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Overlapping transcriptional expression response of wheat zinc-induced

facilitator-like transporters emphasize important role during Fe and Zn stress

Article in BMC Molecular Biology · September 2019

DOI: 10.1186/s12867-019-0139-6

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Sharma et al. BMC Molecular Biol (2019) 20:22
https://doi.org/10.1186/s12867-019-0139-6 BMC Molecular Biology

RESEARCH ARTICLE Open Access

Overlapping transcriptional expression

response of wheat zinc‑induced facilitator‑like
transporters emphasize important role
during Fe and Zn stress
Shivani Sharma1,2, Gazaldeep Kaur1, Anil Kumar1, Varsha Meena1, Jaspreet Kaur2 and Ajay Kumar Pandey1*

Abstract
Background: Hexaploid wheat is an important cereal crop that has been targeted to enhance grain micronutrient
content including zinc (Zn) and iron (Fe). In this direction, modulating the expression of plant transporters involved
in Fe and Zn homeostasis has proven to be one of the promising approaches. The present work was undertaken to
identify wheat zinc-induced facilitator-like (ZIFL) family of transporters. The wheat ZIFL genes were characterized for
their transcriptional expression response during micronutrient fluctuations and exposure to multiple heavy metals.
Results: The genome-wide analyses resulted in identification of fifteen putative TaZIFL-like genes, which were
distributed only on Chromosome 3, 4 and 5. Wheat ZIFL proteins subjected to the phylogenetic analysis showed the
uniform distribution along with rice, Arabidopsis and maize. In-silico analysis of the promoters of the wheat ZIFL genes
demonstrated the presence of multiple metal binding sites including those which are involved in Fe and heavy metal
homeostasis. Quantitative real-time PCR analysis of wheat ZIFL genes suggested the differential regulation of the
transcripts in both roots and shoots under Zn surplus and also during Fe deficiency. Specifically, in roots, TaZIFL2.3,
TaZIFL4.1, TaZIFL4.2, TaZIFL5, TaZIFL6.1 and TaZIFL6.2 were significantly up-regulated by both Zn and Fe. This sug-
gested that ZIFL could possibly be regulated by both the nutrient stress in a tissue specific manner. When exposed to
heavy metals, TaZIFL4.2 and TaZIFL7.1 show significant up-regulation, whereas TaZIFL5 and TaZIFL6.2 remained almost
unaffected.
Conclusion: This is the first report for detailed analysis of wheat ZIFL genes. ZIFL genes also encode for transporter
of mugineic acid (TOM) proteins, that are involved in the release of phytosiderophores to enhance Fe/Zn uptake. The
detailed expression analysis suggests the varying expression patterns during development of wheat seedlings and
also against abiotic/biotic stresses. Overall, this study will lay foundation to prioritize functional assessment of the
candidate ZIFL as a putative TOM protein in wheat.
Keywords: Micronutrient uptake, Triticum aestivum L., Zinc transport, Biofortification, Iron deficiency

*Correspondence: [email protected]; [email protected]

1
National Agri-Food Biotechnology Institute (Department
of Biotechnology), Sector 81, Knowledge City, Mohali, Punjab 140306,
India
Full list of author information is available at the end of the article

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 2 of 17

Background positive co-relation was observed in NA and DMA levels

Cereal crops are important targets for enhancing micro- with the Fe and Zn content especially in embryo tissue
nutrient content, including iron (Fe) and zinc (Zn) in [22]. These functions of TOM family genes suggest their
the developing grains. From human nutritional point importance in the maintenance of micronutrient homeo-
of view, these micronutrients play an important role in stasis through secretion of DMA/MA and thereby help to
growth and development, cognitive and immune impair- translocate them into the sink areas of plants and is char-
ment, and in gene regulation [1–3]. Therefore, researches acteristic of Strategy-II mode of Fe uptake [21]. Further-
worldwide are identifying diverse approaches to generate more, the plant ZIFL genes were also reported for their
micronutrient rich food crops. Apart from a nutritional involvement in potassium homeostasis and their ability
point of view, Fe and Zn are also essential minerals for to transport transition metal such as cesium [23]. These
plant development and various biochemical functions [4, studies provide a valuable clue for ZIFL function that is
5]. Multiple metal specific transporters and regulators not just limited to transport of important micronutrients.
show co-expression and could share common signaling Hexaploid wheat (Triticum aestivum L.) is an impor-
features including the response towards Zn, Fe or other tant crop for the developing countries where, the sub-
metals. Several specific transporters that are involved optimal levels of grain Zn and Fe have been reported.
in the uptake and translocation of Fe and Zn, inside the Initiatives to enhance multiple micronutrients including
plant have been described previously [6–11]. Efficient Zn and Fe are being undertaken by either gain of func-
micronutrient uptake by plants is a concerted effort of tion approach or RNAi mediated gene silencing [24–26].
major genes belonging to the different families of trans- Earlier, using wheat grain transcriptome data it was con-
porters that include, but are not limited to zinc–regu- firmed that a higher proportion of transcripts were pre-
lated transporter, iron–regulated transporter family, the sent in abundant amount that are known to be involved
natural resistance associated macrophage protein family, in transport activity (GO:0005215) [27]. Therefore,
yellow-stripe 1-like (YSL) subfamily of the oligopeptide such studies provided the framework to investigate new
transporter superfamily, ­ Ca2+-sensitive cross comple- resources and genes that could be of immense value to
menter 1 (CCC1) family [12–16]. address the uptake and remobilization of micronutri-
Many evidences are gathering that suggest an impor- ents in cereal grains like wheat. To provide impetus in
tant role of major facilitator superfamily (MFS) clan of this direction, inventory of wheat MFS-ZIFL needs to be
transporters, yet the identification of specific candidate explored to identify important candidate genes.
genes has been always a bottleneck. Earlier, MFS-zinc- In the current work, 35 ZIFL homoeologs representing
induced facilitator-1 like (ZIFL) genes, were identified fifteen genes from hexaploid wheat were identified that
and its role was assessed in different stresses including showed their restricted distribution only at three chro-
Zn homeostasis [17]. Role of ZIFL was largely unad- mosomes viz. 3, 4 and 5. Phylogenetic analysis revealed
dressed in plants until the detailed inventory from model a uniform distribution of wheat ZIFL sequences in mul-
plant Arabidopsis thaliana and in crops like Oryza sativa tiple clades along with rice, Arabidopsis and Zea mays.
[17, 18]. Since, the first identification of the three ZIFL in Detailed characterization of the ZIFL genes for their
Arabidopsis referred to as AtZIF1 (AT5G13740), AtZIFL1 motif composition, promoter sequences and their expres-
(AT5G13750) and AtZIFL2 (AT3G43790) evidences are sion under Fe limiting and Zn surplus condition and
accumulating for their role specifically in Zn homeosta- other heavy metals was also performed. Our data indi-
sis [17]. Subsequently, multiple ZIFL genes from mono- cate that the wheat ZIFL show overlapping expression
cot such as rice were identified and the presence of high response during Fe deficiency and Zn excess condition.
numbers was correlated with the genome duplication Few of the wheat ZIFL gene expression remained unaf-
events. It was also speculated that plant ZIFL proteins fected by the presence of heavy metals. Overall, char-
might perform redundant function that is imperative acterization of crop ZIFL transporters could result in
by their overlapping gene expression response during identifying specific candidate/s that could be used fur-
changed regime of Fe and Zn [19]. In addition to that, the ther to modulate specific Fe-Zn uptake in crop plants
rice ZIFL genes were also functionally characterized to such as wheat.
be a transporter of mugineic acid (TOM), a phytosidero-
phore involved in strategy-II mode of Fe uptake via roots Results
[20, 21]. They are involved in efflux of phytosiderophores Genome‑wide identification and phylogenetic analysis
including deoxymugineic acid (DMA), mugineic acid of wheat ZIFL
(MA) or nicotianamine (NA) have been characterized In order to identify wheat ZIFL genes and to gain
in rice and are reported to be tissue-specific including insight for possible evolutionary relationship, two com-
shoot–root junction or in roots [19, 21]. In rice seeds a plementary approaches were used. This includes, first
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 3 of 17

performing genome-wide sequence search of MFS_1 and AtZIFL2. With rice, the highest percentage identity
family using Pfam BLAST (PF07690), followed by homol- of 87 was observed for wheat ZIFL2.2-3A and OsZIFL2.
ogy-based analysis with previously reported ZIFL genes Divergence was observed among TaZIFL3-4B and
in different plant species using Ensembl database. These OsZIFL13 with percentage identity of 50.
approaches resulted in the identification of one hundred
seventy-nine sequences and to further validate their Molecular structure and genome organization
identity sequences were checked and searched for MFS_1 The predicted protein length of the identified wheat ZIFL
domain through Pfam and conserved domain data- sequence ranged from 300 to 562 amino acids (Addi-
bases (CDD-NCBI) (Additional file 1: Table S1). These tional file 3: Table S2). In general, most of the wheat
sequences were then used to build phylogenetic tree ZIFL showed 10–12 predicted trans-membrane (TM)
with previously known ZIFL protein sequences from dif- domains as reported in rice [18]. Specifically, 16 wheat
ferent plants (Additional file 1: Table S1 and Additional ZIFL proteins were predicted to have 12 TM domains, 15
file 2: Figure S1). The arrangement of tree suggested a proteins have 10–11 predicted TM domains, 3 proteins
distinct clade for the ZIFL cluster when compared to the were found to have 8–9 TM domains and only one wheat
remaining MSF_1 proteins. This indicates that ZIFL is a ZIFL has 4 TM domains (Additional file 3: Table S2).
distinct group of MFS transporters that are tightly clus- Further, the genomic organization analysis revealed the
tered (Additional file 2: Figure S1). Further, this distri- presence of genes in all three A, B and D sub-genomes.
bution was confirmed through signature sequences that Maximum number of genes were found to be present on
are specific to ZIFL proteins. The presence of either of B and D sub-genome with 13 and 12 genes respectively
these signature sequences validated the distribution of (Fig. 2a). TaZIFL1.2, TaZIFL2.2, TaZIFL3, TaZIFL5,
ZIFL proteins. Two wheat specific signature sequences TaZIFL6.2, TaZIFL7.1, TaZIFL7.2 are present in all three
included (i) W-G-x(3)-D-[RK]-x-G-R-[RK] (found in genomes, while TaZIFL2.3 and TaZIFL2.4 are present
all except in TaZIFL2.5-5D) and (ii) S-x(8)-[GA]-x(3)- on only one genome 5B and 5D respectively (Additional
G-P-x(2)-G-G with an exception of A instead of G at file 3: Table S2). The chromosomal distribution mapping
10th position of (ii) signature in TaZIFL2. Furthermore, revealed TaZIFLs to be present only on chromosome 3,
sequences similar to ZIFL specific cysteine (Cys) and 4 and 5 with maximum of 17 sequences on chromosome
histidine (His) signatures were also used for identifying 4 (Fig. 2b, c). Next, the genomic structure was analyzed
TaZIFL. (iii) C-[PS]-G-C, absent in 6 TaZIFL sequences and regions corresponding to intron–exons were marked
(TaZIFL2.5-5D, TaZIFL 3-4B, TaZIFL 4.2-4B, TaZIFL (Fig. 3). TaZIFL clustered into the same group and shared
5-5D, TaZIFL 7.1-4B, TaZIFL 7.2-4B) probably due to almost similar distribution pattern for the number of
missing sequence information and (iv) [PQ]-E-[TS]-[LI]- exon/intron. The intron–exon number varies from 14 to
H-x-[HKLRD] (an insertion of ETLYCRHEHRYSIFISLD 18 in the respective TaZIFL genomic sequences (Fig. 3,
sequence within the motif was found in TaZIFL7.2-4A) Additional file 3: Table S2).
[18]. These signatures guided identification of specific
wheat ZIFL from the rest of the MFS_1 member. Such Protein motif analysis reveals the presence of diverse
analysis resulted in confirmation of a total of thirty-five domains
wheat ZIFL sequences, including individual TaZIFL To have an understanding about the similarity, varia-
genes and their respective homoeologs from differ- tion in motif composition and distribution of TaZIFL,
ent wheat sub-genomes (Additional file 3: Table S2). To 15 sequences representing each ZIFL transcript was
check the distribution along with other plant species, subjected to MEME analysis. Our analysis revealed
ZIFL protein sequences from O. sativa, Z. mays and the presence of fifteen motifs (Fig. 4a, Additional file 4:
Arabidopsis were used to build a rooted phylogenetic tree Table S3). Out of fifteen motifs, six were conserved
through the NJ method (Fig. 1). Because of the genome throughout all ZIFL, while some lacked few motifs. Four
duplication events in wheat, the genes are likely to show unique and exclusive motifs (12, 13, 14, 15) were identi-
multiple alleles of a single gene. Hence the resulted 35 fied, which are specific to the respective group. Motif
putative wheat ZIFL represent 15 genes after distribution 14 and motif 12 (Fig. 4b, Additional file 4: Table S3) are
with respective homoeologs (Fig. 1). To provide the uni- specific to TaZIFL2.1, TaZIFL2.2, TaZIFL2.3, TaZIFL2.4
form nomenclature, TaZIFL genes were named accord- and TaZIFL2.5, which indicated that TaZIFL2 members
ing to their respective closest known orthologs from rice. might share similar functions. Motif 14 was also present
Among the wheat ZIFL proteins TaZIFL4.1 showed high- in TaZIFL6.2. Another set of unique motifs, mentioned as
est homology with TaZIFL4.2 of 95.2 percentage identity. motif 13 and 15 was found in TaZIFL4.1 and TaZIFL4.2,
When a cross species comparison was done, the maxi- which may indicate probable different function from rest
mum identity of 87 percent was shown by TaZIFL2.2-3D of TaZIFLs. The canonical MFS signature WG[V/M/I]
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 4 of 17

Fig. 1 Phylogenetic analysis of wheat ZIFL protein sequences. The analysis was performed by using 35 ZIFL protein sequences from wheat, thirteen
from Oryza sativa, seven from Zea mays and three from Arabidopsis. Rooted phylogenetic tree was constructed by using Neighbour-joining method
using MEGA7 software with 1000 bootstrap replicates

[F/V/A/I]AD[K/R][Y/I//H/L]GRKP was majorly present CPGC reported previously were also present in most of
in the cytoplasmic loop between TM2 and TM3 (Addi- the wheat ZIFL proteins [18]. The absence of these motifs
tional file 2: Figure S2, Additional file 5: Table S4) as well was observed in TaZIFL2.5_5D, TaZIFL 3_4B, TaZIFL
S-x(8)-G-x(3)-G-P-[A/T/G]-[L/I]-G-G as anti-porter 4.2_4B, TaZIFL5_5D, TaZIFL7.1_4B and TaZIFL7.2_4B.
signature mainly in TM5. The results suggest that ZIFL This might be because of missing sequence informa-
proteins share unique signatures and high similarity indi- tion. This motif was found to be present in the cytoplas-
cating they are a distinct group of MFS family. Presence mic N-terminal loop for TaZIFL groups 2, 4, 5, 6 and in
of conserved signatures Cysteine (Cys)-containing motif the non-cytoplasmic N-terminal loop for groups 1, 3
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 5 of 17

Fig. 2 Genomic and chromosomal distribution of wheat ZIFL genes on wheat genome. Distribution of 35 TaZIFL across: a A, B and D sub genomes.
b Wheat chromosomal distribution. c Chromosomal distribution shared on different Chromosomes and sub-genomes

and 7 (Additional file 2: Figure S2 and Additional file 5: respond to Fe deficiency conditions. Few of these pro-
Table S4). Another conserved histidine (His)-containing moters consist of multiple such cis-elements suggest-
motif PET[L/I]H showed its presence in the cytoplasmic ing their diverse function in plants (Additional file 6:
loop between TM domains ranging from 2 and 3 to 6 and Table S5).
7, with highest between 6 and 7 TM domains (Additional
file 2: Figure S2). Expression patterns of wheat ZIFL genes under Zn and Fe
stress
Analysis of conserved cis‑elements in the promoter ZIFL are primarily known to respond towards Zn excess
of wheat ZIFL genes (+Zn), therefore experiments were performed to study
To find the molecular clues that could regulate the the gene expression of wheat ZIFL in roots and shoots.
expression of wheat ZIFL transcripts, the 1.5 kB pro- The qRT-PCR analysis suggested tissue specific expres-
moter region of the all identified wheat ZIFL genes was sion response by wheat ZIFL genes. A total of eight genes,
explored. Our analysis revealed a large number of cis- including TaZIFL1.2, TaZIFL2.2, TaZIFL2.3, TaZIFL4.1,
elements in the promoter of wheat ZIFL. Predominantly, TaZIFL4.2, TaZIFL5, TaZIFL6.1 and TaZIFL6.2 showed
the promoters were enriched with the presence of the significantly higher expression during one of the time
core binding site for iron-deficiency responsive element points under Zn surplus condition (Fig. 5). Among
binding factor 1 (IDEF), iron related transcription factor them, the fold expression level for TaZIFL4.1 was high-
2 (IRO2) and heavy metal responsive element (HMRE) est (~ sevenfold) at 3 days after treatment (DAT) with
(Additional file 6: Table S5). The presence of these pro- respect to control roots (Fig. 5a). Few genes like TaZIFL
moter elements suggests that wheat ZIFL genes might 1.1, TaZIFL7.1 and TaZIFL7.2 remained unaffected
respond towards the presence of heavy metals and to by the +Zn condition in roots. In shoots, TaZIFL1.1,
important micronutrients like Fe and Zn. Interestingly, TaZIFL1.2, TaZIFL6.1 and TaZIFL6.2 showed signifi-
IDE1 cis-element was present on promoters of all the cant transcript accumulation either at 3DAT or 6DAT
respective wheat ZIFL genes suggesting that they could after treatment (Fig. 5b). Notably, TaZIFL1.2, TaZIFL6.1
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 6 of 17

Fig. 3 Intron exon arrangement and protein conservation. The intron-exon structure was obtained using Gene Structure Display Server (GSDS 2.0:)
Pink boxes and black lines depict the introns and exons, respectively

and TaZIFL6.2 show enhanced transcript accumulation TaZIFL4.1, TaZIFL4.2, TaZIFL5 and TaZIFL7.1 show up-
in both the tissues. In contrast, during our experiment regulation only at 3 DAT, suggesting their coordinated
the expression of a few wheat ZIFL genes showed down- response in shoots (Fig. 6b). Under −Fe condition, wheat
regulated in shoots but not in roots. Our expression data ZIFL genes, namely, TaZIFL4.1 and TaZIFL4.2 show
under Zn surplus condition suggested the differential high transcript accumulation in both roots and shoots.
response by wheat ZIFL towards the treatment. Remaining genes remain unaffected by the Fe stress
Previous evidences indicated that plant ZIFL genes not (Fig. 6b). Overall, our expression data suggested that
only respond to Zn excess, but are also affected by the Fe indeed wheat ZIFL respond to the Fe limiting condition,
limiting conditions [19]. Therefore, expression analysis thereby suggesting a common interlink of this gene fam-
of wheat ZIFL genes was checked in roots and shoots of ily during Zn and Fe homeostasis.
seedling under Fe starvation (−Fe). Interestingly, in the
root expression of TaZIFL4.1, TaZIFL4.2 and TaZIFL7.2 Candidate TOM genes show expression response
show up-regulation during Fe limiting condition at both to the presence of heavy metals
at 3 and 6 DAT. Out of the remaining genes, TaZIFL2.3, Phylogenetic arrangement of the wheat MFS-ZIFL
TaZIFL6.2 and TaZIFL7.1 show significant transcript proteins along with the rice also revealed the possible
abundance at one-time point or the other (Fig. 6a). wheat homologs for TOM transporters. Thus, based on
Interestingly, in shoots TaZIFL1.1, TaZIFL1.2, TaZIFL3, the clade distribution and the percentage identity with
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 7 of 17

Fig. 4 Protein sequence analysis for conserved and unique motifs in wheat ZIFL. a Conserved motifs across 15 TaZIFL proteins, as obtained from
MEME. Different colors represent distinct motifs. b Unique motifs found through MEME for group 2, group 4, as well as TaZIFL6.2

the rice TOM (TOM1-OsZIFL4, TOM2-OsZIFL5 and sub-clade along with wheat along with TaZIFL5 and
TOM3-OsZIFL7), wheat TOM transporters were iden- TaZIFL6, therefore we included them to study their
tified as TaZIFL4.1/TaZIFL4.2, TaZIFL5 and TaZIFL7.1/ response in presence of heavy metals.
TaZIFL7.2. Rice TOM1 and TOM2 falls in the same

(See figure on next page.)

Fig. 5 Relative gene expression levels of wheat ZIFL transcripts under +Zn condition. Gene expression profiles of wheat ZIFL genes were studied
in a roots and b shoots. Total RNA was extracted from the wheat seedlings subjected to three and 6 days of treatments +Zn. The roots and shoots
samples were collected, and qRT-PCR was performed on the DNA free RNAs. A total of 2 µg of RNA was used for cDNA synthesis. C ­ t values were
normalized against wheat ARF1 as an internal control. Data represents mean of three biological replicates each treatment containing 12–15
seedlings. Vertical bars represent the standard deviation. # on the bar indicates that the mean is significantly different at p < 0.05 with respect to
their respective control samples
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 8 of 17
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 9 of 17

Fig. 6 Relative gene expression levels of wheat ZIFL genes under −Fe condition. Gene expression profiles of wheat ZIFL genes were studied in a
roots and b shoots. Total RNA was extracted from the wheat seedlings subjected to 3 and 6 days of treatments. The roots and shoots samples were
collected, and qRT-PCR was performed on the DNA free RNAs. A 2 µg of total RNA was used for cDNA synthesis. C ­ t values were normalized against
wheat ARF1 as an internal control. Data represents mean of three biological replicates with each treatment containing 12–15 seedlings. Vertical bars
represent the standard deviation. # on the bar indicates that the mean is significantly different at p < 0.05 with respect to their respective control
treatments
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 10 of 17

Fig. 7 qRT-PCR expression analysis of shoots and roots of wheat seedlings exposed to multiple heavy metals (Ni, Co and Cd). Five days old wheat
seedlings were exposed to the mentioned heavy metals for the period of 14 days. Total RNA was extracted from the treated and control samples
and 2 µg of RNA was used to construct the cDNA. ­Ct values were normalized against wheat ARF1 as an internal control. Fold expression values were
calculated relative to the control tissue of the mentioned wheat ZIFL. Vertical bars represent the standard deviation. # represent the significantly
difference at p < 0.05 with respect to their respective control treatments

Our promoter analysis of wheat ZIFL genes indicates and Cd. During our experiment all the seedlings showed
the presence of multiple HMRE suggesting that few of phenotypic defects when exposed to heavy metals (data
these genes could respond to the heavy metals (Addi- not shown). Our expression analysis suggested that wheat
tional file 6: Table S5). Due to the importance of TOM ZIFL genes show metal specific responses. For example,
genes in micronutrient mobilization the expression of TaZIFL4.2 and TaZIFL7.1 showed significant up-reg-
these transcripts in wheat seedlings (shoots and roots) ulation in both roots and shoots when exposed to any
was studied after exposure to heavy metals such as Co, Ni of the metals tested (Fig. 7). In contrast, the transcripts
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 11 of 17

Fig. 8 Heat map for relative expression of putative TaZIFL genes in different tissues and at multiple developmental stages. Heatmaps were
generated using expression values from expVIP database for grain, leaf, root, spike and shoot tissues. Green to red color change depicts increase in
transcript expression, as shown by the color bar

of TaZIFL5 and TaZIFL6.2 remained unaffected under significant upregulation only in roots upon exposure to
these heavy metals. Expression of TaZIFL7.2 showed Ni and Co (Fig. 7). Overall, these data indicate the influ-
almost no change in the presence of Ni in either of the ence of specific heavy metals on the expression of wheat
tissues, yet it was specifically up-regulated in roots ZIFL genes in a tissue dependent manner.
when exposed to Cd or Co. Similarly, TaZIFL6.1 showed
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 12 of 17

Expression of wheat ZIFL transcripts in different wheat Under heat, drought and heat-drought combined stress
tissues and during stress condition (Additional file 2: Figure S4a, Additional file 7: Table S6),
Analysis of ZIFL genes was also performed in different TaZIFL7.1 (4A, 4D) and TaZIFL7.2-4D were induced
wheat tissues and developmental stages by using qRT- by up to ~ sevenfold and ~ twofold respectively, whereas
PCR and transcript expression data. Developing grains TaZIFL1.2 (3A, 3B, 3D) and TaZIFL5 (5A, 5B) were
are an important reservoir for micronutrients, therefore downregulated by 6 and 7.5-fold, respectively. No sig-
expression studies were carried out for putative wheat nificant changes in the TaZIFL gene expression were
TOM genes during the grain maturation. Transcriptional observed for Fusarium head blight infected spikelets
expression of these genes was checked during grain (Additional file 2: Figure S4b, Additional file 7: Table S6).
development (7, 14, 21 and 28 days after anthesis-DAA). For Septoria tritici infected seedlings, while a ~ two-
Our qRT-PCR analysis of putative TOM genes during the fold induction was observed for TaZIFL1.2 (3A, 3B)
grain development showed differential expression pattern after 4 days of induction, prolonged infection (13 days),
with majority of candidate genes (Additional file 2: Fig- resulted in its downregulation. Other ZIFLs showing
ure S3). In general, most of the putative candidate TOM changed expression were TaZIFL1.2-3D and TaZIFL2.2-
genes showed high expression at the late phase of the 3A (> twofold up-regulation), TaZIFL3-4B (up to 2.7-
grain maturation i.e. 21 and 28 DAA. In addition to this, fold downregulation). TaZIFL1.2 (3A, 3B, 3D) were also
wheat expression browser and expVIP (http://www.wheat​ downregulated (up to ~ fourfold) in seedlings with stripe
-expre​ssion​.com/) was used to extracted the expres- rust infection, while only TaZIFL1.2-3D was downregu-
sion values as Transcript per millions (TPM). TaZIFL lated under powdery mildew infection. These expres-
expression values in different tissues (aleurone-al, starchy sion data suggest that specific ZIFLs are differentially
endosperm-se, seed coat-sc, leaf, root, spike, shoot) and regulated under infection conditions and show perturbed
various developmental stages were extracted (Addi- expression under abiotic stresses.
tional file 7: Table S6) and depicted as a heatmap (Fig. 8).
In reference to grain tissue developmental time course Discussion
(GTDT) [27], highest expression was seen for TaZIFL1.2 Wheat ZIFL proteins as putative phytosiderophore efflux
(3B, 3D) and TaZIFL5 (5A, 5B), with an increase in transporters
expression in “al” at 20 dpa and “al and se” at 30 dpa. In The current work was undertaken to build the inven-
the expression values during grain tissue specific expres- tory of wheat ZIFL. Since wheat is a hexaploid species
sion (at 12 dpa) [28], TaZIFL1.2 was not expressed, but with three genomes therefore, we expect a high number
like GTDT study, TaZIFL5 (5A, 5B) was expressed in “al” of transcripts encoding for a particular gene family. Our
as well as “se” Fig. 8). While for sc tissue, TaZIFL2.2-3D analysis resulted in the identification of 35 ZIFL gene
and TaZIFL7.1 (4A, 4D) had the highest expression when sequences corresponding to fifteen individual genes from
compared to other ZIFL genes. For the tissue specific hexaploid wheat. A unique observation made for the
expression response TaZIFL2.2 was abundant in spike, wheat ZIFL is that all the genes are restricted to chro-
TaZIFL1.2 in leaf and root. TaZIFL5 was predominantly mosome 3, 4 or 5 only (Fig. 2). This study and the pre-
expressed in all the tested tissue, including leaf, shoot, vious preliminary report, led to the identification of a
spike and shoot. The transcripts exclusively expressed in total of fifteen ZIFL genes with TaZIFL1.2, TaZIFL2.2,
root were TaZIFL2.4-5D, TaZIFL2.5-5B, TaZIFL6.1-5A, TaZIFL3, TaZIFL4.2, TaZIFL5, TaZIFl6.2, TaZIFL7.1 and
and TaZIFL7.2-4D, with high induction of TaZIFL4.1 TaZIFL7.2 showing the presence of all the homoeologous
(4B, 4D), TaZIFL4.2-4A, TaZIFL6.2 (4A, 4B, 4D) for (homoalleles) genes [28]. The identified wheat ZIFL pro-
three-leaf and flag leaf stage as compared to the seed- teins belong to the MFS superfamily, thereby containing
ling stage. Highest expression induction was seen for the canonical MFS signature and antiporter sequence
TaZIFL4.2-4D. In addition, the highest expression overall (Additional file 2: Figure S2). Given the high sequence
in five tissues was observed for TaZIFL1.2 (3A, 3B, 3D) homology, they are named from TaZIFL1 to TaZIFL7
in leaves for seedling as well as tillering stage, TaZIFL2.2- according to their clade distribution in the phylogenetic
3D in spike, TaZIFL3-4A in leaf, TaZIFL5-5A in grain, tree that corresponds to the rice genes. Previously, in rice
TaZIFL7.1-4D in grain and leaf. multiple TOM genes were identified as protein belong-
For abiotic stress, while no major changes were ing to the ZIFL sub-family. Subsequent characteriza-
observed for TaZIFLs, TaZIFL4.1 (4B, 4D) and TaZIFL4.2 tion of these rice ZIFL genes led to the identification of
(4A, 4D) were found to be significantly downregu- functionally active OsTOM1, OsTOM2 and OsTOM3 [20,
lated by ~ 14-fold under phosphate deficiency, while 21]. Specifically, TOM2 was shown to be an important
TaZIFL6.2-4D was downregulated by threefold (Addi- internal phytosiderophore efflux transporter whereas,
tional file 2: Figure S4a, Additional file 7: Table S6). TOM1 was demonstrated to efflux into soil from root
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 13 of 17

cells and thereby involved in metal transport in specific also affected by both Fe and Zn in either root or shoot.
tissue [21]. These observations reinforce the impor- RNA-seq expression analysis of Fe deficiency in wheat
tance of TOM family in translocating metals into the roots for 20 days was checked for the expression of wheat
sink areas of the plants [21]. TOM-like genes remain to ZIFL genes. At the late stage of Fe deficiency TaZIFL4.1
be identified from hexaploid wheat. Based on the phylo- and TaZIFL4.2 show highest expression (Additional
genetic arrangement and the percentage corresponding file 2: Figure S5) [32]. These results suggest that few of
homologs for the wheat TOM could be TaZIFL4.1/4.2, these ZIFL genes might be involved in the overlapping
TaZIFL5 and TaZIFL7.1/7.2. Identified wheat ZIFL pro- pathways of Fe and Zn homeostasis. Such commonality
teins show localization on the PM, except for TaZIFL4.2- of Zn and Fe homeostasis were reported earlier and are
4B, TaZIFL5-5D and TaZIFL7.1-4B. Only TaZIFL4.2-4B, also evident from our work. This may suggest the overlap
TaZIFL5-5D are putatively localized in vacuolar mem- of the common network of transcription factors involved
brane, thereby making them a potential candidate to in Fe and Zn homeostasis. In our study, the promot-
assess their tissue specific role in micronutrient mobiliza- ers of TaZIFL1.2, TaZIFL2.3, TaZIFL7.1 and TaZIFL7.2
tion or storage in the cell organelles. showed the presence of IRO2 binding domains that has
In general, very small number of ZIFL genes are being been previously speculated to be the link between Fe
reported from dicot species like Arabidopsis and Vitis and Zn homeostasis [18]. Nonetheless, only TaZIFL1.2
vinifera and Populus trichocarpa [18]. Given the com- respond to −Fe and +Zn stress that is restricted only to
plexity of the wheat genome, we anticipated the presence shoots. Previously, it was also shown that expression of
of multiple possible putative phytopsiderophore efflux Arabidopsis ZIF1 remained unaffected in the presence
transporters that need to be functionally characterized of sub-inhibitory levels of Cd or Cu [17]. Based on the
in the near future. In rice, the high ZIFL numbers have expression response of ZIFL genes during in the presence
been accounted due to the lineage-specific expansion by of heavy metals it seems that TaZIFL5 and TaZIFL6.2
duplication of the gene family [18]. Overall, our analy- could be one of the best candidate genes for the further
sis identified highest number of ZIFL genes reported till studies, as both the genes remained unaffected. Never-
date from any monocot species. theless, careful selection of candidate gene must be done
to minimize the cotransport of other undesired metals
Wheat ZIFL genes display overlapping gene expression during micronutrient uptake.
Plants undergoing metal stress result in series of signaling In addition to their anticipated role in Fe and Zn home-
events that largely include reprogramming of transcripts ostasis, their role in root development has been also
to overcome the toxic effects. In plants, excess of Zn also proven. Plant ZIFL transporters have been reported to
results in the generation of reactive oxygen and nitrogen regulate stomatal movements by means of polar auxin
species [29]. The abundance of multiple membrane pro- transport, thereby modulating potassium and pro-
teins is increased by the presence of either excess Zn or ton fluxes in Arabidopsis [23]. Analysis of the expVIP
Fe deficiency. In line with the previous studies, our data data suggested high expression of wheat ZIFL genes
also confirmed the overlapping expression response of (TaZIFL7.1-4A and 4D) under drought condition. Earlier,
wheat ZIFL genes [30]. Additionally, under Fe limit- zifl-1 and zifl-2 mutants of Arabidopsis showed hyper-
ing conditions, induction of Zn responsive genes could sensitivity to drought stress by disruption of guard cells
be an important step towards limiting the non-specific activity [33]. TaZIFL1.2 is the wheat transporter show-
transport activity of transporters which are primarily ing highest expression in leaf and highest homology with
induced for Fe deficiency. ZIFL are well known for their AtZIFL1, thereby belonging to the same clade in the phy-
response towards the presence of excess Zn [17]. Our logeny tree. In contrast to the expected function of ZIFL
study revealed that multiple wheat ZIFL genes responded in Fe and Zn homeostasis, a putative role has been dem-
to excess Zn, either in shoots or roots in a temporal man- onstrated in plant defense. Maize ZIFL referred as Zm-
ner. Interestingly, most of the wheat ZIFL genes show mfs1 was high induced during plant defense and has been
the presence of Fe responsive cis-element IDE1. IDE1 is implicated its role for export of antimicrobial compounds
one of the primary cis-elements that respond to Fe lim- during its interaction with the bacterial pathogen [34]. In
iting conditions [31]. Therefore, we studied the expres- our study, a strong expression of multiple ZIFL genes was
sion of wheat ZIFL genes under –Fe condition. Multiple observed when infected with multiple pathogens suggest-
ZIFL genes showed specific response towards Fe defi- ing its important role in providing resistance against fun-
ciency, suggesting that the deficiency response shown gal pathogens (Additional file 2: Figure S4b).
by ZIFL transcripts could be mediated by transcription This study concludes that ZIFL transporters are the
factors like IDE1. Expression of the few of the ZIFLs like important players during the crosstalk of Fe and Zn
TaZIFL1.1, TaZIFL1.2, TaZIFL4.2 and TaZIFL6.2 were homeostasis. With the evidence regarding their role as
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 14 of 17

a transporter for NA and MA in crops, ZIFL genes are corresponding ortholog from rice followed by chromo-
certainly a priority candidate to address the uptake and somal and genomic location e.g. TaZIFL3-4A, TaZIFL3-
remobilization of micronutrients in the cereal grains 4B and TaZIFL3-4D represent three homoeologous of
such as wheat. TaZIFL genes in chromosome four of all the three A, B
and D sub-genomes and it is an ortholog of OsZIFL3. To
Conclusion know the evolutionary relationship among ZIFL proteins,
This the first comprehensive study that resulted in the phylogenetic analysis was performed along with other
identification of fifteen putative ZIFL genes from hexa- plant species (O. sativa, Z. mays and Arabidopsis). All the
ploid wheat at the homoeolog level. These are the high- proteins were aligned through MUSCLE algorithm and a
est number of ZIFL genes reported in plant system till rooted phylogenetic tree was used to construct with the
date. Wheat ZIFL were characterized for their expres- Neighbor-joining (NJ) method using MEGA7 software
sion response in seedlings exposed to excess Zn and Fe with 1000 bootstrap replicates [36]. To determine the
deficiency. The contrast expression of these ZIFL in pres- distribution of ZIFL genes in the wheat chromosomes,
ence of heavy metals suggested their functional redun- the position of each ZIFL gene was obtained using wheat
dancy and pinpoint importance of a few candidate ZIFL Ensembl database.
genes for their functional validation. Our work identi-
fied candidate ZIFL from hexaploid wheat that could be Analysis of conserved domains, gene arrangements
the important target to address new means to enhance and subcellular localization of wheat ZIFL
micronutrient uptake by targeting phytosiderophore The divergence and conservation of motifs in wheat
release. ZIFL proteins were also identified by using MEME
(Multiple Expectation Maximization for Motif Elicita-
Methods tion) program version 5.0.2 (http://meme-suite​.org) [37]
Identification of the MFS_1 family in wheat with a maximum motif width, 50; maximum number of
To identify the potential members of ZIFLs from MFS_1 motifs, 15; and minimum motifs width, 6. Gene Struc-
transporter family in wheat genome, we used two inde- ture Display Server (GSDS 2.0) [38] was used to analyze
pendent approaches. In the first approach, the Pfam num- the gene structure. Individual wheat ZIFL CDS and cor-
ber (PF07690) for MFS_1 was used and sequences were responding genomic DNA were aligned to identify the
extracted from wheat using the Ensembl wheat database. intron–exon arrangement. Using Expasy Compute PI/
As a complementary approach, known sequences of ZIFL MW online tool (http://us.expas​y.org/tools​/protp​aram.
genes from A. thaliana, O. sativa and Brachypodium html) the predicted isoelectric points and molecular
distachyon were retrieved and used for BLAST analysis weights of putative TaZIFLs were calculated. To predict
against the wheat database: Ensembl (http://plant​s.ensem​ terminal ends and number of transmembrane domains,
bl.org/Triti​cum_aesti​vum/) to retrieve the sequences. TMHMM (http://www.cbs.dtu.dk/servi​ces/TMHMM​
The identified MFS_1 superfamily was validated through /) was utilized. The putative protein sequences of ZIFL
the domain search in CDD-NCBI database (https​://www. were further in silico analyzed to predict their subcellular
ncbi.nlm.nih.gov/Struc​ture/cdd/wrpsb​.cgi) [35]. localization by WoLF PSORT (https​://wolfp​sort.hgc.jp/)
prediction program [39].
Identification, classification, and chromosomal distribution
of wheat ZIFL genes Plant material and Fe, Zn and heavy metals treatments
To identify putative TaZIFLs genes among MFS_1 super- For giving various Zn and Fe treatments, seeds of Triti-
family sequence, phylogenetic tree was constructed with cum aestivum cv. C306 (provided by Punjab Agriculture
known ZIFLs from different plants that separated the University, Ludhiana, India) were used. Bread wheat cv.
ZIFL cluster from the rest of the MFS_1 superfamily C306, has been undertaken for research purpose since it
members. This distribution of genes in ZIFL cluster was is having a good processing (including chapatti making)
validated through the presence of ZIFL specific signa- quality [40, 41]. Previously this cultivar was also used
ture sequences. To construct the phylogenetic tree one for Fe deficiency related studies to characterize wheat
hundred seventy-nine wheat MFS_1 superfamily pro- YSL transporter [16]. For experiments, the seeds were
tein sequences were retrieved from Pfam no. (PF07690). washed with double autoclaved water for the removal of
In addition to that, thirteen-protein sequence of ZIFL dirt followed by surface sterilization with 1.2% Sodium
from O. sativa, five from Zea mays and three from hypochlorite prepared in 10% ethanol. Seeds were strati-
Arabidopsis were used. Identified TaZIFLs were named fied by keeping them overnight in dark at 4 °C on moist
according to their corresponding rice orthologs having Whatman filter papers in a Petri dish. The stratified seeds
maximum number of ZIFL reported. The name indicates were further allowed to germinate at room temperature.
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 15 of 17

For each experiment, healthy seedlings were transferred using three biological replicates and two to four technical
to phytaboxes (12–15 seedlings/phytabox: in three bio- replicates. The relative mRNA abundance was normalized
logical replicates) and grown in autoclaved water in with wheat ARF1 as mentioned earlier (ADP-Ribosylation
growth chamber for 5 days. The endosperm was removed Factor, AB050957.1) [43, 44]. The relative expression was
after 5 days and plants were kept in their respective media calculated through delta–delta CT-method (­ 2−ΔΔCT) [45].
according to the treatment. Five days old seedlings were The statistical significance of expression data was deter-
subjected to different Zn and Fe conditions and grown mined using student’s t-test (p-value < 0.05).
in Hoagland media [42] in triplicates supplemented
with either 2 µM of Fe(III) EDTA for Fe deficient con-
dition (−Fe) or 200 µM of ­ZnSO4.7H2O [18] for the Zn In‑silico expression analysis
surplus experiment (+Zn). Total S content was adjusted For in-silico expression analysis, a total of 35 TaZIFL
with the macronutrient used in Hoagland media. Seed- genes were selected and wheat expression browser,
lings grown in the Hoagland media containing 20 µM of expVIP [http://www.wheat​-expre​ssion​.com/] was used
Fe(III) EDTA and 2 µM of ­ZnSO4.7H2O were used for to extract the expression values in the form of TPMs.
control experiments. The roots and shoots of plantlets These values were then used to build heatmaps using
(5 days post germination) were collected after 3 and 6 MeV software [http://mev.tm4.org/]. While absolute
DAT along with their respective controls. Every alternate values were used for development and tissue-specific
day, the seedlings in the phyta-boxes were supplemented data, fold change values were used for stress conditions
with the fresh media, replacing the old media. For heavy where a gene was taken to be upregulated if fold change
metal treatment the 5 days old plantlets were subjected was greater than 2 and downregulated if less than 0.66.
to cadmium (50 µM C ­ dCl2), cobalt (50 µM C­ oCl2), nickel For abiotic stress, expression for two studies, phosphate
(50 µM ­NiCl2) treatment. Root and shoot samples from deficiency [46] and heat, drought and heat-drought stress
each replicate were collected after 15 days of treatment. [47] was studied. In case of biotic stress, the studies
To study the gene expression during different stages of considered were [48–50]. For expression of wheat ZIFL
seed development (7, 14, 21 and 28 DAA), tagging was genes under Fe deficiency experiments RNAseq data,
done for the main spikes of the respective biological rep- NCBI Project ID PRJNA529036 was utilized for analysis.
licates at the first DAA. At the indicated time points,
developing grains from the tagged spikes were collected Supplementary information
and frozen in liquid nitrogen for RNA extraction. Supplementary information accompanies this paper at https​://doi.
org/10.1186/s1286​7-019-0139-6.

RNA isolation, cDNA preparation and qRT‑PCR analysis

Additional file 1: Table S1. List of 179 wheat sequence IDs extracted for
Total RNA was extracted from harvested roots and shoot MFS_1 family, Pfam ID: PF07690 using ensembl wheat database.
samples using TRIZOL RNA extraction method. Turbo Additional file 2: Figure S1. Neighbor-Joining (NJ) tree for MFS_1 family
DNAfree kit (Invitrogen, USA) was used to remove the of proteins from Oryza sativa, Brachypodium distachyon, Zea mays and
genomic DNA. RNA samples were then quantified on Triticum aestivum constructed using MEGA7.0 software with a bootstrap
replicate value of 1000. The phylogenetic tree shows ZIFL proteins cluster-
nanodrop and subsequently, 2 µg of total RNA was used ing into a distinct clade within the MFS family. Figure S2. Alignments for
to prepare cDNA by using SuperScript III First-Strand signature sequences specific for ZIFL proteins, namely, ZIFL MFS signature
Synthesis System (Invitrogen, USA). For expression motif, the anti-porter signature, Cys-containing, His-containing signatures.
Figure S3. Gene expression analysis of wheat ZIFL transcript during grain
analysis qRT-PCR primers were designed from the con- development. qRT-PCR analysis of putative wheat TOM genes: TaZIFL4.1,
served region of respective TaZIFL genes to account for TaZIFL4.2, TaZIFL5, TaZIFL7.1 and TaZIFL7.2. qRT-PCR was performed on
the respective additive gene expression response (Addi- cDNA prepared from 2 µg of total RNA as mentioned in methods. Fold
expression levels were calculated relative to 7 days after anthesis (DAA)
tional file 8: Table S7). Amplicons arising from these tissue. Figure S4. Heat-map depicting relative differential expression of
primers were also processed for sequencing to avoid any putative wheat ZIFL genes under various abiotic and biotic stresses: a.
cross amplifications of the ZIFL genes. Sequencing of the Heat maps of TaZIFL genes under various abiotic (Phosphate starvation,
Drought, Heat and Drought-Heat). b. Biotic stresses (Fusarium heat blight,
amplicon revealed gene specific expression except for Septoria tritici, stripe Rust and Powdery mildew). Heat-maps were gener-
TaZIFL2.4 and TaZIFL2.5, for which either no amplicons ated using fold change values obtained after processing the expression
were detected under our condition or were non-specific values from expVIP database. Green color represents down-regulation,
black represents no change and red color represents up-regulation. Fig‑
due to its close homology with other members. Therefore, ure S5. Gene expression analysis of wheat ZIFL transcript in root during

ies. 10X diluted cDNA and SYBR Green I ­(QuantiFast®

these two were not considered for the expression stud- Fe deficiency. Heatmap showing expression for putative TaZIFL genes in
roots of control and 20 days post Fe deficiency. Heatmap was generated

­SYBR® Green PCR Kit, Qiagen, Germany) was used to

using MeV software based on the expression values calculated from NCBI
Project ID PRJNA529036 [33]. Black to red color change depicts increasing
perform qRT-PCR on 7500 Fast Real-Time PCR System expression, as shown by the color bar.
(Applied Biosystems, USA). qRT-PCR was performed
Sharma et al. BMC Molecular Biol (2019) 20:22 Page 16 of 17

Author details
Additional file 3: Table S2. Detailed information for 35 putative wheat 1
National Agri-Food Biotechnology Institute (Department of Biotechnology),
ZIFLs identified. Table includes gene IDs, chromosomal locations, CDS and Sector 81, Knowledge City, Mohali, Punjab 140306, India. 2 University Institute
protein lengths, molecular weight and pI for each of the obtained putative of Engineering and Technology, Panjab University, Sector 25, Chandigarh,
TaZIFLs. Punjab 160015, India.
Additional file 4: Table S3. Conserved motifs identified in putative TaZIFL
proteins using MEME. The consensus sequence logo, e-values and the Received: 1 February 2019 Accepted: 14 September 2019
number of sequences in which each motif was found are listed.
Additional file 5: Table S4. ZIFL Signature motifs (Cys, His, MFS and MFS
antiporter) and their locations in all wheat ZIFL proteins. The motif posi-
tions were mapped to the TM-HMM predicted TaZIFL protein structures to
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