Effects of Deficiency and Excess of Zinc on Morphophysiological Traits

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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.

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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

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 worldwide.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 © XXXX American Chemical Society

Received: January 12, 2013 Revised: April 12, 2013 Accepted: April 16, 2013

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RESULTS AND DISCUSSION Zn-Deficiency Exhibits Morphophysiological Responses. To determine the effects of Zn-deficiency on morphophysiological responses, Arabidopsis seedlings were grown hydroponically under Zn− (0 μM) and Zn+ (3 μM) conditions (Figure 1). Conventionally, model plant species like

vasculature and facilitate not only in effluxing of Zn into xylem for long-distance transport8,14 but also its hyperaccumulation in roots of metal hyperaccumulator Arabidopsis halleri. 15 Whereas, members of the MTP family (MTP1 and MTP3) play a role in the transportation of Zn into the vacuole.8,9 Overall, these studies highlight the coordinated regulation of several members 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 Zndeficiency22,23 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.

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Article

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 according to the work of Malamy et al.27 Real time PCR was done on cDNA prepared from total RNA isolated from the different experimental groups using SYBR Green detection method.28 The list of primers used for real-time PCR is given in Supporting Information Table S1. Plant tissues collected were analyzed by inductively coupled plasma (ICP) analysis for determining the concentrations of various macro and micronutrients. Regulatory region of the ZIPs retrieved from The Arabidopsis Gene Regulatory Information Server, were analyzed in databases AtcisDB for identifying transcription factor binding sites29,30 and PlantCare for cis-regulatory elements, respectively.31 Protein level subcellular localization of the ZIPs was predicted by amino acid sequence analysis at softberry.com. Statistical significance of the difference between mean values was determined using Student’s t test. Detailed description of the methodologies used for this study is provided in the Supporting Information.

Figure 1. Zn-deficiency elicits morphophysiological responses. (A) Lateral roots of Arabidopsis seedlings grown hydroponically under Zn+ (3 μM) and Zn− (0 μM) conditions for 7 d were spread for revealing architectural details (n = 15). Data are presented for (B) primary root length (n = 15), (C) number of first- and higher-order lateral roots (n = 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 histograms indicate that the means differ significantly (P < 0.05).

Arabidopsis and rice are grown on agar23 or phytagel32 for evaluating the effects of Zn deprivation on physiological and molecular responses. However gelling agents are often contaminated with variable levels of elements that could lead to erroneous interpretations on the effects of nutrient deficiencies on morphophysiological and/or molecular responses.33 The efficacy of elemental contamination-free hydroponic system for dissecting various morphophysiological and molecular responses of Arabidopsis seedlings to different nutrient deficiencies have been demonstrated.33 Therefore, it B

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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.

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 crosshomeostasis 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

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 Compared 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 Zndeficiency 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 progressive 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 shortterm treatment (7 d). However, upon prolonged Zn-deficiency treatment extending up to 8 weeks, the plants showed retarded C

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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-deficiencymediated induction of ZIP family members in Arabidopsis11,16,18,21,46,47 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 Zndeficiency 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 Zndeficiency 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

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 Znsupplied 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 cisregulatory 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 Information 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 nonresponsive 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 Zndeficiency. 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 D

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glycosylphosphatidylinositol (GPI) anchor and trans-membrane 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 shown). The effect of Zn-deficiency was also evident on Pi homeostasis as indicated by suppression of Pht1;1 in roots and induction of IPS1 in shoots; the genes playing essential roles in Pi acquisition and mobilization, respectively.21,56,57 However, 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), Pi acquisition (Pht1;457), Pi mobilization (RNS159), and internal allocation of Pi (At460). The results suggested the influence of Zn deprivation only on the part of sensing and signaling cascades that regulate Pi homeostasis. The sulfur level is also influenced by the uptake of Zn.39,45,61,62 Although Zndeficiency resulted in the attenuation of expression of Sultr1;3 in shoots that has been shown to govern the redistribution of S from source to sink,63 the relative expression of some of the other, S-responsive genes (Sult1;2, Sult1,3) remained unaffected (data not shown). Some effect of Zn-deficiency was also evident on the genes involved in K homeostasis. For instance, there were significant suppression in the relative expression of AtHAK5 (in roots and shoot) and AtKC1 (in roots). AtHAK5 is involved in K+-deficiency-induced high-affinity K+ uptake,64 and AtKC1 mediates K+ influx in root hairs.65 However, other genes in the K+ sensing and signaling pathway (AtCHX17, AtKEA5, AtKUP3, AtKUP12) were found to be unaffected. Overall the results indicated partial cross talk of Zn deprivation with several pathways that regulate micro- and macronutrient homeostasis. Excess Zn Triggers Morphophysiological and Molecular Responses. Arabidopsis seedlings grown under Zn++ (75 μM) conditions showed (P < 0.05) 8.4-fold increase in Zn content compared to Zn+ (3 μM) seedlings (Figure 3). There were 35% and 20% reductions in the levels of Fe and Cu, respectively, in Zn++ seedlings compared to Zn+ seedlings. Relatively the effect of excess Zn was less dramatic on the levels of P and K with a marginal though significant (P < 0.05)

Figure 3. Effects of excess Zn on ionomic profile. Arabidopsis seedlings were grown hydroponically under Zn+ (3 μM) and Zn++ (75 μM) conditions for 7 d, and the whole seedlings were used for ICP analysis of micro-(Zn, Fe, and Cu) and macro-(P, K, and S) elements (n = 5). Values are mean ± SE. Different letters on the histograms indicate that the means differ significantly (P < 0.05).

decline of 4%, and 6%, respectively. The values of S content were comparable in Zn+ and Zn++ seedlings. The results clearly suggested a more drastic effect of excess Zn on microelements than on macroelements, and our results were in agreement with earlier studies.22,66,67 Seedlings grown hydroponically under Zn+ and Zn++ conditions were also evaluated for their morphological traits (Figure 4). Under Zn++ conditions, there was about 30% significant (P < 0.05) increases in total shoot area (Figure 4A,B). There was 27% significant (P < 0.05) increase in first- and higher-order lateral root length of Zn++ seedlings compared to that of Zn+ (Figure 4C,D). The results showed that Zn in excess did not exert any inhibitory effect on growth and development of Arabidopsis. These results were found consistent with an earlier study that also did not observe any significant effect on chlorophyll content when grown in presence of excess Zn up to 100 μM; the effect became evident only when plants were grown in excess of 100 μM Zn.22 However the shoots of Zn++ appeared more chlorotic compared to that of the Zn+. Further, we assayed the effects of excess Zn on the relative expression of the genes involved in Zn, Fe, Cu, P, K, and S homeostasis in roots (Figure 4E) and in shoots (Supporting Information Figure S3). There were significant reductions in the relative expressions of ZIP4 (root and shoot), ZIP9 (root and shoot), and ZIP 12 (shoot), and the results were in agreement with earlier studies.16,34 Although excess Zn suppressed the expression of HMA2 in roots, there was about 4-fold induction in shoots. Whereas, induction of ≥2-fold was observed for HMA3 in both roots and shoots of Zn++ compared to the corresponding E

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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 (2.0-fold) or decrease (