Planta (2021) 253:129

https://doi.org/10.1007/s00425-021-03642-z

REVIEW

Zinc toxicity in plants: a review

Harmanjit Kaur1 · Neera Garg2

Received: 6 December 2020 / Accepted: 19 May 2021 / Published online: 27 May 2021
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
Main conclusion This review highlights the most recent updated information available about Zn phytotoxicity at
physiological, biochemical and molecular levels, uptake mechanisms as well as excess Zn homeostasis in plants.

Abstract Zinc (Zn) is a natural component of soil in terrestrial environments and is a vital element for plant growth, as it
performs imperative functions in numerous metabolic pathways. However, potentially noxious levels of Zn in soils can result
in various alterations in plants like reduced growth, photosynthetic and respiratory rate, imbalanced mineral nutrition and
enhanced generation of reactive oxygen species. Zn enters into soils through various sources, such as weathering of rocks,
forest fires, volcanoes, mining and smelting activities, manure, sewage sludge and phosphatic fertilizers. The rising alarm in
environmental facet, as well as, the narrow gap between Zn essentiality and toxicity in plants has drawn the attention of the
scientific community to its effects on plants and crucial role in agricultural sustainability. Hence, this review focuses on the
most recent updates about various physiological and biochemical functions perturbed by high levels of Zn, its mechanisms
of uptake and transport as well as molecular aspects of surplus Zn homeostasis in plants. Moreover, this review attempts to
understand the mechanisms of Zn toxicity in plants and to present novel perspectives intended to drive future investigations
on the topic. The findings will further throw light on various mechanisms adopted by plants to cope with Zn stress which
will be of great significance to breeders for enhancing tolerance to Zn contamination.

Keywords Defence · Heavy metal · Homeostasis · Mechanisms · Phytochelatins · Zinc transporters

Introduction and heavy isotopic enrichment of Zn have been described

Zinc (65.37Zn30) is a group IIB transition element and was
given its name by an alchemist Paracelsus (Swiss Physi-
cian) following the German word Zinke. It ranks next to
the most copious transition element, i.e. iron (Mir et al.
2015). Zn occurs in nature in five stable isotopic forms: 64Zn
(48.63%), 66Zn (27.90%), 68Zn (18.75%), 67Zn (4.90%), and
70
Zn (0.62%) (Broadley et al. 2007). In plants, both light
Communicated by Gerhard Leubner.
* Neera Garg
gargneera@gmail.com; garg_neera@yahoo.com
Harmanjit Kaur
harmanjit.garcha@yahoo.com
1
Department of Botany, Akal University, Bathinda 151302,
Punjab, India
2
Department of Botany, Panjab University,
Chandigarh 160014, India
in shoots and roots, respectively (Caldelas et al. 2010). Zn is
chemically similar to magnesium (Mg), both having similar
size and + 2 oxidation state. ­Zn2+ ions have high binding
affinities for nitrogen (N), oxygen (O) or sulfur (S) of amino
acid residues in proteins/enzymes: N of histidine being the
most common one, followed by S of cysteine, O of aspar-
tate/glutamate and carbonyl O of peptide bond, glutamine/
asparagine and hydroxyl of tyrosine (Leuci et al. 2020).
The normal level of Zn required for well being of the
majority of crops varies between 30 and 200 µg Zn ­g−1 dry
weight (DW) (Marschner 2012). As a vital micronutrient,
Zn takes part in structural and/or catalytic functions in sev-
eral processes like cell division, cell expansion and protein
synthesis (Jain et al. 2010). It has a central role in chroma-
tin structure, gene expression and regulation, metabolism of
nucleic acids, carbohydrates, lipids, proteins as well as pho-
tosynthetic carbon (Gai et al. 2017; Noulas et al. 2018). Zn
is also required for the synthesis of tryptophan, a precursor
amino acid of auxin (Tsonev and Lidon 2012). Additionally,

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Zn is crucial for the working of more than 300 enzymes,
for instance, carbonic anhydrase, aldolases, carboxypepti-
dases, alkaline phosphatases, superoxide dismutase, phos-
pholipase, alcohol dehydrogenase, etc. (Gupta et al. 2016;
Lin et al. 2016). Many molecules connected with the syn-
thesis of DNA as well as RNA are Zn metalloenzymes, for
example, RNA polymerases and reverse transcriptases (Choi
et al. 2018). Zn finger transcription factors participate in the
expansion as well as functioning of flower tissues, such as
pistil, anthers, pollen and tapetum in many plants (Hafeez
et al. 2013) (Fig. 1). Conversely, Zn is involved in the
defence mechanism of plants as well, for example, Zn finger
WRKY transcription factors control various plant processes
including systemic acquired resistance (SAR) (Rushton et al.
2010). Moreover, Zn-binding protein HPP3 is required in
salicylate defence signaling pathway (Zwiesche et al. 2015).
In polluted agricultural fields, total soil Zn concentra-
tions of more than 3000 mg ­kg−1 Zn dry soil (DS) have been
reported (Long et al. 2003; Audet and Charest 2006) which
cause a crucial hindrance to global agricultural productivity.
Human actions have extensively added Zn to soils through
smelting and mining. Additional man-made sources of Zn
input into soils consist of burning of fossil fuels, phosphate
fertilizers (usually 50–1450 µg Zn ­g−1), manure (15–250 µg
Zn ­g−1), limestone (10–450 µg Zn ­g−1), fungicides, rubber
mulches, fragments from Zn-coated surfaces and calcareous
Fig. 1  Effects of optimum and excess level of zinc in plants
13
Planta (2021) 253:129
amendments (10–450 µg Zn g­ −1) (Alloway 2008). Average
Zn content of about 4000 mg ­kg−1DS in Waste Electrical
and Electronic Equipment (WEEE); 1000 mg ­kg−1DS in sew-
age sludge; 3000–4000 mg ­kg−1DS in municipal solid waste
(MSW) and 400–600 mg ­kg−1DS in Recovered Waste Wood
(RWW) has been reported (Jones et al. 2014). Zn input into
the soil through human activities is mobile and can easily
build up in excess in plants (> 400 µg Zn g­ −1 DW), thus
causing toxic effects and metabolic disorders (Bazihizina
et al. 2014). Although Zn toxicity in plants is far less wide-
spread than Zn deficiency and most of the research is focused
on enhancing Zn contents in tissues of crop plants, never-
theless, Zn phytotoxicity is prevalent in acidic and sludge
amended soils. Hence, in the present article, researches on
Zn phytotoxicity carried out at physiological, biochemical
and molecular levels during the last two decades have been
reviewed.
Impact of excess Zn on physiological and functional
aspects of plants
Enhanced levels of Zn in soils are phytotoxic, cause several
structural as well as functional abnormalities, eventually
weakening the plant performance. However, these responses
vary with the type of plant species and their developmental
stages (Khudsar et al. 2004; Vaillant et al. 2005). Various
Planta (2021) 253:129
morpho-anatomical, physiological, biochemical and molecu-
lar responses of crop plants to Zn excess have been sum-
marized in Table 1.
The possible mechanisms responsible for high Zn-
induced plant responses are being discussed in the follow-
ing sub-sections:
Visible symptoms, biomass and auxin activity
The decrease in germination vigour and biomass are the
most apparent physiological responses of plants to Zn tox-
icity which eventually results in decreased yield and prod-
uct quality (Fig. 1) (Garg and Kaur 2013a; Zhang et al.
2017; Garg and Singh 2018). Excess Zn affects the activity
of amylases and transfer of sugars to embryo axis, result-
ing in the decreased proportion of germinated seeds, as
reported in Cicer arietinum (Sharma et al. 2009). Inhibi-
tion of seed germination as well as seedling root growth in
response to enhanced Zn concentrations (40, 80, 160, 240,
320, and 640 mg L ­ −1) has been demonstrated in Cucumus
sativus (Aydin et al. 2012). On the contrary, excess Zn did
not significantly inhibit the seed germination in Coriandrum
sativum L. (0.1, 1 and 2 mM Zn) and Medicago sativa L.
(1.5 mM Zn) but inhibited the seedling root length remark-
ably (Marichali et al. 2014; Yahaghi et al. 2019), indicating
that the response of seed germination and seedling growth to
Zn stress is concentration as well as species-specific.
Chlorosis followed by necrosis in response to Zn stress
(Table 1) has been attributed to the decrease in V-ATPase
levels as well as its activity, as observed in Arabidopsis thal-
iana (Fukao et al. 2011). Excess Zn affects leaves in terms
of both leaf area and leaf number (Table 1 and Fig. 1) which
might result from either inhibition in cell division or elonga-
tion or even both (Glinska et al. 2016). Moreover, high Zn
damages the organization of mitochondria (Tea plant) and
leads to decrease in nicotinamide levels (B-group vitamins),
thereby accelerating N ­ AD+ breakdown and consequently
lowering the energy metabolism (Zhang et al. 2017), which
may account for the decrease in overall plant growth. In
some plants, such as lettuce, coriander and Arabidopsis, Zn
stress results in reddening of leaves and/or shoots which may
be ascribed to increased levels of anthocyanins, validating
that the synthesis of these phenolics is a Zn-induced stress
response (Marichali et al. 2014; Sofo et al. 2018). Though
reports on the role of anthocyanins in plants under Zn stress
conditions are scanty, nonetheless, it seems that Zn-induced
increase in anthocyanins might play a role in enhancing
antioxidant capacity and/or metal chelation in plants. Zn is
usually accumulated in roots which may be an endurance
strategy adopted by plants to maintain lower Zn concen-
tration in the aerial organs (Garg and Kaur 2013b; Tiecher
et al. 2017). Retention of metals (including excess Zn) in
roots is especially desirable in crop plants as these parts
Page 3 of 28 129
are not generally consumed as food or feed, consequently
reducing the risk to humans as well as animals. Black and
decayed root tips, decrease in length, surface area, diameter
and volume of the root system in response to Zn toxicity
have been reported in different plant species (Stoláriková-
Vaculíková et al. 2015; Bernardy et al. 2016) (Fig. 1). Even
root hair morphology got disrupted under Zn stress in A.
thaliana which could be due to reduction in the proteins, for
instance, root hair defective 3 (RHD3) and morphogenesis
of root hair 5 (MRH5/SHV3), responsible for the develop-
ment of root hairs (Fukao et al. 2011). Moreover, reduction
in root growth results in deceleration of water absorption
and mineral elements which in turn affects shoot growth as
well. Zn excess inhibits primary root elongation and stimu-
lates lateral roots to grow more in the vertical direction, as
reported in A. thaliana and Brassica napus, probably as a
compensation mechanism aimed to explore deeper zones of
soil (Zhang et al. 2018; Feigl et al. 2019). High concentra-
tion of Zn has also been demonstrated to cause significant
deposition of callose in root apical meristem of B. juncea
and B. napus which may contribute to growth inhibition
because it lowers cell wall loosening and hampers symplas-
tic transport (Feigl et al. 2015). Moreover, increased pec-
tin contents have also been observed under Zn stress, with
enhanced callose as well as pectin contents appearing to
complement each other. The increased pectin binds excess
Zn in the cell wall and the deposited callose immobilizes it
and ensures that Zn does not enter into the cytoplasm and
this may play a role in growth inhibition (Feigl et al. 2019).
Increased expression of several lignin biosynthesis-related
genes in response to excess Zn has been related to lignin
deposition in root endodermis of A. thaliana (one endoder-
mal layer) and Thlaspi caerulescens (two layers) (Van De
Mortel et al. 2006) which can further hamper root growth
(Li et al. 2012). Excess Zn has been observed to inhibit the
activity of auxins and induced negative geotropism in tomato
roots (Kosesakal et al. 2009). Addition of triidobenzoic acid
(TIBA) (an inhibitor of indole acetic acid [IAA] transport)
induced positive tropism, suggesting that high level of Zn
might play a role in the concentration and/or in the asym-
metric redistribution of endogenous IAA. On the contrary,
excess Zn has also been reported to increase the expres-
sion of auxin biosynthesis-related genes and results in auxin
accumulation in A. thaliana root tips (Zhang et al. 2018).
Taken together, these multiple reports indicate that the
threshold of Zn toxicity sharply varies among the plant
species and Zn concentration applied (for more details,
refer Table 1). Plant growth inhibition under Zn stress
seems to be a non-specific manifestation of alterations in
physio-biochemical traits which might result from direct
effects (toxicity due to accumulation in tissues) and/or
from indirect effects (limitation of minerals and water
acquisition).
13
 Table 1  Effects of zinc toxicity on different morpho-anatomical, physiological, biochemical and molecular traits in crop plants (reports cited 2010 onwards)
Plant
13
Brassica chinensis L
Brassica pekinensis
Citrus reticulata Blanco
Cajanus cajan (L) Millsp.
Hibiscus esculentus cv. Has-
sawi
129
Zn concentration Plant Zn content Type of experiment Effects References
2000–1000 mg ­kg−1 Pot Chlorosis; small sized fronds; Yang et al. (2012)
defoliation; decrease in
Page 4 of 28
photosynthetic pigments
and increase in membrane
permeability
5–10 µM 5.49–7.93 µmol ­g−1 DW (Sh) Hydroponic Shoot biomass affected Stuiver et al. (2014)
more than roots; chlorosis;
organ dependent effect on
concentrations of nutrients;
increased activity of S­ O42−
transporters and non- protein
thiols content
5–10 µM 21.7–61.8 µmol ­g−1 DW (R) Hydroponic Decrease in biomass; increase Aghajanzadeh et al. (2020)
in cysteine, thiols, glucosi-
nolates and transcript levels
of CYP79B3, CYP83B1,
ATPS and APR (genes
catalyzing synthesis of
glucosinolates) in roots than
shoots; enhanced S ­ O42− con-
centration in shoots
20 µM Pot Growth retardation; defolia- Subba et al. (2014)
tion; reduction in photosyn-
thesis, transpiration; reduced
stomatal size; disorgani-
zation of mitochondrial
membranes and random
distribution of cristae; phe-
nols, AsA, sugars and starch
contents decreased; oxida-
tive stress; enhanced antioxi-
dant enzyme activities
500 and 1000 mg ­kg−1 Pot Decreased nodulation, nitro- Garg and Kaur (2012, 2013a,b)
gen fixation, chlorophyll, Kaur and Garg (2017); Garg
yield; oxidative stress, and Singh (2018)
disturbed mineral nutrition,
boost in antoxidant enzy-
matic and non-enzymatic
defence system
20–40 mM Pot Induced lipid peroxidation, Youssef and Azooz (2013)
CAT, APOX, DHAR and
GR activities; reduced non-
enzymatic antioxidants
Planta (2021) 253:129
 Table 1  (continued)
Plant
Hordeum vulgare
Lactuca sativa L.var. longi-
folia
Lactuca sativa cv. Philipus
Brassica oleracea cv. Bronco
Lactuca sativa cv. Philipus
13
Zn concentration Plant Zn content
500–4000 mg ­kg−1 800–2800 mg ­kg−1 (R)
200–300 mg ­kg−1 (Sh)
100 mM 674.5 mg ­kg−1 (R)
100 mM 468 mg ­kg−1 (L)
0.5 mM 34.95 ± 0.376 mg ­g−1 DW (Sh) Hydroponic
218.4 ± 35.7 mg g­ −1 DW (R)
0.5 mM 42.54 ± 0.234 mg
g−1 DW (Sh)
382.4 ± 60.6 mg g­ −1 DW (R)
0.5 mM 34,949 ± 376 μg g­ −1 DW) (R) Hydroponic
218.4 ± 35.7 μg g­ −1 DW) (Sh)
Type of experiment Effects References
Pot Negatively affected length of Kherbani et al. (2015)
roots and leaves, dry weight
of roots, leaves as well as
Planta (2021) 253:129
total biomass
Pot with peat based substrate Decrease in biomass; nutrient Rouphael et al. (2016)
imbalance; polyamines and
polyamine conjugates were
triggered
Pot Increased glycolytic supply of Lucini and Bernardo (2015)
energy substrates; proline,
hydroxycinnamic acids,
ascorbate, sesquiterpene
lactones and terpenoids bio-
synthesis; HSP70, HSP90
were up-accumulated;
down-regulation of MatK
(protein engaged in chlo-
roplast development) and
MYC2 (transcription factor
associated with ABA);
up-accumulation of PAE
and down- accumulation of
CesA (enzymes of lignin
biosynthesis); hormonal
imbalance
Reduced biomass; increase in Paradisone et al. (2015)
proline and glycine betaine
levels
Hydroponic Decrease in proline and gly-
cine betaine levels; increase
in GABA
Decreased biomass; enhanced Barrameda-Medina et al.
anion superoxide, H ­ 2O2 (2014)
and MDA levels; increment
in LOX activity; boost in
SAT, γECS, total GSH,
APX, MDHR, GR whereas
decrease in DHAR
Page 5 of 28 129
 Table 1  (continued)
Plant
13
Brassica oleracea cv. Bronco
Oryza sativa L. cv. TY-167
(Zn-resistant)
and cv. FYY-326 (Zn-sensi-
tive)
Phaseolus vulgaris L
129
Zn concentration Plant Zn content Type of experiment Effects References
0.5 mM 42,540 ± 234 μg g­ −1 DW) (R) Hydroponic Decreased biomass; enhanced
382.4 ± 60.6 μg g­ −1 DW) (Sh) anion superoxide and H ­ 2O2
Page 6 of 28
levels, reduced LOX, MDA
and SAT; increase in γECS,
total GSH, APX and GR
whereas MDHR and DHAR
activities declined
2 µM TY-167: Hydroponic Differential responses in Song et al. (2011)
2500 μg ­g−1 DW (R) genotypes in terms of total
7000 μg ­g−1 DW (L) root surface area and root
FYY-326: length; chlorosis; growth;
2600 μg ­g−1 DW (R) MDA and ­H2O2 levels; root
6000 μg ­g−1 DW (L) plasma membrane integ-
rity; enzymatic antioxidant
activities
50 ppm var. Sel 9: 440 μg ­g−1 DW (L) Hydroponic Decreased biomass; chlorosis; Michael and Krishnaswamy
increase in electrolyte leak- (2011)
age, MDA, ­H2O2, proline,
AsA levels, enhanced SOD,
POX, PPO activities
150–500 μM Hydroponic Decrease dry matter, fresh Nejad et al. (2014)
weights, leaf area, RWC
and increase in total protein
content
Planta (2021) 253:129
 Table 1  (continued)
Plant Zn concentration Plant Zn content
Solanum lycopersicum cv. PKM – 1: 150–
250 mg ­kg−1
50 μM var. Arka Alok: 100 μg ­g−1
DW (R)
140 μg ­g−1 DW (L)
Var. Arka Vikas: 110 μg ­g−1
DW (R)
120 μg ­g−1 DW (L)
50–150 μM 251–495 μg ­g−1 DW (R)
65.43-92.36 μg ­g−1 DW (Sh)
250–500 μM 23,000–44,000 μg ­g−1 DW (R) Hydroponic
5000–10,000 μg ­g−1 DW (L)
Triticum aestivum L 900 mg ­kg−1 160 mg ­kg−1 (Sh)
180 mg ­kg−1 (R)
0.5–3 mM 465.65-1129 μg ­g−1DW (R)
292.03–1124.45 μg ­g−1 DW
(Sh)
300 mg ­L−1 5,553.1±576.8 mg ­kg−1 (Sh) Hydroponic
32,205.2±8928.4 mg ­kg−1 (R)
13
Type of experiment Effects References
Pot Reduced root length, shoot Vijayarengan and Mahalakshmi
length, leaf area, root and (2013)
shoot dry weights
Planta (2021) 253:129
Hydroponic Reduction in shoot dry Pavithra et al. (2016)
weight, total plant biomass
and total chlorophyll content
Hydroponic Shoot growth more affected Cherif et al. (2010)
than root; low total chloro-
phyll content; decrease in
photosynthetic ­CO2 fixation
rate; increase of MDA levels
Reduction in chlorophyll a; Sbartai et al. (2011)
increase in CAT and APOX
activities; decline in GST
activity; higher accumula-
tion of metal in roots than
leaves
Pot Decline in biomass; altered Kanwal et al. (2016)
macro-nutrients and micro-
nutrients; inhibition in
chlorophyll and total sugar
contents; boost in antioxida-
tive enzyme activities (SOD,
CAT, POX, APX) and
proline
Hydroponic Decreased chlorophyll; Li et al. (2013a)
oxidative stress; differential
response of antioxidant
enzymes; enhanced soluble
sugars, OAT but reduced
GK activity; elevated proline
Reduced biomass, water con- Glinska et al. (2016)
tent, mitotic index; nutrient
imbalance; ultrastructural
alterations in organelles of
roots and mesophyll cells
Page 7 of 28 129
 Table 1  (continued)
129

Plant Zn concentration Plant Zn content Type of experiment Effects References

13
Triticum durum 600 μM Hydroponic Leaf necrosis; decrease in Paunov et al. (2018)
relative growth rate, net
Page 8 of 28

photosynthetic rate, pho-

tosynthetic pigments and
electron transport processes;
inactivation of photosystem
II reaction centres
Vigna unguiculata 6-20 mg ­L−1 112.51–142.70 mg ­plant−1 Hydroponic Decline in growth parameters El-Kafafi and Rizk (2013)
(dry weight, number of
roots, root length) and nutri-
ent uptake
Vitis vinifera 60–180 mg ­kg−1 320–490 mg ­kg−1 (Sh) Pot Reduced plant growth; Tiecher et al. (2017)
1500–1700 mg ­kg−1 (R) decreased pigments and
photosynthetic efficiency;
diminished SOD and POD
activities in leaves
14–35 mM 3225.75–7553.35 μg ­g−1 DW Pot Chlorosis; necrosis; suppres- Yang et al. (2011)
(R) sion in daily height growth,
6880.56–7224.84 μg ­g−1 DW daily stem diameter varia-
(St) tion; alterations in nutrient
1007.54–2982.97 μg ­g−1 DW contents; decrease in POD,
(L) CAT and PPO activities;
enhanced ABA and MDA
levels
Planta (2021) 253:129
 Table 1  (continued)
Plant Zn concentration Plant Zn content Type of experiment Effects References

Zea mays 400 and 600 μM Hydroponic Increased EC, MDA, H ­ 2O2 Hosseini and Poorakbar (2013)
contents and non-protein thi-
ols; activities of antioxidant
Planta (2021) 253:129

enzymes also increased

50 μM 150 mg ­g−1 DW (R)
180 mg ­g−1 DW (Sh)
50–750 mg ­kg−1 Light soils: 94.97–
1300.75 mg ­kg−1 DW (Sh)
288.31–2493.33 mg ­kg−1 (R)
Heavy soils: 64.10–
624.40 mg ­kg−1 (Sh)
155.75–1743.94 mg ­kg−1 (R)
Pot Inhibition in plant length, Islam et al. (2014)
leaf area, dry weight; roots
became yellow brown;
modified root distribu-
tion; decreased chlorophyll
and total soluble protein;
enhanced antoxidative
enzyme activities and anti-
oxidant metabolites
Pot (light and heavy soils) Higher metal content in roots Baran (2012)
than aerial parts; bioaccu-
mulation coefficient higher
in roots than above ground
parts; greater phytotoxicity
and phytoavailability in light
than heavy soils

The Zn concentrations are only those in which the phytotoxic symptoms were observed and not all the concentrations assayed in the experiments. Furthermore, only single excess Zn treatments
have been reported in case of multi-metal combinations or Zn- amendments
ABA abscisic acid, APX/APOX ascorbate peroxidase, AsA ascorbic acid, ATPS ATP-sulfurylase, APR adenosine 5′-phosphosulfate reductase, CAT​catalase, CesA cellulose synthase, CO2 carbon
dioxide, DHAR dehydroascorbate reductase, DW dry weight, EC electrical conductivity, γECS γ-glutamylcysteine synthetase, GABA γ-amino butyric acid, GK glutamate kinase, GR glutathione
reductase, GSH reduced glutathione, GST glutathione-S-transferases, H2O2 hydrogen peroxide, HSP heat-shock proteins, L leaves, LOX lipooxygenase, MatK maturase K, MDA malondialde-
hyde, MDHR monodehydroascorbate reductase, OAT ornithine δ-aminotransferase, PAE pectin acetylesterase, POD/POX peroxidase, PPO polyphenol oxidase, R roots, RWC​relative water con-
tent, SAT serine acetyltransferase, Sh shoots, SOD superoxide dismutase, SO42− sulphate, St stem
In addition to the above-mentioned crop plants, similar effects of Zn toxicity have also been reported in other plant species, such as Arabidopsis halleri and A. arenosa (Szopinski et al. 2019),
Camellia sinensis (Zhang et al. 2017), Handroanthus impetiginosus and Tabebuia roseoalba (Gai et al. 2017), Mesembryanthemum crystallinum (Kholodova et al. 2011), Miscanthus × gigan-
teus (Andrejic et al. 2018), Populus × euramericana (Di Baccio et al. 2009, 2011), etc.

13
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129 Page 10 of 28
Nitrogen metabolism and legume–rhizobia symbiotic
association
It has been reported that excess Zn decreases nitrate (­ NO3_)
uptake and translocation as well as total nitrogen content,
thereby disturbing nitrogen metabolism in Brassica species
(Stuiver et al. 2014; Blasco et al. 2019). Additionally, Zn
stress results in chloroplast disorganisation and/or limited
supply of ­NO3_ at the site of nitrate reductase (NR) synthe-
sis, thereby impeding its activity (Sidhu 2016). Decreased
activity of NR reduces the level of free amino acids (for
instance, Glutamate and Asparagine which are required for
ATP and chlorophyll biosynthesis), which in turn hampers
photosynthesis in plants (Sidhu 2016). Moreover, NR activ-
ity relies on photosynthesis or synthesis of photosynthates as
it requires photosynthetic carbon (Solanki et al. 2011). Inhi-
bition of chlorophyll biosynthesis and carbon dioxide ­(CO2)
fixation under Zn stress (more details under “photosynthesis
and respiration section”) may also lead to inefficient nitro-
gen assimilation, consequently reducing NR activity. Low
NR activity could also be due to the direct effect of high Zn
on enzyme/protein synthesis or activity as ­Zn2+ ions have a
high affinity for –SH groups at the active site of the enzyme,
as reported by Khudsar et al. (2008) and Ramakrishna and
Rao (2014) in Zn-stressed Cajanus cajan (Linn.) Huth and
TILLING mutants of B. rapa, respectively. On the contrary,
Zn stress induced the expression of NRT1.1 (nitrate trans-
porter) in the meristematic as well as elongation zones of
the roots of wild-type (Col-0) Arabidopsis plants, leading to
increased ­NO3_ uptake and subsequently Zn accumulation
(Pan et al. 2020). The potential function of other NRTs in
­NO3_-mediated Zn accumulation in plants (both hyperac-
cumulators and non-hyperaccumulators) remains unknown
and future research need to focus on this aspect.
Zn contamination also affects nodulation, i.e. nodule
number and weight as well as nitrogen fixation activity
(nitrogenase activity, leghemoglobin content) in several leg-
umes (Garg and Kaur 2012; Souza et al. 2020) which can be
ascribed to the harmful consequences of Zn on root func-
tioning or associative rhizobia. Excess Zn decreases the free-
living population of rhizobia to less than a critical thresh-
old (Broos et al. 2005), and thus, indirectly affects nitrogen
­(N2) fixation. Reduction in acetylene reduction assay (ARA)
by surplus Zn can be attributed to nitrogenase dysfunction
or outcome of its toxic effects on bacteroid oxygen uptake
(Younis 2007). Furthermore, high Zn-induced reduction in
nodulation could be due to the elimination of Rhizobium or
non-formation of effective ­N2 fixing nodules. Damage to the
root system and inhibition of infection process as a result
of Zn toxicity might be the other reasons for lack of proper
nodule formation. Harmful effects of Zn on the symbiotic
performance might also be linked to a decreased supply of
photosynthates to the nodules by Zn-stressed plants. It is
13
Planta (2021) 253:129
believed that Zn toxicity not only reduces the Rhizobium
population but also their genetic diversity (Lakzian et al.
2002). In this context, the strains of rhizobia that survive in
heavy metal contaminated soils (including excess Zn) have
been reported to have the least number of plasmid types
and increased number of plasmid bands per isolate (Lakzian
et al. 2002). Perhaps Zn stress results in loss of symbiotic
plasmids, thereby affecting the bacterial ability to nodulate
legumes, since the genes essential for nodulation and nitro-
gen fixation are present on this plasmid (Wang et al. 2018).
Hence, it appears that plasmids are involved in metal toler-
ance of rhizobia, nevertheless, the function of these plasmids
is still unclear, suggesting that complex relationship between
the diversity of rhizobia and Zn stress should be revisited.
Plant water status and mineral nutrition
Zn toxicity decreases relative water content (RWC) in plant
tissues or disturbs water relations (Fig. 1) (Garg and Singh
2018) through a reduction in the water transporters, transpi-
ration rates and induces strong dehydration (Ghnaya et al.
2010) which may be due to Zn accumulation in aerial tis-
sues. Reduction in RWC as a result of excess Zn exposure
might also be probably due to reduction in the absorbing
surface of roots or the vascular system mediated by a block-
age in the xylem elements, thereby disturbing the process
of ascent of sap (Koleva et al. 2010). Reduction in water
content implies the loss of turgor/turgidity which in turn
decreases the cell expansion process, ultimately leading to
inhibited growth and biomass production. The decrease in
leaf water potential under high Zn exposure is primarily
because of elevated osmotic potential in the neighbouring
medium and reduction in water absorption (Mir et al. 2015).
Surplus Zn causes physiological drought by altering plant
water balance either by blocking its transport within the
plant body or by restricting the water transport activity of the
tonoplast (Kholodova et al. 2011). In recent times, few work-
ers have tried to explore the molecular basis of Zn-mediated
reduction in root water transport properties. In this context,
Kholodova et al. (2011) demonstrated severe suppression of
aquaporin (AQP) genes whose products were present in the
plasma membrane (McPIP1;1, McPIP2;1, and McPIP2;3)
or tonoplast (McTIP1;2 and McTIP2;2) in Cu/Zn-stressed
Mesembryanthemum crystallinum. Similarly, PIP2 (plasma
membrane intrinsic proteins) AQP was found to be reduced
in lettuce under Zn stress (Lucini and Bernardo 2015). The
decrease in barley root-intrinsic water transport property in
response to high Zn was ascribed to the reduction in root and
shoot hydraulic conductivity which in turn was facilitated
through down-regulation of AQPs (HvPIP1;2 HvPIP2;4 and
HvPIP2;5) activity and gene expression as well as increased
suberization in endodermis (Gitto and Fricke 2018). These
workers further suggested that surplus Zn reduced the water
Planta (2021) 253:129
flow rate in barley plants through a co-ordinated reduction
in the root (AQPs) and shoot (stomata) hydraulic properties,
rather than decreased water-absorbing (root) and -loosing
(shoot) surface (Gitto and Fricke 2018). Recently, Fatemi
et al. (2020) reported a reduction in root hydraulic conduct-
ance and involvement of all PIP1 isoforms as well as PIP2;6
along with PIP2;7 in sensing the long-term response to Zn
treatments in Brassica rapa plants. These reports suggest
that surplus Zn causes both morphological and physiologi-
cal alterations which influence the plant’s ability to absorb
and transport water.
Excess Zn levels hamper specific plant physiological
balances by competing with other ions (Mg, Mn, Cu, Fe
etc.) for binding at numerous sites, for example, principal
absorption region or loading region of roots (Tewari et al.
2008). Furthermore, Zn stress inhibits root growth (already
discussed), injures root elongation regions (Zn-induced
cracking) with reduced capacity to absorb nutrients or even
causes cell death (Vassilev et al. 2007; Simon et al. 2021).
Modifications in nutrient contents under Zn stress could
also be due to alteration in the functioning of membrane
transporters and ion channels as well as membrane depo-
larization in root cells, thereby increasing membrane perme-
ability (Bazihizina et al. 2014). High Zn levels hinder not
only nutrient uptake (N, P, K, Mg, Ca, Fe etc.) but also their
allocation to different plant parts (Sagardoy et al. 2009; Garg
and Singh 2018). Moreover, differential alterations in nutri-
ent uptake and distribution, causing an increase in contents
of some nutrients while a decrease in others depending upon
the plant species/organs have been reported by researchers
(Jain et al. 2010; Di Baccio et al. 2011; Souza et al. 2020).
For instance, in some plants, such as Vitis vinifera, leaves
retained high level of Fe under Zn stress partly due to its
enhanced translocation from the root and shoot to leaves
via xylem, which is crucial for maintaining a high rate of
photosynthesis and carbon assimilation (Yang et al. 2011).
On the contrary, Zn-induced reduction in shoot Fe has also
been reported (e.g. wheat) owing to the interference of Zn in
Fe translocation to shoots (Glinska et al. 2016). This inter-
vention may be partly due to competition in the chelation
processes associated with the translocation of Fe from roots
to aerial parts. For example, Fe in the symplast is chelated
with nicotianamine (NA), and translocation of NA–Fe2+
complexes via phloem determines Fe homeostasis (Kim and
Guerinot 2007). NA takes part in Zn homeostasis also and
hence, competition between Zn and Fe for NA chelation in
phloem may result in Zn-induced decrease in shoot Fe, as
reported in soybean (Ibiang et al. 2017). On the other hand,
in xylem, both Fe and Zn are bound by citrate (Cornu et al.
2015) and therefore competition between both the metals for
citrate chelation in xylem might be another reason for the
decreased shoot Fe content observed in Zn-stressed plants.
These reports indicate that mechanisms responsible for the
Page 11 of 28 129
interference of excess Zn with Fe translocation are distinct
in xylem and phloem. It is worth mentioning here that Zn-
induced Fe deficiency may also be due (at least partly) to the
non-specificity of iron-regulated transporter (IRT-1) which
can transport divalent metals such as Zn, in addition to Fe,
as demonstrated in Arabidopsis (Connolly et al. 2002). On
the contrary, Leskova et al. (2017) argued that Fe-associated
morphological, physiological and molecular responses in A.
thaliana exposed to Zn excess were not caused by Zn-inhib-
ited Fe uptake through IRT-1. Instead, high Zn stimulated
the transcriptional response of Fe-regulated genes: BTS (a
gene encoding Fe sensor) and FRO2 (Ferric reduction oxi-
dase2; gene accountable for ferric-chelate reductase (FCR)
activity), suggesting that Zn disturbs Fe homeostasis at the
level of Fe sensing. As an extension of this work on crop
plants, recently Erenoglu (2019) provided the first evidence
for the presence of IRT-like proteins in wheat. However, this
IRT-1-based Fe deficiency caused by Zn excess is yet to be
established and hence cannot be generalized for all plants.
Excess Zn intervention with the uptake or translocation
of N and Mn results in chlorosis and reduced photosynthetic
rates, clearly evidencing obstruction caused by high Zn in
chlorophyll synthetic pathway (more details under “photo-
synthesis and respiration section”). Similarly, decrease in
K content under Zn stress could probably be a consequence
of disturbed water balance. However, K transport to above-
ground components in Zn-stressed plants (Zygophyllum
fabago) might help in improved photosynthesis and establish
the transmembrane pH gradient essential for ATP synthesis
(Lefèvre et al. 2014). Moreover, Zn-induced deficiency of
Fe and Cu may affect oxidative as well as photophosphoryla-
tion in different plant tissues. The availability of nutrients
in the rhizospheric region may get reduced due to their sub-
stitution by excess Zn at binding sites leading to deficiency
of essential nutrients, further enhancing Zn toxicity. For
example, owing to the similar ionic radius, Zn can replace
Fe and Mg, thus inducing decreased accumulation of pho-
tosynthetic pigments since Fe is required for heme synthesis
while Mg participates in chlorophyll formation (Tripathy
and Pattanayak 2012). Induction of genes, such as FER-
RIC REDUCTASE DEFECTIVE3 (FRD3, AT3G08040)
belonging to MATE (multidrug and toxic compound extru-
sion) efflux family, the metal-associated domain-containing
protein, NRAMP4 (AT3G13100), many Zn finger proteins
(e.g. At5g54040, At3g52800, At3g19580, At5g18550,
At1g76410, At5g04340, At2g19810, and At5g59820), and
C2H2-type Zn finger proteins (ZFPs), may reflect the molec-
ular response to excess Zn-induced P deficiency in A. thali-
ana (Pineau et al. 2012; Bouain et al. 2014). Additionally,
PHR1, PHO1 (phosphate transporter-exporter) and PHO1;
H3 are the genes that link up Zn and P nutrition and homeo-
stasis in A. thaliana (Khan et al. 2014). Although, cross-
talk between Zn and other nutrients has been established, a
13
129 Page 12 of 28
comprehensive evaluation of the exact pathways/mechanism
of mineral nutrition that excess Zn interferes with is still
lacking which needs to be the focus of future research.
Photosynthesis and respiration
Zn toxicity diminishes net photosynthesis, however, it is
unclear whether the decrease is restricted by mesophyll
conductance, stomatal conductance, enhanced respiration,
reduced photochemistry, weakened biochemistry or amal-
gamation of all these aspects (Di Baccio et al. 2009; Vas-
silev et al. 2011). Generally, there is enhanced resistance of
mesophyll to ­CO2 diffusion which could be due to the effect
of drought induced by Zn excess. Augmented Zn levels in
soil resulted in decreased leaf area and stomatal limitations
in Pelargonium graveolens which further reduced the flow
of ­CO2 within the leaves resulting in hindered C­ O2 diffusion
to the chloroplasts, thereby affecting the photosynthetic car-
bon fixation (Misra et al. 2005). At high Zn concentrations,
a reduced stomatal conductance was associated with root
hydraulic reductions and photosynthetic rate decrease in B.
rapa (Fatemi et al. 2020). It seems that excess Zn results in
reduced water uptake and induces stomatal closure, which
together with a decrease in the photosynthesis rate leads to
decline in plant biomass (Fig. 1). Excess Zn declined PSII
(photosystem) efficiency and non-cyclic photophospho-
rylation by disturbing the structure of PSII core complex
and interacting with donor side of PSII or dislocating non-
heme Fe in PSII of Beta vulgaris (Sagardoy et al. 2009).
Similarly, Zn toxicity caused a reduction in the quantum
yield of electron flow through PSII (∆F/F′m) in Z. fabago,
thereby lowering the efficiency of photosynthetic energy
conversion (Fv/Fm) (Lefèvre et al. 2014), which could be
due to Zn-induced perturbation in the structure of reaction
centre complexes (Paunov et al. 2018). Moreover, due to
charge shielding action of Zn ions, thylakoid membranes
swell up and complexes are pulled apart, thereby decreasing
the energy transfer efficiency. According to Paunov et al.
(2018), toxic effects of Zn on photosynthesis in Triticum
durum could be due to decreased rate of electron transfer
from ­QA to ­QB at the acceptor side of PSII, thereby lowering
the electron flow from PSII to PSI. Similarly, Szopinski et al.
(2019) observed a reduction in electron transport flux and
percentage of active reaction centers in A. halleri as well as
A. arenosa under Cd and Zn stress. Precise effects on PSII
photochemistry associated with competitive dislocation of
Mn by Zn at the region of photolysis of water, hampering
the transport of electrons along with evolution of O ­ 2 have
been described in perennial grass, Polypogon monspeliensis
(Ouni et al. 2016). Zn overload hampered C ­ O2 assimila-
tion in Limoniastrum monopetalum by replacing ­Mg2+ in
Rubisco, thereby causing loss of its carboxylation activity
without affecting its oxygenation function (Cambrollé et al.
13
Planta (2021) 253:129
2013). This displacement of M ­ g2+ from Rubisco may result
in under-utilization of ATP and NADPH in C ­ O2 fixation,
followed by an overflow of electrons from oversaturated
electron transport systems to ­O2. Moreover, toxic concen-
trations of Zn cause disorganization of chloroplast, reduc-
tion in thylakoid and grana, augmentation in number of
plastoglobuli and starch grains as well as the disintegration
of chloroplast membrane in plants like Sedum alfredii and
Populus deltoides (Jin et al. 2008; Stoláriková-Vaculíková
et al. 2015). Chlorophyll a: chlorophyll b ratio has also been
found to decrease under Zn toxicity, implying higher sensi-
tivity of chlorophyll a towards Zn stress than chlorophyll
b (Garg and Singh 2018). It could be due to an increase in
PSII light harvesting complexes (LHC II) comparative to
reaction centres, since LHC II has more chlorophyll b than
PSII chlorophyll-binding proteins. The negative correlation
between surplus Zn and photosynthetic pigments might be
attributable to either inhibition of activities of the enzymes
involved in chlorophyll biosynthesis like δ-aminolevulinic
acid dehydratase (ALA-D) and protochlorophyllide reduc-
tase by Zn interference with the functional sulphydryl site,
leading to lower production of 5-aminolevulinic acid (ALA)
or through an increased activity of chlorophyll-degrading
enzyme chlorophyllase or both, as reported by Ouni et al.
(2016) in P. monspeliensis. ERF VII proteins (group VII
Ethylene Response Factors) synthesized under Zn excess
in poplar could also be engaged in suppressing chlorophyll
biosynthetic genes (Carbonare et al. 2019). Furthermore,
interaction of Zn with -SH groups of chlorophyll synthesis/
degrading enzymes affects the integration of chlorophyll
molecules into pigment protein complexes of photosynthe-
sis. Thus, excess Zn-induced inhibition of the photosynthetic
apparatus seems to be coupled with changes in chlorophyll
contents. At the molecular level, pioneering work of Di Bac-
cio et al. (2011) revealed down-regulation of genes encod-
ing products engaged in the light reaction, such as LHC II
proteins as well as polypeptides connected to PSI, PSII, ATP
synthases and cytochrome b6/f complex linked with redox
series, and electron carriers linked with ferredoxin along
with down-regulation of bigger subunit of Rubisco and up-
regulation of smaller subunit under Zn exposure in Popu-
lus × euramericana clone I-214. The decrease in ­Rfd values
(variable chlorophyll fluorescence ratio, an indicator of pho-
tosynthetic capacity) whereas an increase in fluorescence
intensity ratios (FIR) at two maxima (­ F690/F735) in tomato
plants under excess Zn was associated with the decline in
total chlorophyll levels (Cherif et al. 2010).
Respiration and photosynthesis are interrelated and
mutants of A. thaliana and Nicotina tabacum having altered
alternative oxidase (AOX) activity exhibited diminished
photosynthesis (Yoshida et al. 2011; Dahal et al. 2017).
Citrate, fumarase, isocitrate dehydrogenase and citrate syn-
thase activities enhanced under Zn stress in sugar beet plants
Planta (2021) 253:129
(Sagardoy et al. 2011), suggesting an increase in the break-
down of respiratory substrates probably due to the higher
energy requirement of plants to cope with Zn toxicity. There
is a great controversy regarding the consequences of excess
Zn on plant respiration. On one side, in vitro investigations
have indicated that Zn hampers cytochrome bc1 complex
activity in mitochondria, possibly by hindering reaction with
ubiquinol at ­VQH2-binding site which oxidizes ­UQH2, qui-
nol oxidase ­(Q0) site and activity of AOX in Arum macula-
tum (Affourtit and Moore 2004); on the other side, in vivo
experiments suggest that mitochondrial respiration increases
gradually with increase in Zn levels in safflower and sun-
flower plants (Ismail and Azooz 2005). In general, the
cytochrome pathway is connected with an increase in res-
piration rate leading to the elevated synthesis of ATP while
AOX activity is responsible for maintaining respiration
and decreased ATP synthesis (Florez-Sarasa et al. 2007).
Thus, the presence of excess Zn alters mitochondrial activity
resulting in abnormal respiration in plants. We propose that
plant respiration enhances under high Zn levels to increase
the production of various organic acids (oxalic acid, citric
acid, malic acid, fumaric acid) via TCA (Tri-Carboxylic
Acid) cycle since most of them are required for sequestrat-
ing excess Zn into the vacuoles. Recently, Carbonare et al.
(2019) reported that ­Zn2+ competes with Fe (II) for the
active site of plant cysteine oxidase (PCO) and diminishes its
activity, resulting in the stabilization of ERF VII transcrip-
tion factors with subsequent stimulation of anaerobic genes
[Alcohol Dehydrogenase (Pop_ADH), PCO (Pop_PCO), and
Hypoxia Responsive Attenuator1 (Pop_HRA1)] in the roots
of poplar clones as a generic response to Zn excess. The
authors argued that hypoxia-like response triggered under
Zn stress seemed to be a part of the plant adaptive response,
rather than being stimulated by an actual decrease in oxygen
availability in the roots in response to Zn excess.
Thus, it can be inferred from the above that plants
exposed to excess Zn exhibit a plethora of photosynthetic
responses depending upon the species, Zn concentration or
genetic variance (Table 1). Multiple evidence suggests that
there is no single specific target of Zn toxicity on photo-
synthesis, rather a cascade of events might be triggered by
Zn excess. Nevertheless, analyzing the available literature
from a broader perspective, the main basis of Zn toxicity on
photosynthesis seems to be disturbed mineral nutrition (Fe,
Mn, Mg) and thereafter the resultant responses. Moreover,
all the above-described excess Zn-induced effects on photo-
synthesis and respiration are not consistent between the plant
species as most of the effects have been reported in either
shrubs, grasses or hyperaccumulator plants with very few
reports on agriculturally important crops (for more details,
refer Table 1). In addition to this, molecular/genetic studies
focusing on Zn-induced negative effects on these two physi-
ological processes are scanty.
Page 13 of 28 129
Redox balance
Zn is a non-redox heavy metal which is unable to produce
reactive oxygen species (ROS) viz. singlet oxygen (1O2),
superoxide radicals ­(O2•−), hydroxyl radical ­(OH•) and
hydrogen peroxide (­ H2O2) directly via Haber–Weiss reac-
tions. However, over-generation of ROS and manifestation
of oxidative stress in plants as a consequence of Zn toxicity
might be possible due to disturbance in various metabolic
pathways and/or electron transport mechanisms. It has been
reported that phytotoxic Zn concentrations enhance lipoxy-
genase (LOX) activity, stimulate peroxidation of lipids
(malondialdehyde-MDA) and influence membrane integrity
and permeability (Goodarzi et al. 2020) (for more details,
refer Table 1). Excess Zn triggers senescence in plants
through enhanced degradation of proteins which can be
attributed to binding of Zn to sulfhydryl cluster of proteins,
resulting in abnormal protein structure (Singh et al. 2018).
It is believed that excess ROS directly affects the functional-
ity of proteins either by oxidation of amino acid side chains
(histidine, lysine and cysteine) or by secondary reactions
with aldehydic products of lipid peroxidation or glyco-
sylation giving rise to the production of carbonyl groups
in the protein molecule (Madian and Regnier 2010), which
could be the mechanisms behind Zn-induced oxidative
damage to the proteins. Such oxidatively modified proteins
can undergo chemical fragmentation or form aggregates
because of covalent cross-linking reactions and increase
surface hydrophobicity, leading to loss of function (Madian
and Regnier 2010). Moreover, such proteins are selectively
used as target substrates for proteases or additional cata-
bolic enzymatic activities stimulated by Zn excess. In plants,
cystatin/cysteine protease balance plays a critical function
in the turnover of proteins under abiotic or biotic stresses
(Benchabane et al. 2010). Cysteine protease as well as cys-
tatin accumulated to high levels after Zn exposure in lettuce
plants (Lucini and Bernardo 2015). On the contrary, Sid-
diqui and Bano (2019) reported a decrease in the inhibitory
activity of phytocystatin upon Zn-binding and ascribed it to
the functional loss of the protein coupled with the structural
and conformational changes, thereby affecting growth and
development of garlic plants. Zn excess induced increased
protein tyrosine nitration (a post-translational mechanism)
in the roots of B. napus, which was due to an imbalance in
ROS and reactive nitrogen species (RNS) homeostasis (Feigl
et al. 2019). Moreover, the number of cells with active DNA
replication decreased which correlated with the viability of
root meristematic cells (Feigl et al. 2019). Recently, prot-
eomic experiments conducted by Simon et al. (2021) in Zn-
stressed rice roots revealed induction of proteins responsi-
ble for defence mechanisms, redox and ion homeostasis and
proteolysis whereas reduction in those involved in energy
generation and cell wall formation.
13
129 Page 14 of 28
The mitoreductive effects of Zn are attributed to hinder-
ance in the synthesis of nuclear proteins required for cell
cycle or may be as a result of an obstruction in nucleic
acid synthesis, as studied in Nigella sativa and T. aestivum
(El-Ghamery et al. 2002). The stickiness of chromosomes
(characterized by amorphous sticky clumps of chromatin
resulting in sterility) as a result of Zn toxicity has been
ascribed to either physico-chemical attributes of DNA or
proteins or combination of both on the establishment of Zn
complexes with phosphate groups of DNA, or DNA abridg-
ment or development of cross-links between intra- and
inter-chromatids in N. sativa and T. aestivum (El-Ghamery
et al. 2003). The exact pathway(s) of Zn-induced genotoxic-
ity in plants is still unknown, though probable mechanisms
through which Zn induces chromosomal anomalies involve
(i) irregular spreading of chromosomes leading to their scat-
tering, (ii) formation of anaphasic bridges due to unequal
exchange or dicentric chromosomes, (iii) inability of bro-
ken chromosomes to recombine, (iv) failure of the arrival
of chromatids at the poles, and (v) laggard chromosomes
leading to micronucleus formation (Oladele et al. 2013).
Furthermore, Random Amplification of Polymorphic DNA
(RAPD) profiles indicated loss of normal bands due to DNA
damage (e.g. single- and double-strand breaks, modified and
oxidized bases, abasic sites, DNA–protein cross-links, etc.),
point mutations and/or complex chromosomal rearrange-
ments and appearance of new bands because of changes in
some oligonucleotide priming sites, large deletions and/or
homologous recombinations following high Zn exposure in
Phaseolus vulgaris and Zea mays, respectively (Cenkci et al.
2009; Erturk et al. 2013). Thus, experimental evidence indi-
cates that Zn toxicity damages the membranes, proteins and
genetic material of plants which lead to irreparable meta-
bolic dysfunction and severely affect the cell viability.
Defence responses
In spite of the noxious effects of excess Zn, absolute elimi-
nation of Zn is not feasible because of its dual function,
an indispensable microelement on one hand and a poten-
tial toxic environmental contaminant on the other. To cope
with the destruction triggered by ROS, plant cells possess a
broad and combined internal antioxidant defence machinery
comprising of enzymatic and non-enzymatic constituents
in conjunction with certain compounds called osmoprotect-
ants which function as free radical scavengers (Miller et al.
2008). High activities of ROS scavenging enzymes (for more
details, refer Table 1), for instance, superoxide dismutase
(SOD), catalase (CAT), guaicol peroxidase (GPOX), ascor-
bate peroxidase (APOX) and glutathione reductase (GR)
in plants under Zn stress suggest positive adaptive defence
response not only against Zn toxicity and maintaining ROS
balance but also preventing further oxidative damage (Jain
13
Planta (2021) 253:129
et al. 2010; Michael and Krishnaswamy 2011). However,
decrease in enzymatic antioxidant activities has also been
witnessed in Zn exposed plants, indicating excessive oxida-
tive load (Song et al. 2011). The primary isozyme among the
superoxide scavenging enzymes, i.e. copper–zinc superoxide
dismutase (CuZnSOD) is directly related to stress resistance
in plants (Song et al. 2006). As a co-factor of CuZnSOD,
increased Zn levels may lead to augmented activity of this
enzyme (Gomes et al. 2013). Genetic adaptation to high
Zn tolerance through stimulation as well as deactivation of
genes belonging to SOD and POX family has been observed
in Zn-treated A.thaliana and Thlaspi caerulescens plants
(Van de Mortel et al. 2006). Although an active antioxida-
tive metabolism does not represent Zn tolerance mechanism
in a strict sense, it is beneficial for plant performance under
heavy metal stress.
Glutathione (GSH) and phytochelatins (PCs) assist in the
exclusion of excess cytosolic Zn via formation of complexes
with Zn, resulting in reduced toxicity. Binding of Zn to -SH
groups of cysteine and thiol-peptide compounds weakens the
generation of ROS and this may be considered as a critical
method for decreasing oxidative injury to plant cells. A posi-
tive correlation of non-enzymatic antioxidants viz. ascorbic
acid (AsA), carotenoids, non-protein mercaptans, and phe-
nols with high Zn has been reported (Mishra and Prakash
2010). Genetic evidence for enhanced tolerance to Zn stress
through the expression of A. thaliana AtPCS1 in B. juncea
resulting in PCs synthesis has been provided by Gasic and
Korban (2007). GSH production, utilization and redox level
played an important role in response to Zn excess as expres-
sion of GSH reductase and γ-ECS genes augmented in Zn-
treated poplar plants (Di Baccio et al. 2005). Furthermore,
up-regulation of 8 out of 17 genes engaged in AsA/GSH
metabolism (γ-ECS, GR, GSTs, GPXs and their isoforms)
and nine being down-regulated explained the genetic basis
for Zn tolerance in Populus x euramericana I-214 clone (Di
Baccio et al. 2011). In addition to PCs and GSH, metal-
lothioniens (MTs) have been implicated in Zn homeostasis,
defence, Zn transport and translocation in plants (Pramanick
et al. 2017). The cysteine-loaded amino (N-), as well as, car-
boxyl (C-) terminal sites of MTs are engaged in the differen-
tial binding of metals. The N-terminal plays a role in estab-
lishing stable equilibria with vital elements like Cu and Zn,
while C-terminal has an affinity for noxious metals and assist
in their sequestration (Barbosa et al. 2016). In some plants,
for instance, Populus alba L. cv. Villafranca, differential
response of MT expression profiles in response to excess Zn
has been observed, with ­MT1 and ­MT3 mRNA levels being
enhanced in leaves while ­MT2 transcripts being unaffected
(Castiglione et al. 2007). Furthermore, a unique set of pro-
teins ‘plant defensins type I (PDF1s)’ have been identified
and described owing to their contribution in imparting Zn
tolerance to plants (Mirouze et al. 2006). PDF1s are minute
Planta (2021) 253:129
cysteine-rich peptides whose up-regulation in Zn hyperac-
cumulators; T. caerulescens, as well as, A. halleri provides
direct molecular evidence of their role in increasing plant Zn
tolerance (Van de Mortel et al. 2006; Shahzad et al. 2013).
The precise means through which PDF1s are involved in Zn
tolerance are not much known. Recently, Zhang et al. (2019)
characterized a heat-shock transcription factor A4a (PuHS-
FA4a) that functions as a positive regulator of excess Zn
tolerance in transgenic Populus ussuriensis. The authors evi-
denced that PuHSFA4a activates ROS scavenging and root
development-related genes by directly targeting PuGSTU17
(glutathione-S-transferase U17) and ­PuPLA2 (phospholipase
A), respectively, under excess Zn. In total, these studies con-
firm that increased contents of non-enzymatic antioxidant
compounds are genetically correlated with the ability of
plants to tolerate Zn-induced oxidative stress. Plants adapt
to a new metabolic equilibrium of these compounds through
their co-ordinated synthesis and complexation to combat Zn
toxicity. Information acquired from studies on hyperaccu-
mulator species to combat the effects of Zn toxicity can be
used for breeding programs aimed at developing tolerant
genotypes of crop species.
Apart from the enzymatic and non-enzymatic defence
machinery, Zn stress initiates accumulation of a set of
“stress metabolites” (proline, glycine betaine (GB), solu-
ble sugars and free amino acids) which have an essential
function in osmotic adjustment and continued existence
as well as the survival of plants under heavy metal stress,
including Zn (Kaur and Garg 2017). Literature indicated
differential response of plant organs (B. napus) towards the
accumulation of osmolytes under Zn toxicity (Ghnaya et al.
2010). Kholodova et al. (2011) proposed that proline did
not function as a compatible solute for osmotic adjustment
in M. crystallinum; in fact, it behaved as a low-molecular
weight chaperone. Besides being a metal chelator along-
with osmolyte, proline scavenges ­OH. radicals and singlet
oxygen, thereby imparting protection to plants against oxi-
dative stress. Accretion of proline under Zn stress may be
due to a decline in the activity of enzyme causing proline
degradation, resulting in enhanced proline accumulation or
augmented activity of some enzymes responsible for the bio-
synthesis of proline. In Lactuca sativa, an increase in proline
and GB did not signify a resistance mechanism, rather it was
possibly a symptom of Zn stress (Paradisone et al. 2015).
Many more osmotic stress-associated metabolites enhance
under Zn stress (e.g. lettuce), for example, sesquiterpene
lactones, hydroxycinnamic acids and terpenoids (Lucini and
Bernardo 2015). Accumulation of soluble sugars in response
to Zn toxicity facilitates regulation of osmotic stress in
plants which results in stabilization of membranes as well
as biomolecules. In addition to this, accumulated sugars
act as a scavenger of ROS, and also induce antioxidative
gene expression in plants, for instance, cucumber (Sun et al.
Page 15 of 28 129
2012). Increase in free amino acids in response to metal
stress can be considered as a defence mechanism as they
function in maintaining the cellular osmotic equilibrium or
as metal chelators. Leaves of Mimosa caesalpiniaefolia and
Erythrina speciosa (Brazilian native legume trees) accumu-
lated enhanced soluble free amino acid contents, implying a
tolerance response to Zn stress in these species (Souza et al.
2020). Overall, these reports suggest that accumulation of
osmoprotectants as a Zn tolerance mechanism might be con-
sidered as a mere correlation or coincidence with no direct
genetic evidence in favour of it reported so far, though they
play an important role in adaptation and ultimate survival of
plants during Zn stress.
Zn uptake, root‑to‑shoot translocation
and allocation within plants
Plant roots take up Zn primarily in the form of hydrated Zn
as well as ­Zn2+ free ions and then it is allocated within the
plant in an intricate manner (Kisko et al. 2015). Zn transpor-
tation commencing from root epidermal to cortical cells and
then to xylem can take place through the symplast pathway
from where Zn is pumped into the stellar apoplast (Caldelas
and Weiss 2017). Zn can be transported extracellularly to
stellar apoplast in areas where endodermal casparian band is
not completely developed, i.e. close to root apex and lateral
root initiation regions (White et al. 2002). Both symplastic
and apoplastic pathways function together in Zn fluxes to
the aerial parts (Noulas et al. 2018). Zn movement through
plasma membrane occurs in the direction of higher negative
electrical potential via cation channel in dicots and mono-
cots except for Poaceae. In Poaceae, “phytometallophores”
or non-protein amino acids called “phytosiderophores (PS/
MAs)” form complexes with Zn and transport them to the
external surface of the root (Suzuki et al. 2006). Increased
uptake of Fe (III) through secretion of PS is well established
for Fe acquisition by Poaceae, but till date this has not been
conclusively proved for Zn acquisition. There is conflicting
evidence for use of Zn–PS complexes as the main uptake
system in members of Poaceae. For example, Suzuki et al.
(2006) reported that Zn-deficient barley absorbed more Zn
(II)-DMA (deoxymugineic acid) than Z ­ n2+ ions from the
soil. In another study, Suzuki et al. (2008) revealed that
­Zn2+ was favoured for root uptake while Zn-DMA com-
plexes were preferred over ­Zn2+ for internal transport and
seed loading in rice. On the contrary, results of Arnold et al.
(2010), Widodo et al. (2010) and Ptashnyk et al. (2011) pro-
vided strong evidence in support of bulk uptake of Zn as
Zn–DMA complexes in rice. These inconsistencies in the lit-
erature suggest that Zn might share the same uptake pathway
as Fe in graminaceous plants under Zn deficiency. Addition-
ally, ZIP transporters have been identified in Poaceae species
13
129 Page 16 of 28
(rice, maize, etc.) and their role in Zn uptake cannot be ruled
out either (more details under “ZIP section”).
Concentration-dependent uptake of free Zn ions is con-
sidered to be saturable which takes place via a carrier-medi-
ated system. Uptake experiments carried out by Hacisali-
hoglu et al. (2001) showed the occurrence of two distinct
low- and high-affinity transport systems for Zn uptake in
wheat plants. Zn can be loaded into the vasculature (xylem)
of Zn hyperaccumulator plants (N. caerulescens and A.
halleri) where it forms complexes with several molecules
comprising organic acids, for example, citrate and malate,
amino acids like histidine in addition to NA (Cornu et al.
2015). MTs and phytate also assist in chelating bulk of the
toxic Zn in vacuoles, for instance, in N. caerulescens (Mon-
sant et al. 2011). Seclusion of Zn in the vacuoles of root
cells of hyperaccumulator plants restricts its root-to-shoot
transfer, while its unloading in the shoots and sequestration
in the vacuoles of leaf cells may be responsible for this pro-
cess (Verbruggen et al. 2009). Zn-rhamnolipid complexes
have been identified in B. napus roots (Stacey et al. 2008).
Zn–malate and Zn–citrate complexes dominate in the leaf
epidermis whereas Zn–NA is the major complex found in
the mesophyll of N. caerulescens (Schneider et al. 2013).
In rice, Zn was present in the xylem in the form of free
ions and to some extent in bound form whereas in phloem
sap Zn was principally bound to chelator NA (Yoneyama
et al. 2015). The major form of Zn accumulated in spongy
mesophyll and vascular bundle of Z. fabago was Zn bound to
O/N-ligands/chelators (Lefèvre et al. 2014). In the xylem of
strategy I plants (non-graminaceous), Zn is primarily loaded
as ­Zn2+ ions and the remaining proportion is associated with
organic acids (Lu et al. 2014). In plants belonging to strategy
II (Poaceae family), Zn–DMA complexes have been traced
in wheat root extracts (Xuan et al. 2006) and in japonica rice
shoots under Fe-deficient conditions (Tsednee et al. 2016),
indicating that Zn is transported in xylem as Zn–MA com-
plexes. Studies have revealed differential localization of Zn
in non-accumulators as well as hyperaccumulators. In cow-
pea, Zn was confined mainly in the root meristematic region
(Kopittke et al. 2011; Wang et al. 2013). Zn accumulates at
the base of trichomes in non-accumulators, such as Arabi-
dopsis lyrata (Sarret et al. 2009) and tobacco (Nicotiana
tabacum L.) (Sarret et al. 2006; Straczek et al. 2008). How-
ever, Weremczuk et al. (2017) found high Zn concentrations
in clusters of “Zn-accumulating mesophyll cells” of tobacco
that experienced programmed cell death, ultimately resulting
in formation of necrotic lesions. In hyperaccumulators like
A. halleri, Mishra et al. (2017) reported restriction of Zn at
the base of trichomes and also in mesophyll cells whereas
N. caerulescens stored it in the leaves’ epidermis. Similarly,
in Sedum alfredii, Zn accumulated in the epidermis of stems
and leaves (Tian et al. 2009). Widening of cell wall along-
with augmented cation exchange capacity (CEC), functions
13
Planta (2021) 253:129
as an external barrier restricting entry of Zn into protoplasm
(Liu et al. 2014). It has been established that Zn binds to
hydroxyl groups of cellulose, hemi-cellulose, carboxyl and
hydroxyl groups of pectin as well as to proteins in cell walls
of plants (Sarret et al. 2009). ­Zn2+ toxicity altered the meris-
tematic cells of T. aestivum roots through an increase in the
number of vacuoles, accumulation of granular substances
within the vacuoles and thickening of cell walls (Glinska
et al. 2016). These modifications indicate that the process of
formation of cell wall components accelerates under excess
Zn to increase the ability of apoplast to sequester the over-
loaded metal in the regions where they are less harmful.
Moreover, an increase in the number of vacuoles filled with
granular deposits suggests that excess Zn is trapped inside
the vacuoles as a strategy to prevent metal toxicity by plants.
Zn transport through phloem has also been proved
through research findings. Page et al. (2006) demonstrated
the xylem-mediated transfer of 65Zn to the aerial parts of
white lupin and its subsequent allocation through the phloem
from mature to young leaves. Crop plants show variations to
a large extent in the symplastic allocation of Zn. Erenoglu
et al. (2002) demonstrated that variations in the manifesta-
tion of Zn competency were not associated with realloca-
tion of Zn in wheat plants. Ozturk et al. (2006) however,
reported increased Zn concentrations in grains of wheat sub-
sequent to foliar Zn applications, implying high Zn mobility
in plants. Interaction between xylem and phloem transport
systems plays a significant role in regulating Zn transport
to maturing grains. In this context, Zn transfer from stem to
the vascular bundle of rice grains via xylem was believed
to be uninterrupted throughout grain filling (Krishnan and
Dayanandan 2003). Nevertheless, control of Zn packaging
into rice grains differs from its loading to other plant parts
(Jiang et al. 2008). Furthermore, Zn contents in grains are
directly correlated with concentrations of Zn supplemented
to plants. Most importantly, knowledge of Zn mobility in
phloem is crucial not only for the management of Zn nutri-
tion but also for assessing manifestation of potential Zn tox-
icity symptoms in different plant species.
Molecular insights into Zn homeostasis in plants
Plants normalize Zn levels to deal with indispensable
Zn requirements and at the same time shield themselves
against its toxicity via synchronization of particular trans-
porters connected with Zn import, export and intracellular
compartmentalization (Stephens et al. 2011). Plants have
various types of membrane Zn transporters: ZRT-1/IRT-1
like protein (ZIP) (recognized as zinc-ion permease), metal
tolerance protein (MTP), zinc-induced facilitator (ZIF)
genes [belonging to Major Facilitator Superfamily (MFS)
transporters], heavy metal ATP-ase (HMA) (belonging to
­P1B subgroup of P-type ATPase family), yellow stripe like
Planta (2021) 253:129
(YSL) [Oligopeptide Transporter (OPT) superfamily] and
vacuolar iron transporters (VIT) (Fig. 2a) (Kramer et al.
2007; Neeraja et al. 2018).
ZIP (zinc‑ion permease) transporters
ZIP associates take up Zn via pH gradient caused by
­H+ATPase of the cell membrane. The expression of a num-
ber of ZIP family members is regulated by transcription
factors of the basic-region leucine zipper (bZIP) family
(Lira-Morales et al. 2019), mostly bZIP19 and bZIP23 from
Arabidopsis thaliana under Zn deficiency (Assunção et al.
2010). Only a very little research is done on the characteriza-
tion of Zn-responsive and ZIP-related TFs in crops and most
of the reports available are confined to Arabidopsis under
Fig. 2  a Various transporters are involved in Zn uptake, transport and
sequestration in plants exposed to high Zn. Zn is actively taken up
by root cell membrane transporters as a free cation by ZIP19, ZIP23,
IRT1, IRT3 and/or by means of ZNT2 and ZNT5. Transport of free
Zn into the xylem is believed to occur via HMA2, HMA4, YSL and
FRD3. Xylem–phloem Zn allocation occurs with the assistance of
YSL transporters. The YSLs also translocate Zn to the leaf and seed
tissues (YSL 6, 9, 12) with the assistance of NA-Zn complexes. Zn
can also enter inside leaf cells as Zn-free form via ZIP4 and ZIP6. b
After transport inside the leaf symplast, Zn may be stored in certain
organelles and vacuoles. Zn transport into mitochondria has not been
Page 17 of 28 129
Zn-deficit conditions. We are not aware of any transcrip-
tion factor regulating ZIPs in crop plants under high Zn lev-
els. Moreover, although ZIP family transporter genes have
been identified in many plants, few reports concerning their
functional characterization under Zn excess are available in
the literature compared to those under Zn deficiency. Stud-
ies on metal hyperaccumulating dicot species, for instance,
Noccaea caerulescens (formerly known as Thlaspi caerule-
scens) and A. halleri, showed high expression of NcZNT1,
AtZIP4 and AtIRT3, thus playing a vital role in increased
accumulation of Zn (Hanikenne et al. 2008; Lin et al. 2009;
Milner et al. 2012). Van de Mortel et al. (2006) isolated
NcZNT1, a Zn transporter across the endodermal cell in
N. caerulescens. Overexpression of NcZNT1 in A. thali-
ana enhanced the plant metal tolerance with a simultaneous
characterized. Zn influx to the chloroplast is unknown, but its efflux is
mediated by HMA1. HMA3, HMA4, MTP1, MTP8, MTP11, MHX,
VIT1, VIT2, NRAMP3 and NRAMP4 transporters mediate the influx
of Zn across the tonoplast into the vacuole. Furthermore, MTs have
been implicated in Zn homeostasis, transport and sequestration in
plants. (FRD ferric reductase defective 3; HMA heavy metal ATP-
ase; IRT Fe-regulated transporter; MHX metal/proton exchanger; MT
metallothioniens; MTP metal tolerance protein; NA nicotianamine;
NRAMP natural resistance-associated macrophage protein; VIT Vacu-
olar Iron Transporters; YSL yellow strip like; ZIP zinc-ion permease;
ZNT zinc transporter)
13
129 Page 18 of 28
increase in accumulation of Zn relative to wild-type plants
(Lin et al. 2016). Moreover, NcZNT 2 and 5 (orthologs of
A. thaliana IRT3 and ZIP5, respectively) are engaged in the
transportation of Zn in the roots (Van de Mortel et al. 2006;
Plaza et al. 2007; Fasani 2012). Furthermore, IRT3 was dis-
covered in the root plasma membrane of A. halleri exposed
to excess Zn (Talke et al. 2006; Lin et al. 2016; Caldelas and
Weiss 2017). High expression of IRT3 was detected in the
roots of A. halleri and N. caerulescens (Talke et al. 2006;
Moreira et al. 2018). The study conducted by Shanmugan
et al. (2013) revealed the localization of IRT-1 in the plasma
membrane of A. halleri and its involvement in Zn transport.
Lin et al. (2016) also detected the location of this transporter
in the plasma membrane, which validated its function in the
passage of Zn ions into the cytoplasm. In the presence of
Zn, ZIP19 and ZIP23 expression was considerably induced
in roots of A. halleri and N. caerulescens (Lin et al. 2016).
ZIP6 elevates Zn uptake as its expression was predominant
in the shoots of A. halleri (Becher et al. 2004; Gupta et al.
2016), suggesting its contribution in increasing noxious Zn
concentrations. Recently, Spielmann et al. (2020) concluded
that ZIP6 is a duplicated gene (encoding a Zn and Cd trans-
porter) expressed primarily in the vascular tissues of A. hal-
leri shoots (AhZIP6-1) and roots (AhZIP6-2). Yang et al.
(2018) discovered SaZIP4 gene as an essential Zn uptake
transporter involved in Zn hyperaccumulation in roots and
shoots of Sedum alfredii. NtZIP11 has been recognized as
the first tobacco gene playing an important role in Zn accu-
mulation in mature leaves of tobacco plants under surplus
Zn concentrations (Kozak et al. 2019). Recently, Weremczuk
et al (2020) provided evidence highlighting NtZIP11 and
NtZIP1-like as Zn uptake transporters responsible for Zn
loading into “Zn-accumulating mesophyll cells” of tobacco
leaves.
In lower and/or above ground portions of A. thaliana,
the ZIP lineage includes 15 associates, ZIP1 to ZIP12 and
IRT-1 to IRT3 (Lin and Aarts 2012), which are mostly
augmented in Zn-deficient conditions, with three being
recently described: IAA-alanine resistance protein 1-
IAR1, Zinc transporter 29-ZTP29 and PutZnT. Amongst
these, ZIP4 and IRT3 are believed to be involved in trans-
location of Zn from root-to-shoot. A subcellular localiza-
tion analysis revealed ZIP4 to be located in the plasma
membrane and involved in Zn uptake in the shoots as well
as in the roots. Previously, it has been shown that overex-
pression of AtIRT3 enhanced the accumulation of Zn in A.
thaliana shoots, indicating its possible involvement in the
loading of Zn for long-distance transport (Lin et al. 2009).
Barberon et al. (2014) explained that non-Fe substrates
of IRT-1 (i.e., Zn, Mn, and Co) control its localization
between the outer polar domain of the plasma membrane
and early endosomes/trans-Golgi network compartments
(EE/TGN) in root epidermal cells of A. thaliana and
13
Planta (2021) 253:129
further confirmed relation between the dynamics and the
lateral polarity of IRT-1 and transport of its substrates.
ZIP transporters show extremely regulated expres-
sion pattern as established in the model plant A. thaliana
whose data on genome sequences and knockout mutants
facilitated characterization and classification of ZIPs in
other species. For instance, nine ZmZIPs (ZmZIP 1–8
and ZmIRT1) confined to the endoplasmic reticulum and
plasma membrane in maize (Li et al. 2013b), 14 TaZIP
genes in bread wheat (Triticum aestivum) (Evens et al.
2017), 13 members of ZIP gene family PtIRT1, PtZIP1-
PtZIP3 and PtZIP5-PtZIP14 in Poncirus trifoliata (Fu
et al. 2017) and seven SiZIPs from Setaria italic (Ala-
garasan et al. 2017) have been identified and character-
ized, majority of them being induced under Zn deficiency.
ZmZIP7 encodes a Zn and Fe transporter and ectopic over-
expression of ZmZIP7 in Arabidopsis stimulated Fe and
Zn uptake (Li et al. 2016). The expression of ZmZIP4,
ZmZIP5, ZmZIP7, and ZmZIP8 reduced in shoots whereas
ZmZIP3 was down-regulated in maize roots under high
Zn supply (Li et al. 2013b). Moreover, in response to
Zn excess, accumulation of ZmIRT1 increased in shoots
while it was suppressed in maize roots (Li et al. 2013b).
Majority of the available evidence for Zn transport by ZIPs
has been acquired from the overexpression studies, e.g.
OsZIP7 (Ricachenevsky et al. 2018) and HvZIP7 (Tiong
et al. 2014). 16 members of the ZIP family have been iden-
tified in rice (Tiong et al. 2015) but only a few have been
examined to determine their biological roles, particularly
under surplus Zn conditions. Earlier OsZIP1 was believed
to be responsible for Zn uptake in rice, but recent evidence
suggests that OsZIP1 is a metal-detoxifying transporter
located in both endoplasmic reticulum and plasma mem-
brane that checks excessive accumulation of Zn in roots,
rather than Zn uptake (Liu et al. 2019). Tan et al. (2020)
described two duplicated genes, OsZIP5 and OsZIP9,
which function redundantly in Zn uptake but have diverged
in terms of expression regulation. HvZIP7 is a close
homolog of NcZNT1, AtZIP4 and AtIRT3 from dicot spe-
cies whose overexpression in barley enhanced the root Zn
uptake and/or root-to-shoot translocation at more or less
high concentrations of Zn (Tiong et al. 2014), signifying
it to be a low-affinity Zn transporter. Zn toxicity resulted
in enhanced MsZIP2 expression which is believed to be a
detoxification mechanism involving storage of excess Zn
in xylem parenchyma cells (Cardini et al. 2021). Down-
regulation of MsZIP3 under high foliar Zn supplementa-
tion was a detoxification mechanism to reduce Zn uptake
by Medicago sativa shoot cells (Cardini et al. 2021) which
is in accordance with the findings of Sasaki et al. (2015)
regarding involvement of OsZIP3 in unloading Zn from
the vascular tissues under excess Zn supply in rice. These
reports suggest that although, there is confirmation for the
Planta (2021) 253:129
contribution of ZIPs towards Zn homeostasis under high
Zn levels, lack of functional data makes it complicated to
designate precise physiological roles.
MTP (metal tolerance protein) transporters
Overloaded Zn is transported from cytoplasm to vacuoles by
transporters belonging to metal protein (MTP1 and MTP3)
family which are involved in Zn sequestration and detoxifica-
tion (Martinoia 2018). MTPs confined to vacoular membrane
operate as ­Zn2+/H+ exchanger and are related to CDF (cation
diffusion facilitator) family. ZAT1 was the foremost MTP gene
categorized in A. thaliana which was later renamed as MTP1.
A. thaliana plants altered with AtMTP1 showed improved Zn
tolerance with increasing Zn levels in roots, whereas plants
without AtMTP1 exhibited hypersensitivity to Zn in response
to decreased Zn content in stems and leaves (Desbrosses-Fonu-
rage et al. 2005). N. goesingense has three allelic variations
of TgMTP1 which accumulate at elevated levels in vacuolar
membranes of shoots and is believed to take part in both Zn
tolerance and enhanced uptake (Gustin et al. 2009). A. halleri
has five gene replicas of MTP1 whose transcript amounts of
MTP1-A1, MTP1-A2 along with MTP1-B are tremendously
high at normal and toxic Zn levels (Shahzad et al. 2010). Up-
regulation of MTP8 and MTP11 by excess Zn has also been
proved in the shoots of N. caerulescens and A. halleri (Hani-
kenne and Nouet 2011). Expression of AtMTP1 is found to be
high in root vascular tissue and leaves of juvenile A. thaliana
seedlings, while AtMTP3 activity is unnoticeable in aerial
parts (Desbrosses-Fonrouge et al. 2005; Arrivault et al. 2006).
On the other hand, expression of MTP3 was extremely low in
a natural state but its transcripts mount to elevated amounts
in epidermal as well as cortical cells of root hair region of
A. thaliana seedlings exposed to surplus Zn (Arrivault et al.
2006). Moreover, both AtMTP1 and AtMTP3 exhibit con-
trasting results on Zn accumulation. AtMTP3 decreases shoot
Zn contents, whereas AtMTP1 enhances Zn levels in roots
(Desbrosses-Fonrouge et al. 2005; Arrivault et al. 2006). The
major function of AtMTP3 seems to be the exclusion of Zn
from root-to-shoot transport conduit under elevated Zn levels
in roots (Arrivault et al. 2006). On the contrary, TgMTP1 may
operate to sequester Zn in dividing and enlarging tissues or
it can accumulate Zn in specific aerial plant tissues (Gustin
et al. 2009). This reflects the differential expression pattern of
MTP1 in roots and shoots of non-hyperaccumulator (A. thali-
ana) and hyperaccumulator (T. goesingense) plants.
ZIF (zinc‑induced facilitator) genes (Major Facilitator
Superfamily)
ZIF1 and two additional Zn-induced facilitator-like genes,
namely AtZIFL1 and AtZIFL2, constitute a different fam-
ily (MFS) of Arabidopsis membrane proteins engaged in
Page 19 of 28 129
controlling Zn homeostasis (Haydon and Cobbett 2007).
ZIF1 is believed to be engaged in transferring NA from
root cytoplasm into the vacuole, thereby assisting in the
formation of NA–Zn complexes inside vacuoles (Haydon
et al. 2012). The ZIF1 gene in A. thaliana was up-regulated
under excess Zn and plants had a tendency to store Zn in
root vacuoles (Haydon and Cobbett 2007). Similarly, ZIF2
also mediates root vacuolar sequestration of Zn and prevents
its translocation to the shoot, thereby contributing towards
Zn tolerance in A. thaliana (Remy et al. 2014). In Arabi-
dopsis, ZIFL1 displays H ­ +-coupled ­K+ transport activity
whereas ZIFL2 functions in ­Cs+ and ­K+ homeostasis (Remy
et al 2013; 2015). It is worth mentioning that AtZIF2 is
not similar to AtZIFL2 as AtZIF2 is considered to be more
distantly related to the other Arabidopsis ZIFs. ZIFL genes
are considered to be involved in carrying out the task of Zn
allocation to the seeds. Moreover, a higher number of ZIFL
family paralogs have been reported in monocot species than
dicots (Ricachenevsky et al. 2011). Through quantitative
trait loci (QTLs) studies in A. thaliana, ZIF1 and ZIFL1 are
considered to be responsible for high Zn concentrations in
seeds (Waters and Grusak 2008). Likewise, ZIF1-like gene
was found to express in the seed aleurone of barley whose
expression was stimulated after foliar Zn supply (Tauris
et al. 2009). Amongst eight ZIFL genes described in wheat
by Pearce et al. (2014), TaZIFL1 was down-regulated dur-
ing senescence, whereas TaZIFL2-like1 and TaZIFL7 were
significantly up-regulated. Recently, Sharma et al. (2019)
identified fifteen TaZIFL-like genes in wheat and proposed
their tissue specific expression under excess Zn. In rice,
several of the thirteen ZIFL genes are expressed in the flag
leaves during senescence (Ricachenevsky et al. 2011). This
indicates that in monocots, some ZIFL genes are involved
in nutrient remobilization during senescence. Moreover, rice
ZIFL genes also encode for the transporter of mugineic acid
(TOM) proteins which release PS, thereby increasing Fe/
Zn uptake (Nozoye et al. 2015). Characterization of ZIFL
genes in rice led to the identification of three TOM proteins-
OsTOM1, OsTOM2 and OsTOM3 (Nozoye et al. 2015).
Similarly, wheat TOM transporters have also been identi-
fied through qRT-PCR analysis: TaZIFL4.1, TaZIFL4.2,
TaZIFL5, TaZIFL7.1 and TaZIFL7.2 (Sharma et al. 2019).
However, further studies are required to better characterize
the function of ZIFL transporters in plants.
HMA (heavy metal ATP‑ase) transporters
Root-to-shoot translocation of Zn requires proteins belong-
ing to the heavy metal ATPase “HMA” family. Two mem-
bers of HMA family, HMA2 as well as HMA4 are expressed
in vascular tissues of A. halleri and play a key role in con-
tributing to Zn hyperaccumulation and hypertolerance
(Hanikenne et al. 2008; Frérot et al. 2018). Both of them are
13
129 Page 20 of 28
located in the plasma membrane of the root pericycle cells
and facilitate ­Zn2+ efflux from these cells and subsequent
loading into the root xylem (Moreira et al. 2018). HMA2 is
considered to be a Z­ n2+-ATPase which may also be triggered
by other metals like ­Cd2+ and to a lesser degree by other
divalent cations whereas HMA4 transports Zn, Cd and Pb.
In hyperaccumulator plants, the transcriptional level analy-
sis revealed a high expression of these genes. For instance,
expression of HMA4 was 4–10 fold higher in roots and
30-fold higher in shoots of A. halleri compared to A. thali-
ana (Talke et al. 2006). The enhanced expression of HMA4
was due to tandem triplication and cis-regulatory altera-
tions that stimulated the promoters of all three HMA4 cop-
ies (Hanikenne et al. 2008). These three HMA4 homologs,
i.e. HMA4-1, HMA4-2 alongwith HMA4-3 are accountable
for elevated transfer of Zn from roots to shoots in A. hal-
leri and are vital features for Zn tolerance (Hanikenne et al.
2008). Furthermore, by means of overexpressing HMA4,
Hanikenne et al. (2008) suggested the function of this gene
in the allocation of Zn to different organs of A. thaliana.
Similarly, Medicago sativa plants subjected to Zn toxicity
exhibited enhanced HMA4 expression in roots and shoots,
clearly indicating augmentation in the transport of Zn in the
xylem (Cardini et al. 2021). AtHMA1 has been categorized
in the chloroplast membrane of A. thaliana where it mobi-
lizes Zn from the stroma of chloroplast into the cytosol (Kim
et al. 2009) under conditions of high levels of Zn, thereby
participating in detoxification of Zn by decreasing its content
in the plastid (Fig. 2b). Nevertheless, the role of HMA1 in
the transportation of Zn in hyperaccumulator species has
not been confirmed yet. Thus, we put forward the potential
involvement of HMA1 in restricting the accumulation of Zn
in the chloroplasts of hyperaccumulator species.
Noteworthy, HMA3 present in vacuolar membrane,
transports excess Zn from the cytoplasm into vacuoles
(Fig. 2b) which seems to be a mechanism for avoiding tox-
icity (Jain et al. 2013). Recently, HMA3 and HMA4 were
examined at the tissue and cellular level concerning their
role in Zn/Cd sequestration in shoots of A. halleri and N.
caerulescens (Mishra et al. 2017). Considerable up-regula-
tion of HMA3 and HMA4 was observed in the mesophyll of
A. halleri, whereas in N. caerulescens their highest expres-
sion was in the bundle sheath of the vein which could be
described by the ultimate storage sites; the mesophyll for
A. halleri and epidermis for N. caerulescens (Mishra et al.
2017). Limited reports indicate the role of OsHMA3 and
AtHMA3 in Zn homeostasis. In a recently published study,
Cai et al. (2019) compared rice natural accessions having
functional OsHMA3 with those containing loss-of-func-
tional alleles, and witnessed that former were more toler-
ant to surplus Zn and accumulated more Zn in roots, while
maintaining similar Zn concentrations in shoots. This was
because of up-regulation of Zn uptake genes (ZIPs) in roots
13
Planta (2021) 253:129
of rice accessions with higher expression of HMA3. Pita-
Barbosa et al. (2019) also dealt with the same hypothesis
implicating HMA3 in Zn transport but in different species,
A. thaliana and investigated the influence of AtHMA3 on
the Zn homeostasis transcriptional network. They employed
Col-0 accession (having a natural non-functional AtHMA3
allele), altered it with a functional allele from the CSHL-5
A. thaliana accession, produced several independent Col-
0::HMA3CSHL-5 lines and compared them with wild-type
Col-0 plants. The authors observed significant positive
correlations between HMA3 and ZIP3, ZIP4, MTP1 and
bZIP19 expression and suggested that functional modifica-
tions in AtHMA3 alter the expression of other genes of Zn
homeostasis intricate system in A. thaliana. They further
concluded that A. thaliana plants transcriptionally protect
alterations in the compartmentalization of Zn in root vacu-
oles due to changes in AtHMA3 function, to regulate con-
stant Zn concentration in leaves. Thus, based on these two
reports, we strongly support the inference that enhanced Zn
compartmentalization into vacuoles by HMA3 stimulates the
expression of certain Zn-deficiency-related genes to main-
tain cytosolic Zn homeostasis.
YSL (yellow stripe like), VIT (vacuolar iron
transporter) and other transporters
YSL transporters are involved in xylem loading and unload-
ing of Zn (Fig. 2a). In endospermic aleurone layer as well
as embryo of barley grains, uptake of Z ­ n2+ takes place by
members of YSL family transporters, viz. YSL 6, 9, and 12
with the assistance of Zn complexes (NA-Zn) (Tauris et al.
2009; Caldelas and Weiss 2017). It is pertinent to mention
here that, even when Zn-dependent transcriptional regula-
tion is absent, Zn transport via YSL transporters rely on
the concentration of Zn-NA complexes that might enhance
when NA levels increase under Zn excess, Zn deficiency or
Fe deficiency (Haydon et al. 2012). In A. thaliana, YSL1
and YSL3 are involved in the remobilization of Zn from age-
ing leaves as well as in the development of pollen (Waters
et al., 2006). Analogous to YSL, FRD3 (ferric reductase
defective 3), a MATE (multidrug and toxin efflux) trans-
porter, is a crucial regulator of excess Zn and is necessary
for Fe/Zn translocation by xylem (Pineau et al. 2012). This
protein can be of key significance in the allocation of met-
als between the cells. The VIT family mediates transport
of heavy metals (­ Fe2+, ­Mn2+ and Z ­ n2+) across tonoplast
and hence assists in their vacuolar sequestration (Fig. 2b).
Two VIT1 transporters [The Arabidopsis orthologue of the
CCC1 ­(Ca2+-sensitive Cross-Complementer1) gene] have
been identified in barley. VIT1_1 is strongly expressed in
leaf transfer cells of barley, where it helps in mobilization
of ­Zn2+ into vacuoles whereas VIT1_2 is highly expressed in
the aleurone cells (Tauris et al. 2009). Two members of VITs
Planta (2021) 253:129
family found in rice, i.e. OsVIT1 and OsVIT2 play a role in
decreasing Zn and Fe in flag leaves through storage inside
vacuoles, thereby regulating the allocation of metals from
flag leaves to emerging grains (Zhang et al. 2012). AtMHX
(metal/proton exchanger) is an additional transporter situated
in the vacuolar membrane of A. thaliana and engaged in Zn
homeostasis (David-Assael et al. 2006). A novel transporter,
plant cadmium resistance 2 (PCR2) operates as a Zn efflux
transporter at the root plasma membrane and contributes
to Zn translocation and detoxification in A. thaliana (Song
et al. 2010). Nevertheless, its role in Zn hyperaccumulation
is not known. Recently, Wang et al. (2021) identified and
characterized a Golgi-localized transporter, ZINC NUTRI-
ENT ESSENTIAL1 (AtZNE1, At3g08650) in A. thaliana
and suggested its role in mediating sequestration of excess
­Zn2+ into the Golgi and in plant adaptation to Zn toxicity
or Fe deficiency. Transporters, such as NRAMP1 (natu-
ral resistance-associated macrophage protein) NRAMP3,
NRAMP4, and NRAMP5, are believed to facilitate Zn com-
partmentalization in the vacuole of leaf cells (Fig. 2b). High
expressions of NRAMP1 and NRAMP5 were found in the
shoots of N. caerulescens whereas NRAMP3 was expressed
in the leaves of A. halleri (Weber et al. 2004; Krämer et al.
2007). NcNRAMP4 played a key role in Zn hypertolerance
(Oomen et al. 2009), however, the exact function of NcN-
RAMP3 is yet to be determined. Distinctively expressed
transporters, namely NRAMP, VIT, POT (proton-coupled
peptide transporter), PHO and MATE, have been established
for elevating Zn levels in rice (Neeraja et al. 2018). Passive
uptake of ­Zn2+ can take place by means of a depolarization-
activated non-selective cation channel (DA-NSCC) found
in T. caerulescens (Piñeros and Kochian 2003) as well as
voltage-independent NSCCs (VI-NSCCs) (Demidchik and
Maathuis 2007).
In general, these studies underline the co-ordinated role
of diverse transporter families involved in controlling Zn
homeostasis in plants. Strict maintenance of intracellu-
lar Zn concentrations is necessary for a functional cel-
lular metabolism, which requires finely tuned regulation
of these Zn transporters. While maintaining Zn homeo-
stasis, some plant transporters transport Z ­ n2+ ions, while
others transport a Zn-ligand complex. It seems that those
membrane transporters which transport only ligands,
could make an additional contribution to Zn homeostasis,
whereby ligands may function following their transport by
binding to ­Zn2+ ions on the other side of membrane. Most
of the studies on molecular aspects of Zn homeostasis have
been carried out in either hyperaccumulator or model plant
species, which are still in early stages and therefore can-
not be generalized for all crop plants. Moreover, at times,
the concept of hyperaccumulation is overly-stated, mixing
high tolerant species with hyperaccumulators, which could
Page 21 of 28 129
be due to the difference in the nature of experiments or
bioaccumulation factors or uptake rates. Nevertheless, it
is essential to ascertain a detailed picture of the pathway
of Zn all the way through different organs of the plant.
Moreover, detailed histological analyses are necessary
to determine the link between various Zn transporters/
proteins to establish their redundancy (if any) in a par-
ticular tissue while expression in other tissues. Therefore,
complete understanding of the molecular aspects of Zn
homeostatsis in plants can pave the way to several promis-
ing molecular applications with a holistic view to improve
crop management and food quality.
Conclusion and future prospects
Zn is a critical nutrient required by plants for carrying out
numerous metabolic functions. However, high Zn levels in
plants affect various physiological and biochemical param-
eters. Information regarding bioavailable forms of Zn and
soil–Zn-binding capability is crucial in considering the
potential risk of its transfer in soil–plant–human chain.
Various transporters take part in Zn homeostasis and trans-
location in plants. No doubt, in this omic era, proteomic
and genomic approaches have facilitated our understand-
ing of the functions of Zn transporters, however, despite
this, significant knowledge gaps still exist. Expression of
membrane proteins may contribute to understand the inter-
face between transporter proteins and other components
engaged in cellular Zn trafficking which may further assist
in understanding the plant mechanisms of Zn transport in
a better manner. Furthermore, the revelation of secondary
genes and messengers in regulating Zn uptake, accumula-
tion and translocation might represent a promising way
to acquire additional deep insights into various processes
involved in the absorption of Zn by plants. Lastly, com-
plete knowledge of Zn signalling would be of immense
scientific relevance and important for accomplishing sus-
tainable agricultural growth on a global basis.
Author contribution statement Both the authors have con-
tributed equally to the preparation of this review article.
Acknowledgements This work was supported by Department of Bio-
technology, Government of India, New Delhi, India [Grant Number
42-945/2013(SR)].
Declarations
Conflict of interest The authors declare that they have no conflict of
interest.
13
129 Page 22 of 28 Planta (2021) 253:129

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