were
multiple putative phosphorylation sites in these protein se-
quences, which might have acted as substrates for several
kinases in the form of casein kinase II, protein kinase C,
tyrosine kinase, and cAMP- and cGMP kinases (Table 2)for
these proteins, and this is also well-documented in the litera-
ture for the ZIP genes (Aydemir et al. 2012). Further, it had
been found that N-Myristoylation was one of the major post
transcriptional modifications, the value of which varied from a
maximum of 23 to a minimum 7 with no myristoylation with
the ZmZIP11 protein. Myristoylation can influence the con-
formational stability of individual proteins, as well as their
ability to interact with membranes or the hydrophobic do-
mains of other proteins. Thus, it plays a critical role in many
cellular pathways, especially in the areas of signal transduc-
tion, apoptosis, and extracellular export of proteins and mem-
brane transport (Zaun et al. 2012). Therefore, we concluded
that it might play an important role in the transport of zinc in
maize. Locations of the members in the chromosome are very
important and depend on the gene duplications, linkages, and
recombinations. To provide a simplified nomenclature for
each identified gene, names were given from ZmZIP1 to
ZmZIP12 to distinguish each ZmZIPs (and corresponding
proteins) that were denominated as ‘ZmZIP
’, and the followed
by a number to represent the gene number according to their
locations in the chromosomes 1 to 10 in descending order.
Based on available information, standard ZmZIP genes were
positioned on maize chromosomes (Fig. 1). Although the 3rd,
5th and 9th chromosomes did not have any ZmZIPs, yet the
maximum of three genes (ZmZIP1 , ZmZIP2 and ZmZIP3 )
were each located on chromosome 1 and three (ZmZIP7,
ZmZIP8 and ZmZIP9 ) on chromosome 6. Two genes, namely
ZmZIP5 and ZmZIP6, were located on chromosome 4. On the
other hand, ZmZIP10 and ZmZIP12 were located on chromo-
somes 8 and 10, respectively.
Phylogenetic and Synteny Analysis of ZmZIPs
In order to analyze the evolutionary relationship of the ZIP
family, a phylogenic tree was constructed. The rooted tree
topologies of ZmZIPs generated by the two methods were
comparable without modifications at branches, and supported
by their high bootstrap values, sugge sting tha t we had
constructed a reliable rooted tree topology, in which the 12
ZmZIPs were grouped into three distinct classes
(Supplementary Fig. 1) that were generated by their evolu-
tionary divergence, mostly corresponding to the subgroups
identified by motif analysis. On the other hand, the global
phylogenetic rooted tree comprised of 5 different monocot
species indicated that all the plants had larger numbers of ZIP
genes than maize. For instance, Setaria italic, Brachypodium
distachyon, Sorghum bicolor,andOryza sativa had 16, 16,
17, and 17, respectively (Fig. 2). Syntenic analysis indicated
that all ZmZIPs were found to have orthologous sequences in
other monocots analyzed and possessed similar ZIP domain.
Comparative multiple alignment of amino acid sequences of
these monocot ZIP genes common to each orthologous group
shared 63 % identity (data no t shown). Considering that
Tabl e 2 Number of predicted post-translational modification sites of
ZmZIP genes
Gene name cAMP Casein N-Myr PKC N-gly Ami Tyk
ZmZIP1 3 9 7 6 2 ––
ZmZIP2 1 1 13 5 2 1 –
ZmZIP3 – 3103–––
ZmZIP4 – 61052––
ZmZIP5 – 2153–––
ZmZIP6 – 4134–––
ZmZIP7 1 2 12 3 2 1 –
ZmZIP8 – 78311–
ZmZIP9 – 3123– 1 –
ZmZIP10 – 117111–
ZmZIP11 – 2 – 311––
ZmZIP12 2 3 23 5 –––
cAMP cAMP - and cGMP-dependent protein kinase C phosphorylation
site; caesin casein kinase II; N-Myr N-myristoylation site; PKC protein
kinase C phosphorylation site; N-gly N-glycosylation site; Tyk tyrosine
kinase phosphorylation site; Amid amidation site
Plant Mol Biol Rep
orthologs often retained equivalent functions in the course of
evolution (Altenhoff and Dessimoz 2009), we examined the
orthologous relationships between ZmZIPs and SbZIPs of
sorghum genes using a local synteny-based method. A total
of 12 genes from ZmZIPs had one or more putative orthologs
in sorghum. All of them were classified into the same group as
their orthologs in sorghum, further supporting the results of
the phylogenetic analysis. Although the reason is not clear,
certainly gene duplication might have played an important
role in a succession of genomic rearrangements and expan-
sions of this gene family among the different species (Zaun
et al. 2012) as indicated by the synteny analysis. It is a
I
II III IV V VI VII VIII XI XII
ZmZIP1
ZmZIP2
ZmZIP3
ZmZIP4
ZmZIP5
ZmZIP6
ZmZIP7
ZmZIP8
ZmZIP9
ZmZIP10
ZmZIP11
ZmZIP12
301 Mb
(198 )
(258 )
(300)
(10 )
237 Mb
232 Mb
241 Mb
217 Mb
169 Mb 176 Mb
175 Mb
156 Mb
150 Mb
(26)
(233)
(104)
(132)
(153)
(21)
(125)
(10)
Fig. 1 Locations of the different
ZmZIP genes in the maize
genome. Roman numerals
indicates different chromosomes
of maize and numbers in
parentheses indicate the position
ofthegeneinMb
I
II
III
IV
V
Fig. 2 A rooted, neighbor-joining
(NJ)-based tree of the ZIP proteins
in selected monocots. The analysis
was performed as described in
“Materials and Methods”
Plant Mol Biol Rep
common phenomenon that has been reported in several stud-
ies (Vision et al. 2000; Lynch and Conery 2000; Simillion
et al. 2002;Raesetal.2003). Further, we also compared the
chromosomes between maize and sorghum which revealed
interesting syntenic relationships among the ZIP genes of both
species. Genes of chromosome 1 from maize showed co-
linearity with sorghum chromosomes 1 and 7, whereas chro-
mosome 6 revealed colinearity with sorghum chromosomes 1,
6, 9, and 10 (Supplementary Fig. 2). Additionally, it is known
that gene family expansion occurs through three mechanisms.
They are t andem duplication, segmental duplication, and
transposition events (Maher et al. 2006). In this study, we
found several segmental duplication events occurred among
the ZmZIPs rather than the other two methods. For an exam-
ple, ZmZIP2 an d ZmZIP10 had se gmental duplication.
Similarly, ZmZIP8 as well as ZmZIP5 and ZmZIP1 and
ZmZIP3 had segmental duplication. It was found that the
coding sequences of all the ZmZIPs genes were disrupted by
introns. Therefore, we concluded that intron loss might ac-
company the recent evolution of maize ZmZIP genes which is
also observed in the case of the aldehyde dehydrogenase gene
in maize (Zhou et al. 2012). Although gene-order conserva-
tion is widely used as the benchmark for orthology prediction
(van der Heijden et al. 2007), all 12 ZmZIPs genes (100 %)
were revealed by the synteny analysis. This findings were also
supported by phylogenetic analysis to provide support to
interpret putative orthologous or paralogous genes. Although
the bootstrapping values for some nodes were not exception-
ally high, the reliability of our phylogenetic trees was sup-
ported by gene structure and synteny analyses. Similar results
have been reported in rice (Nakano et al. 2006), and grape
(Zhuang et al. 2009). Genome analysis indicated that whole
genome duplication in the ancestral grass genome occurred
around 70 million years ago, predating the divergence into
panicoid, oryzoid and pooid sub-families (Vision et al. 2000).
Analysis of ZIP gene sequences from maize, rice, sorghum
and brachypodium in the plaza database (www.
bioinformatics.psb.ugent.be/plaza) indicated that these genes
might have undergone segmental duplications. The genes in
the segmentally duplicated regions were found to b e
congruent with the orthologous sequences in the phylogeny
tree and had high sequence similarity, suggesting that these
genes were evolutionarily conserved and may have functional
redundancy.
The global phylogenetic tree was divided into five different
groups which mostly corroborated the presence of the con-
served motif. Although we found 10 conserved motifs as
generated by MEME, which is one of the most widely used
tools for observation of new sequence patterns in biological
sequences and analysis of their significance, yet among them
only the 1st, 2nd, 3rd, 4th and 9th motifs were found to have
ZIP domains (Supplementary Fig. 3) and the rest did not have
any hit in the MOTIF search database (www.genome.jp). This
indicate d that the 1 st, 2nd, 3rd, 4th an d 9th motifs were
directly involved in Zn metal binding and transport. We
found that group I was composed of 35 ZIP genes, all of
which contained 8 motifs, i.e. 1st, 2nd, 3rd, 4th, 5th, 6th,
7th, and 10th motif. Similarly, group II contained 25 ZIP
genes, each of which contained 5 motifs (3rd, 5th, 6th, 7th
and 8th) and hence formed a different group. Group III
consisted of small number of 5 genes with 7 different motifs
(1st, 2nd, 4th, 5th, 6th, 7th and 10th). Simultaneously, group
IV consisted of 10 ZIP genes with mostly 4 motifs, namely 3rd,
5th, 6th and 7th, except for two genes,
BrpZIP6 and OsZIP1,
that had one additional 1st motif. Finally, group V was the
smallest one with 3 genes (SiZIP2 , OsZIP7 and SiZIP1)and
motif analysis revealed that they were composed of
heterogeneous motifs. For example, while SiZIP2 consisted of
4 motifs (3rd, 5th, 6th and 7th), the other two, OsZIP7 and
SiZIP1 , had 7 different motifs (1st, 2nd, 3rd, 4th, 5th, 6th and
10th), which might be the reason that both of them were in same
clade. Similar phylogenetic classification of the gene family
based on the conserved motifs in plants a re well-documented
in the literature (Jami et al. 2011; Ricachenevsky et al. 2011).
Detection of Exon–intron and Cis-elements in the Promoters
of ZmZIPs
Structural diversity among the members of a gene family de-
pends on the number of exons and introns as well as their length.
We also tried to understand the identity among the ZmZIPs and
found that, in general, the ZmZIPs were highly diverse, varyimg
from a minimum of 11 % identity between ZmZIP7 and
ZmZIP1 to a maximum 72 % identity between ZmZIP2 to
ZmZIP3 genes (Supplementary Table 3). Perhaps the var ying
length of peptides as indicated for a minimum of 279 to a
maximum 573 amino acids (T able 1) might contribute to the
structural diversity . These findings also corroborated the earlier
finding of Chen et al. (2008) who reporte d the iden tify of OsZIP
genes of rice varied widely, from 17 to 70 %, indicating the
highly diverse nature of ZIP genes. The high structural diversity
of ZIP genes might be involved in the transport of a variety of
cations (Belouchi et al. 1997). Secondly, it has also been ob-
served that the length as well as the number of introns may also
contribute to the structural diversity. It has also been revealed
that ZmZIP6 had a maximum 12 introns, whereas ZmZIP3 had
a minimum 2 (Supplementary Fig. 4). Therefore, we concluded
that the length as well as the number of introns and correspond-
ing peptide length contributes to the structural diversity of
ZmZIPs as well documented in the literature (Jain et al. 2006;
Nakano et al. 2006; Terol et al. 2006).
The cis -elements are important molecular switches in-
volved in the transcriptional regulation of genes during gene
expression and may be induced through ABA-dependent and
ABA-independent signal transduction pathways (Yamaguchi-
Shinozaki a nd Shinozaki (2005). Previous studies i n
Plant Mol Biol Rep
Arabidopsis showed the presence of cis-elements ZDRE
(Zinc Deficiency Responsive Elements) that could respond
to zinc deficiency stress (Assunção et al. 2010). Having seen
the differential expression of ZmZIPs, we analyzed the puta-
tive promoter sequence of 1,000 bp from the translational start
site to search for stress-responsive cis-elements in the PLACE
database (Higo et al. 1999). In silico sequence a nalysis
showed that the promoter of each gene contained at least
one of the four related putative cis-elements, such as zinc
deficiency-related elements (ZDRE) (GTCGAC), ABA re-
sponsive elements (ABRE) (ACGTG), dehydration respon-
sive elements (DRE/CRT) (G/ACCGCC), and low tempera-
ture responsive element (LTRE) (CCGAC) motifs. The pro-
moters of 6 genes, namely ZmZIP2, ZmZIP5 , ZmZIP7,
ZmZIP8 , ZMZIP9 and ZmZIP11, contained ZDRE cis-ele-
ments which are responsible for activation during zinc defi-
ciency stress (Assunção et al. 2010) (Supplementary Table 4).
Analysis of Conserved Motif
Motifs are the most distinctive features of the proteins. A total
of 10 motifs containing 6–53 amino acid residues were iden-
tified (Supplementary Fig. 1). While most of the ZmZIPs were
found to have 6–8 TM domains, a typical characteristic feature
of Zn transporter protein (Eide 1998), ZmZIP12 had a maxi-
mum of 13, while ZmZIP1 had 7 TM domains
(Supplementary Fig. 5). It is known that Zn transporters can
be basically classified into two categories: the cation diffusion
facilitator (CDF) transporter family and the ZIP family. The
CDF family has common structural characteristics with 6 TM
domains containing histidine-rich motifs, which are predicted
to be exposed to the cytosol (van der Zaal et al. 1999;Eng
et al. 1998). On the other hand, the proteins of the ZIP family
are predicted to have 8 TM domains in which the C terminal of
peptide is found inside the surface of to plasma membrane,
and the N terminal ends of the protein are located on the
outside surface of the plasma membrane. However, the im-
portant feature shared by most of the ZIP proteins is a long
hydrophobic loop located between TM domains III and IV.
This region is referred to as the ‘variable region’ because both
its length and sequence showed little conservation among the
family members (Guerinot 2000), which has also been found
in our analysis (Fig. 3). Additionally, variable region that is
shared by several of the ZIP proteins is characterized by the
presence of many conserved histidine residues i.e. H-x-H-x-H
which is reported to be a putative metal binding sequences
I
II III
IV
V
VI
VI
I
VIII
Q
ZmZIP3
ZmZIP2
ZmZIP4
ZmZIP5
ZmZIP9
ZmZIP10
ZmZIP8
ZmZIP7
ZmZIP11
ZmZIP6
ZmZIP12
ZmZIP1
ZmZIP3
ZmZIP2
ZmZIP4
ZmZIP5
ZmZIP9
ZmZIP10
ZmZIP8
ZmZIP7
ZmZIP11
ZmZIP6
ZmZIP12
ZmZIP1
Z
mZIP3
Z
mZIP2
Z
mZIP4
Z
mZIP5
Z
mZIP9
Z
mZIP10
Z
mZIP8
Z
mZIP7
Z
mZIP11
Z
mZIP6
Z
mZIP12
Z
mZIP1
ZmZIP3
ZmZIP2
ZmZIP4
ZmZIP5
ZmZIP9
ZmZIP10
ZmZIP8
ZmZIP7
ZmZIP11
ZmZIP6
ZmZIP12
ZmZIP1
Fig. 3 Multiple sequence alignment of deduced peptides of ZmZIP
genes obtained by ClustalW. The TM domains indicated as roman nu-
merals at the top of the alignments. The red bar indicates the ‘variable
region’ between III and IV TMs. The square indicates the conserved
histidin motif (H-x-H-x-H) in the ‘variable’ region. The dotted square
indicates the ZIP motifs
Plant Mol Biol Rep
(Gaither and Eide 2001), similar to the present study (Fig. 3).
It is interesting to note that a fairly well-conserved histidyl
residue, substituted by polar or semipolar residues, and adja-
cent to another polar residue, was found at the beginning of
the TM domain of Vamong the ZmZIPs. Eide et al. (1996)as
well as Zhao and Eide (1996) identified regions in the IRT1,
ZRT1 ,andZRT2 proteins that exhibited the ‘HX
3
’ sequence
between putative spanners III and IV. Therefore, it was pos-
tulated that HN
3
migh t be an extra membrane metal ion
binding site. Similar sequences have been identified in mem-
bers of another heavy metal ion transport family, the cation
diffusion facilitator (CDF) family (Paulsen and Saier 1997).
Further, we also identified two ZIP motifs within the ZIP
domain i.e. [LM] GIV [VS] HS VIIG [LVIM] SLG [AV] S
and [SA] FH [QN] [VMLF] FEG [MIF] [GA] LGGCI which
are present in TM domains IV and V, respectively. While the
former one found in the present study was reported earlier as a
typical ZIP signature in the TM domain IV in rice (Chen et al.
2008) and various organisms (Eng et al. 1998), the latter has
not been documented for any plant system in the literature
until now.
In silico Expression Patterns of the ZmZIP Genes
After integrating and analyzing all expression data, we found
that all the ZmZIP genes were divided into 9 groups based on
Dev In Silk Husk Ear Pollen Tussle Embryo Root Leaf
Z
mZIP1
Z
mZIP2
Z
mZIP3
Z
mZIP4
Z
mZIP5
Z
mZIP6
Z
mZIP7
Z
MZIP8
Z
mZIP9
Z
mZIP10
Z
mZIP11
Z
mZIP12
Fig. 4 In silico expression
analysis of ZmZIP genes across
the different tissues. The heat map
displaying the transcript
abundance was produced from
integrating and analyzing of all
expressed data (Dev In
developing inflorescence). Green
to deep red indicate the relative
increase of abundance
0
500
1000
1500
CM-COB VQL-COB CM-FL VQL-FL
ZmZip1
0
100
200
300
CM-COB VQL-COB CM-FL VQL-FL
ZmZip2
0
1
2
3
CM-COB VQL-COB CM-FL VQL-FL
ZmZip3
0
50
100
CM-COB VQL-COB CM-FL VQL-FL
ZmZip4
0
2000
4000
CM-COB VQL-COB CM-FL VQL-FL
ZmZip5
0
200
400
600
CM-COB VQL-COB CM-FL VQL-FL
ZmZip6
0
5
10
15
20
CM-COB VQL-COB CM-FL VQL-FL
ZmZip7
0
500
1000
1500
2000
CM-COB VQL-COB CM-FL VQL-FL
ZmZip8
0
500
1000
1500
2000
CM-COB VQL-COB CM-FL VQL-FL
ZmZip9
0
500
1000
1500
2000
CM-COB VQL- COB CM-FL VQL-FL
ZmZip10
0
2000
4000
6000
8000
10000
12000
CM-COB VQL-COB CM-FL VQL-FL
ZmZip11
0
5
10
15
20
CM-COB VQL-COB CM-FL VQL-FL
ZmZip12
Fig. 5 Relative transcript abundance (on y-axes)ofZmZIP genes as revealed by qPCR analysis (COB 10-day-old kernel, FL Flag leaf sampled at 10-
day-old kernel, CM CM-145, VQL VQL-2)
Plant Mol Biol Rep
types of tissue that were found as hits in the database search.
Differential tissue-specific expression was found among the
different ZmZIP genes. While maximum expression of
ZmZIP5 , ZmZIP11 , ZmZIP1 and ZmZIP10 was found in leaf,
very little expression was found for developing inflorescence,
silk, and husk. It had also been found that, while expression of
ZmZIP1 was maximum across a wide range of tissues, the
expression of ZmZIP12 was found to be very low (Fig. 4),
which corroborated with low in vivo expression detected by
real-time PCR (Fig. 5). Interestingly, ZmZIP5 and ZmZIP11
were highly expressed in flag leaf which again was similar
with the real-time PCR analysis in the present study (Fig. 5).
Tissue-specific Expression of ZmZIPs
Finally, to understand the physiological function of the
ZmZIPs , the expressio n pattern was investigated by real-
time PCR in some selected tissues. Flag leaves are the major
source of phloem-delivered photo-assimilates for developing
seeds, and are believed to be one of the sources of remobilized
metals for the seeds (Narayanan et al. 2007; Sperotto et al.
2009), which has been experimentally demonstrated in vari-
ous plants (Uauy et al. 2006; Waters et al. 2009). Similarly, in
maize, 10-day-old baby kernels have been found to be meta-
bolically very active (Seebauer et al. 2004), and Zn plays an
essential role in embryo and endosperm development (Vallee
and Falchuk 1993). Therefore, we selected both tissues i.e.
flag leaf and baby kernel, to determine the expression levels of
the genes. Our data revealed that, except for ZmZIP3 and
ZmZIP12, the overall expression of the other 10 genes was
higher in the flag leaf. The ZmZIP2 transcripts were not
detected or were detected below a confidence threshold. The
expression levels of ZmZIP genes varied considerably, with
some genes reaching higher expression levels (ZmZIP5 and
ZmZIP11 ) and others showing very low expression
(ZmZIP7). Similar behavior of the Zn genes has also been
reported in rice, another strategy II plant like maize. In rice,
several Zn transporters have been functionally characterized,
e.g., OsIRT1 , OsIRT2, OsZIP1 , OsZIP3 , OsZIP4 ,and
OsZIP5 (Ishimaru et al. 2006; Lee et al. 2010a; Ramesh
et al. 2003). Among them, OsZIP1, OsZIP3, OsZIP4
,and
OsZIP5 have been found to be rice Zn transporters induced by
Zn deficiency (Ramesh et al. 2003; Lee et al. 2010b;Ishimaru
et al. 2006). In situ hybridization analysis has revealed that
OsZIP4 in Zn-deficient rice was expressed in the meristem of
Zn-deficient roots and shoots, and also in vascular bundles of
the roots and shoots. These results suggest that OsZIP4 is a Zn
transporter that may be responsible for Zn translocation to the
plant parts that require Zn. A few members of the ZIP genes of
maize have also shown high ti ssue specificity, i.e. they
expressed at very low level (ZmZIP2 , ZmZIP5 , ZmZIP6 ,
ZmZIP8 and ZmZIP11 ) in kernel but expressed at a compar-
atively higher level in the flag leaf. Similar results were also
found in rice. The OsZIP3, OsZIP4 and OsZIP10 gene have
shown significantly high levels of expression in flag leaf tissue
(Ramesh et al. 2003; Ishimaru et al. 2006), thus suggesting the
role of ZmZIP2 , ZmZIP5, ZmZIP6 , ZmZIP8 and ZmZIP11
genes in grain partitioning of Zn ions in maize. Flag leaves are
the major source of remobilized metals for developing seeds
(Sperotto et al. 2009). We also found that certain genes such as
ZmZIP2 , ZmZIP3 , ZmZIP9 , ZmZIP9 were expressed more in
VQL-2. This type of genotypic-dependent expression of Zn
transporter is also observed in rice. The OsZIP4 gene, known
as a functional transporter of Zn
2+
ions in rice (Ishimaru et al.
2006), showed genotype-specific variation in the level of
expression in leaf tissue with a higher level of expression in
high Zn rice genotypes and a lower level expression in low Zn
rice lines. The expression patterns of ZmZIP2 , ZmZIP3,
ZmZIP9 , ZmZIP9 genes suggested that the genes might play
a role in governing the movement of Zn ions in leaf tissue. The
expression patterns of ZmZIP2, ZmZIP3, ZmZIP9, ZmZIP9
genes indicated that acquisition of higher amounts of Zn ions
in leaves of high Zn use in an efficient inbreed of maize might
enhance its remobilization to developing grains and hence
contribute to higher grain Zn values. Similarly, it had been
demonstrated that OsNAC5, a novel senescence-associated
ABA-dependent NAC transcription factor, was highly
expressed in flag leaf of rice, and were primarily responsible
for mobilizing the Fe and Zn from the flag leaf to the growing
kernel (Sperotto et al. 2009). Similarly, in the present study,
ZmZIP5 and ZmZIP11 were highly expressed in the flag leaf
of the VQL-2 inbreed which leads us to conclude that they
might play a vital role for the mobilization of Zn from flag leaf
to developing kernel for accumulation of high zinc contents in
the kernels of VQL-2 (Fig. 5). Therefore, it can be assumed
that over-expression of these two ZmZIP genes may provide
an alternative strategy for the biofortification of crops with Zn.
Acknowledgments The authors are grateful to Dr. K.V. Bhat, Head,
Division of Genetic Resource, NBPGR, New Delhi for his encourage-
ment to conduct this work and Dr. P.K. Agrawal, Head, Plant Improve-
ment Division, VPAS, Almora, India, for providing the maize inbred
seeds. We are also grateful to anonymous authors for making genome
sequence data available in the public domain. T.R.S. is thankful to ICAR,
New Delhi, for financial assistance under the NPTC project. T.K.M is
thankful to DBT, New Delhi for financial assistance.
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