Article

pubs.acs.org/est

Effects of Deficiency and Excess of Zinc on Morphophysiological

Traits and Spatiotemporal Regulation of Zinc-Responsive Genes
Reveal Incidence of Cross Talk between Micro- and Macronutrients
Ajay Jain,†,Δ Bhaskaran Sinilal,‡,Δ Gurusamy Dhandapani,† Richard B. Meagher,§ and Shivendra V. Sahi*,‡
†
National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110012, India
‡
Department of Biology, Western Kentucky University, Bowling Green, Kentucky 42101-1080, United States
§
Department of Genetics, University of Georgia, Fred C. Davison Life Sciences Complex, Athens, Georgia 30602, United States
*
S Supporting Information

ABSTRACT: Zinc (Zn) is an essential micronutrient which

affects plant growth and development in deficiency and can be
toxic when present in excess. In Arabidopsis thaliana, different
families of cation transporters play pivotal roles in Zn
homeostasis. In the present study, we evaluated the effects of
Zn in its deficiency (0 μM; Zn−) and excess (75 μM; Zn++) on
various morphophysiological and molecular traits. Primary root
length was reduced in Zn− seedlings, whereas there were
significant increases in the number and length of lateral roots
under Zn− and Zn++ conditions, respectively. Concentration of
various macro- and microelements showed variations under
different Zn regimes and notable among them was the reduced
level of iron (Fe) in Zn++ seedlings compared to Zn+. Certain
members of the ZIP family (ZIP4, ZIP9, and ZIP12) showed significant induction in roots and shoots of the Zn− seedlings.
Their suppression under Zn++ condition indicated their transcriptional regulation by Zn and their roles in the maintenance of its
homeostasis. Zn-deficiency-mediated induction of HMA2 in roots and shoots suggested its role in effluxing Zn into xylem for
long-distance transport. Attenuation in the expression of Fe-responsive FRO2 and IRT1 in Zn− roots and their induction in Zn+
+ roots provided empirical evidence toward the prevalence of a cross talk between Zn and Fe homeostasis. Variable effects of
Zn− and Zn++ on the expression of subset of genes involved in the homeostasis of phosphate (Pi), potassium (K), and sulfur
(S) further highlighted the prevalence of cross talk between the sensing and signaling cascades of Zn and macronutrients.
Further, the inducibility of ZIP4 and ZIP12 in response to cadmium (cd) treatment could be harnessed by tailoring them in
homologous or heterologous plant system for removing pollutant toxic heavy metals from the environment.

■ INTRODUCTION
Zinc (Zn) is an essential micronutrient for all living organisms
and biofortification of edible crops is considered to be a viable
remedy.1,2 But, Zn-deficiency affects cultivated soils world-
wide.3 It often causes oxidative damage to biomolecules
triggering several morphophysiological adaptive responses.4,5
Whereas, excess Zn can be toxic to plants due to its ability to
compete with other metal ions.6 Therefore, to resolve both the
problems of deficiency and toxicity of Zn, it is essential to
decipher the molecular mechanisms governing its homeostasis.
This would expedite the process of engineering plants with
higher Zn-use efficiency to serve as a rich dietary source of Zn
and potentially tailoring for phytoremediation of sites
contaminated with toxic levels of Zn.
Zn is taken up from soil by root membrane transport
mechanisms, transported into xylem and stored within vacuoles
of leaf cells.7−10 Zn homeostasis is achieved by the specific and
coordinated actions of several members of the families
comprising Zn-regulated transporter (ZRT) Fe-regulated
transporter (IRT)-like proteins (ZIPs),8,11,12 P-type ATPases
heavy metal transporters,13,14 and metal tolerance protein
(MTP).15 Among the members of ZIP family, ZIP1 and ZIP3
showed induction only in Zn-deprived roots, whereas ZIP4 was
induced in both Zn-deprived roots and shoots.11 Different Zn
regimes also exerted differential effects on the expression of ZIP
family members.16 Although IRT1, another member of the ZIP
family, plays a key role in iron (Fe) uptake, reduced level of Zn
in the mutant irt1 suggested its likely role in the transport of Zn
as well.17 Likewise, yeast complementation and mutant analysis
in Arabidopsis found ZIP1 and ZIP2 transporting both Zn and
Mn.18 These studies suggested a lack of functional redundancy
across the members of the ZIP family. Further, heavy metal
ATPases HMA2 and HMA4 are expressed largely in the
Received: January 12, 2013
Revised: April 12, 2013
Accepted: April 16, 2013

© XXXX American Chemical Society A dx.doi.org/10.1021/es400113y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology

■
Article

vasculature and facilitate not only in effluxing of Zn into xylem RESULTS AND DISCUSSION
for long-distance transport8,14 but also its hyperaccumulation in Zn-Deficiency Exhibits Morphophysiological Re-
roots of metal hyperaccumulator Arabidopsis halleri. 15 Whereas, sponses. To determine the effects of Zn-deficiency on
members of the MTP family (MTP1 and MTP3) play a role in morphophysiological responses, Arabidopsis seedlings were
the transportation of Zn into the vacuole.8,9 Overall, these grown hydroponically under Zn− (0 μM) and Zn+ (3 μM)
studies highlight the coordinated regulation of several members conditions (Figure 1). Conventionally, model plant species like
of different cation transporter families in maintaining the Zn
homeostasis. Also, there is evidence of cross talk of Zn
homeostasis with that of Fe,19 copper (Cu),20 phosphorus
(P),21 and toxic heavy metal cadmium (Cd).15 However,
comprehensive evaluation of the effects of different Zn regimes
on morphophysiological traits, different members of cation
transporter families, and their cross talk with micro- and
macroelements are not available. Further, different gelling
agents used for assessing molecular responses of plants to Zn-
deficiency22,23 are often prone to elemental contamination
thereby influencing the responses to various nutrient
deficiencies.21 The aim of the present study was, therefore, to
use an element-contamination-free hydroponic system21 for (i)
evaluating the effects of deficiency and excess of Zn on various
morphophysiological traits and the responses of different
members of Zn-responsive cation transporter families, (ii)
cross talk of Zn with the homeostasis of other micro- and
macronutrients, and (iii) responsiveness of the members of ZIP
family to heavy metal Cd. We hypothesize that the study would
help in the identification of specific Zn-responsive genes that
could be potentially used for engineering plants for Zn
biofortification and environmentally friendly remediation of
the sites contaminated with heavy metals.

■ MATERIALS AND METHODS

Seeds were germinated and grown for 5 d in one-half-strength
of liquid MS medium.21 Plants were then transferred to media25
with Zn− (0 μM), Zn+ (3 μM), and Zn++ (75 μM) and grown
for 7 d under standard growth conditions. For Cd treatment,
the seeds were germinated on one-half-strength MS medium
supplied with agar and kept vertically oriented in growth room
for 2 weeks and transferred to liquid medium in falcon tubes
supplied with Cd and grown for 3 d. Morphological analysis of
the plants were carried out on the images of shoot and root
spread separately on agar plate.26 Meristematic activity in
CycB1;1::uidA24 seedlings were verified by GUS staining
Figure 1. Zn-deficiency elicits morphophysiological responses. (A)
according to the work of Malamy et al.27 Real time PCR was Lateral roots of Arabidopsis seedlings grown hydroponically under Zn+
done on cDNA prepared from total RNA isolated from the (3 μM) and Zn− (0 μM) conditions for 7 d were spread for revealing
different experimental groups using SYBR Green detection architectural details (n = 15). Data are presented for (B) primary root
method.28 The list of primers used for real-time PCR is given in length (n = 15), (C) number of first- and higher-order lateral roots (n
Supporting Information Table S1. Plant tissues collected were = 15), and (D) ICP analysis of micro-(Zn, Fe, Cu) and macro-(P, K,
S) elements (n = 5). Values are mean ± SE. Different letters on the
analyzed by inductively coupled plasma (ICP) analysis for
histograms indicate that the means differ significantly (P < 0.05).
determining the concentrations of various macro and micro-
nutrients. Regulatory region of the ZIPs retrieved from The
Arabidopsis and rice are grown on agar23 or phytagel32 for
Arabidopsis Gene Regulatory Information Server, were analyzed
evaluating the effects of Zn deprivation on physiological and
in databases AtcisDB for identifying transcription factor binding molecular responses. However gelling agents are often
sites29,30 and PlantCare for cis-regulatory elements, respec- contaminated with variable levels of elements that could lead
tively.31 Protein level subcellular localization of the ZIPs was to erroneous interpretations on the effects of nutrient
predicted by amino acid sequence analysis at softberry.com. deficiencies on morphophysiological and/or molecular re-
Statistical significance of the difference between mean values sponses.33 The efficacy of elemental contamination-free
was determined using Student’s t test. Detailed description of hydroponic system for dissecting various morphophysiological
the methodologies used for this study is provided in the and molecular responses of Arabidopsis seedlings to different
Supporting Information. nutrient deficiencies have been demonstrated.33 Therefore, it
B dx.doi.org/10.1021/es400113y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology Article

Figure 2. Zn-deficiency-mediated differential regulation of Zn-responsive genes. Real-time PCR analyses of the members of ZIP family and HMA2 in
roots and shoots of Arabidopsis seedlings grown hydroponically under Zn+ (3 μM) and Zn− (0 μM) conditions for 7 d. At-ACT2 was used as an
internal control, and Zn+ values were normalized to 1. Data presented are means of six technical replicates ± SE. Except ZIP1 (roots) and ZIP7
(shoot), all the Zn-responsive genes showed significant (>2.0-fold) increase in the relative level of their gene expression in Zn-deprived roots and
shoots compared with their corresponding tissues grown under Zn+ condition.

was anticipated that the use of a similar sterile hydroponic
system in the present study would provide a system free of
residual Zn for dissecting the effects of Zn-deficiency on
different traits. Another notable inconsistency across various
studies has been the use of different Zn concentration ranging
from 0.5 to 30 μM for making Zn+ medium.11,16,23,32−35 Since
3 μM ZnSO4 was used earlier for making nutrient-rich solution
for hydroponic system,21 we used this concentration for making
Zn+ in the present study. Among the morphological traits, root
system architecture (RSA) exhibits extensive plasticity toward
environmental cues27 and nutrient deficiencies.21,26,36 Com-
pared to Zn+ seedlings, Zn− seedlings revealed an altered RSA
(Figure 1A) due to significant (P < 0.05) reduction in primary
root length (Figure 1B) and significant (P < 0.05) increase in
first- and higher-order lateral root number (Figure 1C). The
differential responses of primary and lateral roots to Zn-
deficiency could be attributed to their distinct ontogeny with
the former being embryonic in origin and the latter developed
post-embryonically. Further, there was a strong CycB1;1::uidA
expression in the primary root tips of both Zn+ and Zn−
seedlings (data not shown) suggesting that Zn-deficiency does
not effects meristematic cells in the primary root. This is in
contrast with phosphate (Pi) deficiency that triggers pro-
gressive loss of meristematic cells in the primary root causing a
shift from an indeterminate to determinate developmental
program. The present study thus reiterates the specific
responses of RSA to different nutrient deficiencies.37 On the
contrary, the effect of Zn-deficiency was not perceptible on the
total shoot area (data not shown). This suggests that root
system of Arabidopsis is relatively more sensitive to Zn
deprivation compared to aerial parts at least during short-
term treatment (7 d). However, upon prolonged Zn-deficiency
treatment extending up to 8 weeks, the plants showed retarded
C dx.doi.org/10.1021/es400113y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
growth and abnormalities during flowering and fruiting as
well.38 Further, ICP analysis revealed a significant (P < 0.05)
decline in Zn content in Zn− seedlings compared to Zn+
seedlings (Figure 1D). Levels of other micro-(Fe, Cu) and
macro-(P, K, S) elements were also compared between Zn+
and Zn− seedlings to examine any incidence of cross-
homeostasis between Zn and these elements. The level of
iron was comparable between Zn+ and Zn− seedlings. This is
contrary to earlier studies that demonstrated reduction in an
accumulation of Fe in shoots of A. thaliana grown under excess
Zn and alleviation of Zn toxicity by excess Fe,19 and subsequent
study showed the role of glutathione in mediating cross talk
between Zn and Fe.39 However in another study, the level of Fe
was shown to be significantly reduced in Zn− roots compared
to Zn+ roots.16 Therefore, the effect of Zn status on Fe
homeostasis could be influenced by several other variables
including growth conditions and duration of the treatment.
However, there was a significant (P < 0.05) reduction in Cu
content in Zn− seedlings compared to Zn+ seedlings and was
consistent with a previous study.20 Zn-deficiency also triggered
reductions in the contents of macronutrients (P, K, and S) and
was in agreement with several earlier reports highlighting the
cross talk of Zn with Pi,21,40−43 K,44 and S.45 Overall, ICP
analysis suggests a key role for Zn not only in maintaining
essential cellular functions but also its potential influence on
homeostasis of some of the essential macro- and microelements
in Arabidopsis.
Differential Effects of Zn-Deficiency on ZIPs and
Heavy Metal ATPases. Roots and shoots of Arabidopsis
seedlings grown hydroponically under Zn + (3 μM) and Zn−
(0 μM) conditions for 7 d were analyzed by real-time PCR for
the relative expression levels of different members of the ZIP,
heavy metal ATPase, and MTP families (Figure 2). Several
Environmental Science & Technology
members of the ZIP family showed Zn-deficiency-mediated ≥
2.0-fold induction in roots (ZIP7), shoots (ZIP1), or in both
roots and shoots (ZIP2, ZIP3, ZIP4, ZIP5, ZIP8, ZIP9, and
ZIP12). Whereas, the relative expressions of ZIP6, ZIP10, and
ZIP11 were comparable in both Zn+ and Zn− roots and shoots
(data not shown). The study thus demonstrated differential
effects of Zn-deficiency on the relative expression of different
members of the ZIP family. The relative expression of ZIP2,
ZIP3, ZIP4, ZIP5, ZIP9, and ZIP12 was relatively higher in Zn−
shoots compared to Zn− roots suggesting their roles not only
in acquisition of Zn from rhizosphere but also in its
mobilization to aerial tissues. For instance, ZIP9 showed
14.24-fold induction in Zn− roots and 196.78-fold induction in
Zn− shoots. Likewise ZIP12 also exhibited 19.48-fold induction
in Zn− roots and 136.47-fold induction in Zn− shoot. The
results suggested the vital roles of ZIP9 and ZIP12 in Zn
homeostasis. Several studies have also reported Zn-deficiency-
mediated induction of ZIP family members in Arabidop-
sis11,16,18,21,46,47 and rice.23,35,48 However, these studies reveal
several discrepancies in the reported effects of Zn-deficiency on
the inducibility of one or more members of the ZIP family in
roots and/or shoots. For instance, Grotz et al.11 reported the
expression of only ZIP1 and ZIP3 in roots and ZIP4 in both
roots and shoots of Zn-deprived Arabidopsis. Notably, in their
study ZIP2 mRNA could not be detected in both Zn+ and Zn−
plants. Whereas in another study, in addition to ZIP4 up
regulation of ZIP9 was also observed in Zn-deprived roots but
the latter could not be detected in leaves under different Zn
regimes.34 Contrary to these reports,11,34 a significant induction
was observed for both ZIP3 and ZIP9 in Zn-deprived roots and
shoots along with several other members of the ZIP family
(Figure 2). Observed incongruities on the effects of Zn-
deficiency on the transcript levels of different members of the
ZIP family in roots and shoots could be due to the use of
elemental-contamination prone gelling medium,32,23 variable
Zn concentrations (0.5−30 μM) that were used for making Zn
+ (control),11,16,23,33−35 and/or the techniques (Northern
blot,11,35 semiqunatitative RT-PCR,16,34 microarray,16,35 and/
or Real time PCR23,32,35) that were used for detecting them.
Our study thus demonstrated the efficacy and reliability of
hydroponic systems in elucidating the effect of different Zn
regime on the transcript levels of different members of ZIP
family. The same system was also used for identifying Zn-
deficiency responses of PCR2,48 and the members of heavy
metal ATPases (HMA214,16,49 and HMA450) that have been
shown to play distinctive roles in the maintenance of Zn
homeostasis. There was 6.75- and 3.88-fold increase in the
relative expression of HMA2 in Zn-deprived roots and shoots,
respectively compared to their corresponding tissues grown
under Zn+ condition (Figure 2). Zn-deficiency-mediated
elevated expression of HMA2 has been suggested to be a
response to increased requirement of Zn by shoot by facilitating
its export through xylem from root to shoot and/or its
reallocation from shoot to root via phloem for meeting its
higher demand by the latter.14,16,49 Although PCR2,48 a
membrane protein, has been implicated in the translocation
of Zn from root to shoot, and metal pump HMA450 has been
shown to facilitate hyperaccumulation of Zn, in the present
study neither of them showed Zn-deficiency-mediated
increased expression. In rice, the ZIP family members
OsZIP1, OsZIP4, OsZIP5 have also been shown to play a
substantial role in acquisition of Zn by roots, its mobilization to
shoots, and/or its redistribution.23,35,47,51,52 Zn deprivation was
D dx.doi.org/10.1021/es400113y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
further extended up to 2 weeks for assessing the specificity of
Zn-deficiency on the induction of Zn-responsive genes
(Supporting Information Figure S1). The effect of long-term
Zn-deficiency treatment was pronounced on the relative
expression of ZIP4 (2.09-fold in shoot), ZIP9 (9.39-fold in
roots), and ZIP12 (6.53-fold in roots and 5.24-fold in shoot)
compared to their relative expression in roots and shoots of Zn-
supplied seedlings (Figure 2). Temporal treatments thus clearly
demonstrated the specific responses of ZIP4, ZIP9, and ZIP12
to different Zn regime and could be used as potent indicators of
Zn status in Arabidopsis.
Predicted cis-Regulatory Elements, Transcription
Factor Binding Motifs and Subcellular Localization of
ZIPs. To determine the basis for the differential responses of
the members of the ZIP family to Zn-deficiency, the cis-
regulatory elements in the promoter region of ZIPs were
analyzed in silico in ZIP4, ZIP9, and ZIP12 that were
responsive (RZnD) and ZIP6, ZIP10, and ZIP11 that were
nonresponsive (NRZnD) to Zn-deficiency (Supporting In-
formation Table S2). Even though the members of RZnD and
NRZnD were analyzed extensively for finding out the reason
for specificity in expression of ZIPs in response to Zn level in
the medium, none of the regulatory sequences found relevant
in targeting the expression pattern due to their nature of
response and distribution. Further, the promoter region of the
ZIPs were analyzed for putative transcription factor (TF)
binding motifs (Supporting Information Table S3). Since
binding sites for TFs such as WRKY, ABI3VP1, bZIP, etc. were
present in the promoters of one or more members of both
RZnD and NRZnD, their role in differential responsiveness
toward different Zn regime could not be assumed. Two closely
related members of basic-region leucine zipper (bZIP) TFs,
bZIP19, and bZIP23 have been shown to regulate the
adaptation to zinc deficiency.53 The target genes of these TFs
have one or more copies of a 10 bp imperfect palindrome
(RTGTCGACAY) in their promoter region to which bZIP
proteins can bind and was referred to as the zinc deficiency
response element (ZDRE). Screening of the promoters of the
ZIP family members revealed the presence of one or more
copies of ZDRE in all the three members of RZnD and also in
ZIP1, ZIP3, and ZIP5. The present study is consistent with this
assumption as all these ZIPs show significant induction in roots
and/or shoots (Figure 2). Whereas, ZIP2 does not have ZDRE
sequence in its promoter53 and was reported to be non-
responsive to Zn-deficiency.11 However, in the present study
Zn-deficiency triggered significant induction of ZIP2 in both
roots (∼2.0 fold) and in shoots (5.6 fold). In addition, ZIP10
has one ZDRE sequence in its promoter; it is not responsive to
Zn-deficiency. Whereas, ZIP6 and ZIP11 do not have ZDRE
sequence in their promoter and are also not responsive to Zn-
deficiency. Therefore, ZDRE apparently plays a pivotal role in
regulating responses to Zn-deficiency, but its role as a sole
regulator could be speculative and argued.
Amino acid analysis of the 12 ZIPs was performed for
predicting the metal binding domains and the sites of
intracellular localization (Supporting Information Table S4).
Most of them were found to be localized on plasma membrane
with 6−7 trans-membrane domains. The loop region in
between trans-membrane domains III and IV contains a
potential metal-binding domain rich in His residues that is
predicted to be cytoplasmic.54,55 Our analysis predicted nuclear
localization of ZIP2 and the association of ZIP6 with organelles
like endoplasmic reticulum and chloroplast. The absence of
Environmental Science & Technology Article

glycosylphosphatidylinositol (GPI) anchor and trans-mem-

brane domains in these proteins suggest their role in mediating
intracellular signaling rather than metal transporting across
membranes. In ZIP5, prediction favored plasma membrane
localization due to the presence of seven trans-membrane
segments. However, the absence of a GPI anchor may hinder its
functioning on a lipid bilayer membrane.
Zn-Deficiency Affects Responses of Genes Involved in
Homeostasis of Other Micro- and Macroelements. Earlier
studies have shown the prevalence of cross talk between Zn
homeostasis with that of Fe,19 Cu,20 and P.21 Therefore, in the
present study we evaluated the effect of Zn-deficiency on the
expression of genes involved in the uptake and mobilization of
Fe, Cu, P, K, and S in roots and shoots of Arabidopsis seedlings
(Supporting Information Figure S2). Zn-deficiency elicited
significant and differential effects on the relative expression of
genes involved in Fe homeostasis as evidenced by the
attenuation in the expression of FRO2 and IRT1 in the roots.
Significant inductions were observed for IRT2 in shoots and of
IRT3 and FRD3 in both roots and shoots. However, such an
effect was not obvious in the relative expression of AHA1,
AHA2, AHA7, VIT1, NRAMP3, NRAMP4, and FER1 (data not
shown) that also have been shown to play specific roles in the
maintenance of Fe homeostasis.8 This suggested the likely
effect of Zn-deficiency only on the subset of genes that govern
Fe homeostasis in Arabidopsis, and it is consistent with earlier
studies.19,39 There was no apparent influence of Zn-deficiency
on the relative expression of Cu transporter COPT18 (data not Figure 3. Effects of excess Zn on ionomic profile. Arabidopsis seedlings
shown). The effect of Zn-deficiency was also evident on Pi were grown hydroponically under Zn+ (3 μM) and Zn++ (75 μM)
conditions for 7 d, and the whole seedlings were used for ICP analysis
homeostasis as indicated by suppression of Pht1;1 in roots and of micro-(Zn, Fe, and Cu) and macro-(P, K, and S) elements (n = 5).
induction of IPS1 in shoots; the genes playing essential roles in Values are mean ± SE. Different letters on the histograms indicate that
Pi acquisition and mobilization, respectively.21,56,57 However, the means differ significantly (P < 0.05).
Zn-deficiency did not exert any influence on the relative
expression of Pi-starvation-responsive genes involved in root
development and maintenance of Pi homeostasis (PLDZ258), decline of 4%, and 6%, respectively. The values of S content
Pi acquisition (Pht1;457), Pi mobilization (RNS159), and were comparable in Zn+ and Zn++ seedlings. The results
internal allocation of Pi (At460). The results suggested the clearly suggested a more drastic effect of excess Zn on
influence of Zn deprivation only on the part of sensing and microelements than on macroelements, and our results were in
signaling cascades that regulate Pi homeostasis. The sulfur level agreement with earlier studies.22,66,67 Seedlings grown hydro-
is also influenced by the uptake of Zn.39,45,61,62 Although Zn- ponically under Zn+ and Zn++ conditions were also evaluated
deficiency resulted in the attenuation of expression of Sultr1;3 for their morphological traits (Figure 4). Under Zn++
in shoots that has been shown to govern the redistribution of S conditions, there was about 30% significant (P < 0.05)
from source to sink,63 the relative expression of some of the increases in total shoot area (Figure 4A,B). There was 27%
other, S-responsive genes (Sult1;2, Sult1,3) remained unaffected significant (P < 0.05) increase in first- and higher-order lateral
(data not shown). Some effect of Zn-deficiency was also root length of Zn++ seedlings compared to that of Zn+ (Figure
evident on the genes involved in K homeostasis. For instance, 4C,D). The results showed that Zn in excess did not exert any
there were significant suppression in the relative expression of inhibitory effect on growth and development of Arabidopsis.
AtHAK5 (in roots and shoot) and AtKC1 (in roots). AtHAK5 These results were found consistent with an earlier study that
is involved in K+-deficiency-induced high-affinity K+ uptake,64 also did not observe any significant effect on chlorophyll
and AtKC1 mediates K+ influx in root hairs.65 However, other content when grown in presence of excess Zn up to 100 μM;
genes in the K+ sensing and signaling pathway (AtCHX17, the effect became evident only when plants were grown in
AtKEA5, AtKUP3, AtKUP12) were found to be unaffected. excess of 100 μM Zn.22 However the shoots of Zn++ appeared
Overall the results indicated partial cross talk of Zn deprivation more chlorotic compared to that of the Zn+. Further, we
with several pathways that regulate micro- and macronutrient assayed the effects of excess Zn on the relative expression of the
homeostasis. genes involved in Zn, Fe, Cu, P, K, and S homeostasis in roots
Excess Zn Triggers Morphophysiological and Molec- (Figure 4E) and in shoots (Supporting Information Figure S3).
ular Responses. Arabidopsis seedlings grown under Zn++ (75 There were significant reductions in the relative expressions of
μM) conditions showed (P < 0.05) 8.4-fold increase in Zn ZIP4 (root and shoot), ZIP9 (root and shoot), and ZIP 12
content compared to Zn+ (3 μM) seedlings (Figure 3). There (shoot), and the results were in agreement with earlier
were 35% and 20% reductions in the levels of Fe and Cu, studies.16,34 Although excess Zn suppressed the expression of
respectively, in Zn++ seedlings compared to Zn+ seedlings. HMA2 in roots, there was about 4-fold induction in shoots.
Relatively the effect of excess Zn was less dramatic on the levels Whereas, induction of ≥2-fold was observed for HMA3 in both
of P and K with a marginal though significant (P < 0.05) roots and shoots of Zn++ compared to the corresponding
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Environmental Science & Technology Article

Figure 4. Excess Zn-mediated modulated morphophysiological and molecular responses. Arabidopsis seedlings were grown hydroponically under Zn
+ (3 μM) and Zn++ (75 μM) conditions for 7 d. Seedlings were dissected into (A) shoots (n = 10) and (C) roots with their lateral roots spread for
revealing architectural details (n = 10). Data are presented for (B) total shoot area (n = 10) and (D) length of first- and higher-order lateral roots (n
= 10). Values (B, D) are means ± SE, and different letters on the histograms indicate that the means differ significantly (P < 0.05). (D) Real-time
PCR analyses of Zn, Fe, P, K, and S responsive genes in the roots. At-ACT2 was used as an internal control, and Zn+ values were normalized to 1.
Data presented are means of six technical replicates ± SE. The relative level of the gene expression was considered significant when its value showed
either increase (>2.0-fold) or decrease (<0.5) in Zn++ roots compared with Zn+ roots.

tissues of Zn+. The role of HMA2 in vasculature uploading and
the efflux of the metal into extracellular compartment have
been reported.49 Hence the reduction in transcript levels
observed in roots is likely to impede entry into the vasculature
and the corresponding induction observed in shoots could
possibly accentuate the mobilization of the ions into the
extracellular compartment thereby preventing cellular accumu-
lation and the resultant toxicity. HMA3 is involved in vacuolar
storage of Zn.68 Induction of HMA3 in roots and shoots in
excess Zn confirms its role in the mobilization of the excess Zn
from cytoplasm to vacuole for preventing toxicity. Among the
Fe-responsive genes, ferric chelate reductase FRO2 mediates
the reduction of Fe3+ into Fe2+ which is then subsequently
transported into the symplast of the epidermis by high affinity
transporter IRT1 and both the genes are induced in response to
Fe deficiency.8 Significant increase in the relative expression of
both FRO2 and IRT1 in roots and shoots of Zn++ seedlings
F dx.doi.org/10.1021/es400113y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
compared to Zn+ seedlings confirmed the Fe stress being
experienced by the seedlings when grown under excess Zn.
Whereas, there were significant reductions in the relative
expression of IRT2 and IRT3 in both roots and shoots of Zn++
seedlings compared to Zn+ seedlings. The results revealed an
incidence of a cross talk between Zn and Fe homeostasis. The
effect of excess Zn was also evaluated on the expression of
genes involved in homeostasis of Pi (Pht1;1, IPS1), K
(AtCHX17, AtHAK5, AtKC1), and S (Sultr1;3) in both roots
(Figure 4E) and shoots (Supporting Information Figure S3).
The attenuating effects of Zn++ on expression of these genes
were more pronounced in shoots than in roots. Overall the
results demonstrated the cross talk of Zn++ with genes
involved in the maintenance of both micro- and macronutrient
homeostasis and was in agreement with earlier studies.19,39,69
Cadmium Induces Changes in Expression of ZIPs and
IRTs. Cadmium (Cd) is a toxic heavy metal which competes
Environmental Science & Technology
with Zn for absorption and mobilization.15 However, the exact
mechanism and the specific role of Zn transporters in these
processes are not yet confirmed.62 Hence, expression of few
ZIPs and IRTs were analyzed in presence of Cd (Figure 5).
Figure 5. Heavy metal cadmium induces ZIPs. (A) Arabidopsis
seedlings were grown on vertically oriented agar plates containing full
nutrient solution comprising one-half-strength MS + 1.5% sucrose for
2 weeks. (B) A pool of five seedlings were transferred to falcon tubes
containing full nutrient solution without cadmium (Zn+ Cd−) and
nutrient medium supplemented with 10 ppm cadmium (Zn+ Cd+)
and grown for 3 d. (C) Roots of Zn+ Cd− and Zn+ Cd+ were
harvested for real-time PCR analysis of ZIP4, ZIP12, IRT1, and IRT2.
At-ACT2 was used as an internal control, and Zn+ Cd− values were
normalized to 1. Data presented are means of six technical replicates ±
SE. The relative level of the gene expression was considered significant
when its value showed either increase (>2.0-fold) or decrease (<0.5) in
Zn+ Cd+ roots compared with Zn+ Cd− roots.
ZIP4 and ZIP12 were induced significantly in presence of Cd,
whereas Fe transporters IRT1 and IRT2 were suppressed.
Similarity of Cd induced expression pattern of ZIPs and IRTs
to that of Zn-deficiency suggested the utilization of Zn
transporting pathway by Cd for its uptake and mobilization.
This suggests the utility of ZIPs in monitoring Cd uptake.
■
ASSOCIATED CONTENT
*
S Supporting Information
Figure S1: long-term Zn-deficiency on expression of Zn-
responsive genes. Figure S2: expression of genes involved in
homeostasis of other nutrients in Zn-deficiency. Figure S3:
changes in gene expression in response to excess Zn. Table S1:
G dx.doi.org/10.1021/es400113y | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
list of primers used for real-time PCR. Table S2: prediction
results of putative transcription factor binding motifs. Table S3:
results of prediction of cis-regulatory elements of the promoter
region of selected ZIPs. Table S4: prediction results of
subcellular localization of the 12 ZIPs in Arabidopsis. Section
Materials and Methods: details of the experimental procedures
used for this study. This material is available free of charge via
the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: shiv.sahi@wku.edu. Phone: 1-270-745-6012. Fax: 1-
270-745-6856.
Author Contributions
Δ
A.J. and B.S.: Both the authors contributed equally to this
study.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was carried out at Western Kentucky University
(WKU) with support from U.S. Department of Agriculture
(Grant 58-6406-1-017) awarded to S.V.S. The award of
Ramalingaswamy Fellowship (2009) to A.J. by the Ministry
of Science and Technology, Department of Biotechnology,
Government of India, is duly acknowledged.
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