Improving the zinc-use efficiency in plants: A review

Abstract

Zinc (Zn) deficiency causes large scale yield losses in major crops such as rice, wheat and maize. Zn is an important micronutrient and the only metal ion acting as a co-factor for all six classes of enzymes in plants and other organisms. In this review, we have identified the phenotypic and biochemical changes associated with Zn deficiency in plants. We also present the current understanding on uptake and translocation of Zn and provide details on various approaches made to improve Zn-use efficiency (ZUE) in plants. The details of membrane transporters involved in acquisition of Zn from soil and its translocation in plants have been explained. Molecular markers and transgenic tools have been used for improving ZUE in various plants. Over expression of various Zn transporters through transgenic modification improved the Zn uptake in deficient Zn condition. Several quantitative trait loci (QTL) related to Zn content in rice grain have been identified. Further studies based on molecular markers and functional genomics will help improve ZUE and yield in crop plants grown in low Zn soil. Key findings: In this review, we present the Zn-plant relationship, mechanism of Zn transport in plants and efforts made so far to improve Zn-use efficiency in plants using various complementary approaches.

Full text

Ajeesh Krishna et al. (2017)
211
SABRAO Journal
of Breeding and Genetics
49 (3) 211-230, 2017
IMPROVING THE ZINC-USE EFFICIENCY IN PLANTS: A REVIEW
T.P. AJEESH KRISHNA1, S. ANTONY CEASAR1, 2, T. MAHARAJAN1,
M. RAMAKRISHNAN1, V. DURAIPANDIYAN1, 3, N.A. AL-DHABI 3 and
S. IGNACIMUTHU1*
1Division of Plant Molecular Biology, Entomology Research Institute, Loyola College, Chennai, 600 034, India
2Centre for Plant Sciences and School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds,
Leeds, LS2 9JT, United Kingdom
3Addiriyah Chair for Environmental Studies, Department of Botany and Microbiology, College of Science, King Saud
University, P.O. Box.2455, Riyadh-11451, Kingdom of Saudi Arabia
*Corresponding author’s email: eriloyola@hotmail.com
Email addresses of co-authors: ajeeshkrishnatp@gmail.com, antony_sm2003@yahoo.co.in, susirajan143@gmail.com,
ramkyeri@gmail.com, avpandiyan@gmail.com, naldhabi@ksu.edu.sa
SUMMARY
Zinc (Zn) deficiency causes large scale yield losses in major crops such as rice, wheat and maize. Zn is an important
micronutrient and the only metal ion acting as a co-factor for all six classes of enzymes in plants and other
organisms. In this review, we have identified the phenotypic and biochemical changes associated with Zn deficiency
in plants. We also present the current understanding on uptake and translocation of Zn and provide details on various
approaches made to improve Zn-use efficiency (ZUE) in plants. The details of membrane transporters involved in
acquisition of Zn from soil and its translocation in plants have been explained. Molecular markers and transgenic
tools have been used for improving ZUE in various plants. Over expression of various Zn transporters through
transgenic modification improved the Zn uptake in deficient Zn condition. Several quantitative trait loci (QTL)
related to Zn content in rice grain have been identified. Further studies based on molecular markers and functional
genomics will help improve ZUE and yield in crop plants grown in low Zn soil.
Key words: Zinc, zinc deficiency, zinc transporters, zinc-use efficiency, molecular markers.
Key findings: In this review, we present the Zn-plant relationship, mechanism of Zn transport in plants
and efforts made so far to improve Zn-use efficiency in plants using various complementary approaches.
Manuscript received: March 19, 2017; Decision on manuscript: June 5, 2017; Manuscript accepted: July 27, 2017.
© Society for the Advancement of Breeding Research in Asia and Oceania (SABRAO) 2017
Communicating Editor: Bertrand Collard
INTRODUCTION
Plants are irreplaceable resources of foods that
provide energy and nutrients to both humans and
animals. Plants need various nutrients in
different amounts for their development and
reproduction. In agriculture, nutrients are
essential for growth and yield of crop plants
(Sadeghzadeh, 2013). These nutrients play major
roles in physiological and metabolic activities of
plants. Unavailability of one or more of these
elements prevents plants from completing their
life cycle (Ramesh et al., 2004).
Zinc (Zn) interacts with both the macro
and other micro-nutrients that are involved in
many physiological functions of plants (Brar and
Sekhon, 1976; Slamet-Loedin et al., 2015). Zn is
an important micronutrient for cellular
REVIEW ARTICLE

SABRAO J. Breed. Genet. 49 (3) 211-230
212
metabolism and biological activities such as
anti-oxidative defense, carbohydrate
metabolism, protein synthesis and auxin
metabolism (Broadley et al., 2007). Zn also
plays a crucial role in enzyme activity; it is the
only element necessary to activate all six classes
of enzymes (oxidoreductases, transferases,
hydrolases, lyases, isomerases and ligases)
(Sadeghzadeh, 2013). More importantly Zn is an
essential co-factor for approximately 300
individual enzymes in plants (Rathore and
Mohit, 2013).
Plants require low concentration of Zn
(0.5 to 2 µM) from the soil (based on our
unpublished study). Both lower and higher
quantities of Zn cause adverse effects to plants.
The Food and Agriculture Organization (FAO)
reported that 50% of the world’s agricultural soil
is low in Zn (Das and Green, 2013). Zn
deficiency occurs in many crop species such as
Oryza sativa (rice), Sorghum bicolor (sorghum),
Zea mays (maize), Hordeum vulgare (barley)
and Arachis hypogaea (peanut). Low availability
of Zn also significantly affected the nutritional
quality and yield in Triticum aestivum (wheat)
(Cakmak et al., 1996). Rice, sorghum and maize
are categorized as highly sensitive to Zn
deficiency, and barley, wheat, Avena sativa (oat)
and Secale cereale (rye) as less sensitive (Viets
Jr et al., 1954; Clark et al., 1990).
Zn-use efficiency (ZUE) has been
defined as the efficient acquisition of Zn and
utilization or (re)-translocation within a plant in
Zn deficient condition (Graham and Rengel,
1993; Erengolu et al., 2000). Certain genotypes
are able to grow and yield well under Zn
deficiency, which has been termed Zn efficiency
(Graham and Rengel, 1993). This may be due to
some genetic variations present in plants. Only
limited information is available on the genetic
control of Zn efficiency mechanisms, its
molecular backgrounds and genes responsible
for Zn efficiency (Sadeghzadeh, 2013). The
physiological mechanisms involved in Zn
efficiency have been documented by many
researchers (Hacisalihoglu and Kochian 2003).
In this review, we present the details on Zn
deficiency in crop plants, phenotypic and
genotypic differences, biochemical changes,
agronomic aspects of managing low Zn in soil.
Apart from this, we also cover Zn transport and
efforts to improve ZUE in various plants.
Zn status in relation to water status
In soil, Zn solubility and availability to plants
varies between water logged soil and dry land
soil (Gao et al., 2012). Zn deficiency symptoms
are more noticeable in plants grown under dry
land soils as compared to flooded soils (Huaqi et
al., 2002). It is evident that the Zn
bioavailability was lower in dry land soils as
compared to flooded rice cultivation systems, as
indicated by decreased Zn concentration not
only in plant tissue but also in Zn uptake and Zn
harvest index (Gao et al., 2006). The
bioavailability of Zn in soil is controlled by both
absorption-desorption reactions and solubility
relation (Gao et al., 2012). And the soil solution
and solid phase are mainly involved in the
absorption-desorption and dissolution-
precipitation reactions of Zn in soil. The factors
such as organic matter, soil texture, soil pH,
redox potential, pedogenic oxide and sulfur
contents affect the amount of Zn dissolved in
soil (Alloway 2009; Mandal et al., 2000).
Aerobic rice cultivation may increase soil pH,
leading to reduction in Zn availability than
anaerobic condition (Gao et al., 2006). The
status of Zn in relation to anaerobic (flooded
condition) and aerobic (dry land condition) soil
has been recorded by many researchers (Gao et
al., 2006; Gao et al., 2012). The reduction of the
soil moisture content significantly restricts the
transport of Zn towards the plant root (Yoshida,
1981).
Zn deficiency symptoms in crop plants
Hacisalihoglu et al. (2001) defined Zn
deficiency as the condition in which only
insufficient Zn is available for the optimal
growth that may cause dramatic reductions in
crop yield. The low organic matter, high level of
P, calcareous soils, low temperature, and
repeated application of N fertilizer can cause Zn
deficiency (Bogdanovic et al., 1999; Lindsay,
1972; Mousavi et al., 2012). Plants generally
show morphological changes in response to
nutrient deficiency. Details on various symptoms
associated with Zn deficiency in some plants are

Ajeesh Krishna et al. (2017)
213
listed in Table 1. Several studies have been
performed in crop plants to assess the
phenotypic responses to Zn deficiency (Hafeez
et al., 2013). Zn deficient plants show stunted
growth, delayed fruit maturity, chlorosis,
shortened internodes and petioles and
malformed leaves (Das and Green, 2013; Hafeez
et al., 2013). In rice, Zn deficiency caused
seedling mortality, stunted growth, leaf
bronzing, and delayed flowering (Widodo et al.,
2010). Similarly, wheat showed brown spots on
upper leaves under Zn deficiency; chlorosis was
seen on the midrib and base of the younger
leaves. Other symptoms in wheat included loss
of leaves, decreased tillering ability, inhibited
growth of shoot and root and more spikelet
sterility (Cakmak et al., 1997b). Zn deficiency
also affected proper formation of panicles in
some cereal crops (Alloway, 2004). We also
observed that Zn deficiency affected panicle
formation, grain setting and other parameters in
sorghum (Figure 1).
Phenotypic studies
Assessment of genotypes in deficient Zn for
phenotypic variation may be helpful for
choosing genotypes for further breeding works.
Several studies have been performed in major
crop plants such as rice and barley to assess their
phenotypic variation due to Zn stress (Genc et
al., 2007; Nanda and Wissuwa, 2016;
Sharifianpour et al., 2014). Changes in root
architecture have also played essential role in
capturing Zn during deficiency. The low
availability of Zn significantly affected the root
architecture (Fageria, 2004). Induction of longer
root types viz. nodal, primary, and lateral roots
with less induction of adventitious roots has
been found in barley in Zn deficiency (Genc et
al., 2007). Early formation of crown roots was
affected by Zn deficiency in a Zn-inefficient
genotype than in Zn-efficient genotype of rice
(Nanda and Wissuwa, 2016). In Arabidopsis, Zn
deficiency reduced the length of primary root
and increased the number and length of lateral
roots (Jain et al., 2013). The importance of root
traits associated with P and Zn uptake has been
highlighted (reviewed in Rose et al., 2013).
The genotypes with better ZUE are
believed to utilize Zn more efficiently. For
example, the Zn-efficient genotypes of rice have
the ability to translocate Zn from older to
actively growing younger tissue compared to
Zn-inefficient genotypes (Impa et al., 2013).
However, contradictorily, such relation between
Zn re-translocation and Zn efficiency does not
exist in bread and durum wheat genotypes
(Erenoglu et al., 2002). Wissuwa et al. (2008)
found that the grain Zn concentration was also
dependent on the genotype in rice. Zn-efficient
genotypes were characterized by higher Zn
uptake efficiency in low Zn soil which helps
gain higher biomass and yield (Wissuwa et al.,
2006). The grain yield efficiency index (GYEI)
was also used to sort the genotypes into Zn-
efficient and inefficient, in rice (Hafeez et al.,
2010). Kumar (2001) studied GYEI in ten
lowland rice genotypes for ZUE. These studies
are helpful to identify the best performing
genotype with efficient ZUE based on GYEI in
rice. Similar studies in other crop plants will
help find efficient genotypes from the
germplasm collection which can be grown in
low Zn. Efforts need to be initiated for such
studies in other crop species too.
Plant biomass was also used as a crucial
diagnostic tool for determining the ZUE in some
plants. Zn-inefficient rice genotypes produced
less biomass compared to Zn-efficient genotypes
(Hoffland et al., 2006; Gao et al., 2009; Widodo
et al., 2010). Similarly, Gao et al. (2005) grew
23 genotypes of rice in low Zn soil. Zn content
and dry matter of seed, root and shoot were
taken for the characterization of the genotypes.
Several genotypes of oilseed rape (Brassica
napus and B. juncea) (Grewal et al., 1997) and
Arabica coffee (coffee) (Pedrosa et al., 2013)
were screened to find the best performing
genotypes in Zn deficient condition based on
biomass production. Genotypic responses to Zn
deficiency were also demonstrated in field
growth experiments in rice. Two rice genotypes
(IR55179 and KP) were grown in Zn deficient
condition and IR55179 accumulated higher Zn
in grain than KP (Impa and Johnson-Beebout,
2012). These basic studies may be helpful to
choose the genotypes for use in further studies
like marker assisted selection and breeding to
produce new varieties with improved ZUE.

SABRAO J. Breed. Genet. 49 (3) 211-230
214
Table 1. Details of various diagnostic symptoms of Zn deficiency in important crop plans.
Name of the species
Zn deficiency symptoms
Reference
Hordeum vulgare
Leaves show uniform chlorosis and drying, appearance of white spots
on leaves, collapsed mid-leaf and decreased tip growth.
Singh et al.
(2005)
Singh et al.
(2005)
Triticum aestivum
Brown spots on upper leaves, midrib becomes chlorotic, mostly seen in
leaf base of younger leaves, loss of leaves and lower leaves have brown
blotches and streaks appear, decreased tillering, inhibited growth of
shoot and root, spikelet sterility.
Gossypium hirsutum
Chlorotic spots between the main veins of topmost leaves. Youngest
leaves became equally chlorotic except a dark green area around the
petiole. Shortened internodes, red spots developed on the leaf blade and
on parts of the veins.
Ghoneim and
Bussler (1980)
Carica papaya
Depression in growth, appearance of chromatic spots in the interveinal
areas. Flowers do not form in severe condition.
Samant (2009)
Samant (2009)
Samant (2009)
Brassica nigra
Younger leaves show deep purple colour which moves towards midrib.
Small circular spots of purple colour are also seen and with
advancement of time these spots become bleached. The plant becomes
stunted and flowering/maturing is delayed.
Arachis hypogaea
Reduced leaf size with light yellow colour. Rosette appearance of
internodes. Plants become stunted and kernel becomes shrivelled.
Oryza sativa
Leaves develop brown blotches and streaks that may fuse to cover older
leaves entirely, stunted growth and in severe cases may die, delay in
maturity and reduction in yield.
Neue and Lantin
(1994)
Avena sativa
The leaves become pale green.
Alloway (2004)
Zea mays
Yellow striping of leaves, formation of white bud, stunted due to
shortened internodes and the lower leaves show a reddish or yellowish
streak.
Alloway (2004)
Figure 1. Zn deficiency symptoms in sorghum (APK-1). The sorghum plant grown under Zn deficient
(0.00 µM) and Zn sufficient (1.00 µM) condition. A, B and C show characteristic features of sorghum
grown under Zn sufficient condition. D, E and F show characteristic features of sorghum grown under Zn
deficient condition. Under Zn deficient condition sorghum shows stunted growth especially reduction in
plant height, number of leaf, length and width (D), youngest leaves with light yellow colour (E), small-
sized flowers with poor grain filling, retarded development and maturation of seed (F) compared to Zn
sufficient condition (A, B and C).

Ajeesh Krishna et al. (2017)
215
Biochemical studies
Zn is an essential structural component of
enzymes like Cu/Zn superoxide dismutase
(SOD) and carbonic anhydrase (CA) (Singh et
al., 2005). The activities of these enzymes may
be used as indicators of Zn deficiency in plants.
Generally, lower levels of Zn decreased the
activities of these enzymes in many species
(Kabir et al., 2014). A Zn-efficient wheat
genotype showed decreased activity of CA
compared to a Zn-inefficient genotype in Zn
deficiency condition (Rengel, 1995). The
expression and activities of the Zn requiring
enzymes Cu/Zn SOD and CA were also
associated with Zn-efficient genotypes of wheat
(Hacisalihoglu et al., 2003). Expression levels of
Cu/Zn SOD were elevated in Zn-efficient
genotypes of wheat (Hacisalihoglu et al., 2003).
The activities of these enzymes were also
decreased in Vinga mungo (black gram) during
Zn deficiency (Pandey et al., 2002). Similar
responses were obtained in enzyme studies in
bread wheat, durum wheat and rye in Zn
deficiency (Cakmak et al., 1997a). Further
molecular studies confirmed that expression of
Cu/Zn SOD genes was induced in Zn-efficient
wheat genotypes compared to Zn-inefficient
genotypes in Zn deficiency (Hacisalihoglu and
Kochian, 2003).
Plant root exudates can help overcome
Zn deficiency by increasing the bioavailability
of Zn to plants. The genotypic difference in Zn
acquisition from the soil may be linked to
composition of root exudates released by each
genotype (Marschener, 1998). The low
molecular weight organic acids such as citrate,
malate, nitric oxide, oxalic acid, acetic acid and
amber acid are involved in the mobilization of
Zn under Zn deficiency (Li et al., 2012).
Similarly, citrate efflux also helps uptake higher
amount of Zn in low Zn, and the process is
genotype dependent in rice (Hoffland et al.,
2006). Studies in rice confirmed that release of
low molecular weight organic acid anion like
malate was increased by up to 64% in low Zn
supply compared to adequate Zn supply (Gao et
al., 2009). These studies provided evidence that
root exudates helped improve Zn uptake during
Zn deficiency. Identification of genotypes with
efficient release of organic acid anion may help
uptake Zn more efficiently in low Zn conditions.
Smilarly, phytosiderophores helped uptake Zn
more efficiently in low Zn conditions in barley
(Erenoglu et al., 2000). Crop plants like
sorghum and wheat significantly increased
phytosiderophore efflux in response to Zn
deficiency (Hopkins et al., 1998).
Mechanism of Zn uptake and translocation in
plants
Zn is absorbed from soil as Zn2+ and transported
through xylem to shoot (Clemens, 2001; Hart et
al., 1998). Zn is transported from soil through
the root plasma membrane. The rate of Zn
uptake depends on uptake efficiency of the root
system, Zn concentration at the root surface and
permeability of the cell membrane (Shukla et al.,
2014). Zn enters the plant from the soil through
membrane bound transporters (Hacisalihoglu
and Kochian, 2003). These transporters are
involved in absorption of Zn from the soil,
transport within the plant, xylem loading and
unloading, vacuolar sequestration and
remobilization from the vacuole. Many types of
Zn transporters have been identified and their
function has been characterized in plants (Figure
2; Table 2). These include Zn-regulated, iron-
regulated transporter-like protein (ZIP), plasma
membrane type ATPase (P-type ATPase), cation
diffusion facilitator (CDF), plant cadmium
resistance (PCR) and cation exchanger (CAX).
Apart from Zn, most of these transporters are
also involved in the transport of other cations
viz. manganese (Mn), iron (Fe), cadmium (Cd),
cobalt (Co) and copper (Cu). The details of some
of these transporters are discussed below.
ZIP
The ZIP family comprises more than a hundred
transporters found at every phylogenetic level
(Grotz and Guerinot, 2006). The ZIP
transporters have eight transmembrane (TM)
domains, with their amino and carboxyl ends
exposed to the external surface of the plasma
membrane (Guerinot, 2000). Besides Zn, ZIP
also transports various other metal ions (Mn, Fe,
Cd, Co, Cu and Ni) (Pedas and Husted, 2009).
The functions of ZIP transporters have been

SABRAO J. Breed. Genet. 49 (3) 211-230
216
Table 2. Details of various Zn transporters reported in plants.
Plant name
Transporter
family
Transporter name
Metal
transport
function
References
Medicago
truncatula
ZIP
MtZIP1, MtZIP5, and MtZIP6
Zn
Lopez-Millan et al. (2004)
Manihot
esculenta
ZIP
MeZIP
Zn
Bamrungsetthapong et al.
(2010)
Triticum
aestivum
ZIP
TaZIP1
Zn
Durmaz et al. (2011)
Hordeum
vulgare
ZIP
HvZIP7
Zn, Fe, Mn
and Cu.
Tiong et al. (2009)
CDF
HvMTP1
Zn and Co
Podar et al. (2012)
ZIP
HvIRT1, HvZIP5
Zn
Pedas and Husted (2009)
P-type
ATPase
HvHMA2
Zn and Cd
Mills et al. (2012)
ZIP
HvZIP7
Zn
Tiong et al. (2014)
Zea mays
ZIP
ZmZIP1, ZmZIP2, ZmZIP3, ZmZIP4,
ZmZIP5, ZmZIP6, ZmZIP7, ZmZIP8 and
ZmIRT1
Zn and Fe
Li et al. (2013)
Arabidopsis
ZIP
AtZIP1, AtZIP2, AtZIP3, AtZIP4,
AtZIP5, AtZIP6, AtZIP7, AtZIP8,
AtZIP9, AtZIP10, AtZIP11 and AtZIP12
Zn
Jain et al. (2013)
ZIP
AtIRT1 and AtIRT2
Zn and Fe
Henriques et al. (2002)
CDF
AtMTP1
Zn
Tanaka et al. (2013);
Kobe et al. (2004)
CAX
AtMHX1
Zn
Shaul et al. (1999)
ZIP
AtZIP1, AtZIP2, AtZIP3 and AtZIP4
Zn
Grotz et al. (1998)
CDF
AtZAT1
Zn
Bloß et al. (2002)
ZIP
AtIRT3
Zn and Fe
Lin et al. (2009)
PCR
AtPCR2
Zn
Song et al. (2010)
P-type
ATPase
AtHMA2 and AtHMA4
Zn
Hussain et al. (2004)
Oryza sativa
ZIP
OsZIP8
Zn
Yang et al. (2009)
P-type
ATPase
OsHMA3
Zn
Sasaki et al. (2014)
ZIP
OsZIP4
Zn
Ishimaru et al. (2005)
ZIP
OsZIP1 and OsZIP3
Zn
Ramesh et al. (2003)
ZIP
OsZIP1, OsZIP3 and OsZIP4.
Zn
Chen et al. (2008)
ZIP
OsZIP8
Zn
Lee et al. (2010)
Populus spp.
CDF
PtdMTP1
Zn
Blaudez et al. (2003)
Vitis vinifera
ZIP
VvZIP3
Zn
Gainza-Cortés et al.,
(2012)
Glycine max
ZIP
GmZIP1
Zn
Moreau et al. (2002)

Ajeesh Krishna et al. (2017)
217
Figure 2. Localization of various Zn transporters in plant cell. Zn transporter family is actively involved
in uptake, transport, detoxification and homeostasis of Zn within plants. Depending on the Zn
concentration in soil, various types of Zn transporters are expressed. During deficient concentration of Zn,
ZIP (ZIP1, ZIP2 and ZIP4) and P-Type ATPase (HMA2) families of Zn transporters are induced which
transport Zn into the cell through plasma membrane from the soil, and then CAX (MHX1), CDF (MTP1
and ZAT1), P-Type ATPase (HMA2 and HMA4) and ZIP (ZIP4) families of transporters are involved in
mobilization of Zn into organelles. The PCR family member PCR2 is important for redistribution and
detoxification Zn. The P-Type ATPase family member HMA1 is involved in detoxification of Zn in
chloroplast. Studies on localization and transport activity of ZIP transporters are still under progress.
investigated in several species, such as
Medicago truncatula (barrel medic) (Lopez-
Millan et al., 2004), barley (Tiong et al., 2009;
Pedas and Husted, 2009; Tiong et al., 2014),
maize (Li et al., 2013), rice (Lee et al., 2010;
Chen et al., 2008; Ishimaru et al., 2005; Ramesh
et al., 2003; Yang et al., 2009), Arabidopsis
(Grotz et al., 1998; Henriques et al., 2002; Lin et
al., 2009), Manihot esculenta (cassava)
(Bamrungsetthapong et al., 2010) wheat,
(Durmaz et al., 2011), Vitis vinifera (grape)
(Gainza-Cortés et al., 2012) and Glycine max
(soybean) (Moreau et al., 2002). The ZIP
transporters are highly expressed in roots in Zn
deficiency. In Arabidopsis, 15 ZIP family
members were identified (Mäser et al., 2001)
and most of these were induced in response to
Zn deficiency. Functions of AtZIP1, AtZIP2,
AtZIP3 and AtZIP4 were tested in the Zn uptake
deficient yeast mutant (zrt1 zrt2) and all these
four genes were able to complement yeast
mutant (Grotz et al., 1998). ZIP1 and ZIP3
genes are induced in roots and ZIP4 is induced
in root and shoots in Zn deficiency (Grotz et al.,
1998; Wintz et al., 2003). ZIP9 is also induced
in Zn deficiency in Arabidopsis (Talke et al.,
2006). Another ZIP family transporter, iron-
regulated transporter 3 (IRT3), was also
identified in Arabidopsis halleri and A. thaliana
and its function was characterized (Lin et al.,
2009). IRT3 from both species of Arabidopsis
was able to complement zrt1, zrt2 and Fe-uptake
deficient mutant (fet3, fet4) confirming the
transport ability of Zn and Fe. Localization
studies confirmed the expression of IRT3 in
plasma membrane (Lin et al., 2009).

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CDF
The CDF is a large and ubiquitous metal
transporter family. It is another Zn transporter
found in both prokaryotes and eukaryotes
(Guffanti et al., 2002). CDF family of
transporters plays a vital role in heavy metal
homeostasis in plants (Blaudez et al., 2003). It is
a proton antiporter, and transports metals such as
Zn, Fe, Co, Ni, Cd, and Mn (Gustin et al., 2011).
Many members of CDF family have been
implicated in the transport of Zn in plants
(Gaither and Eide, 2001). The CDF family
proteins have six putative TM domains (Eide,
2006). Metal tolerance proteins (MTPs) are
another group of metal transporters belonging to
the CDF family and are highly specific to Zn
(Krämer, 2005). Some members of CDF family
are found on both plasma membrane and
vacuole membrane and are involved in the
uptake and redistribution of heavy metals. The
identification of CDF family members like Zinc
Arabidopsis transporter (ZAT)/MTP1 on the
vacuolar membrane revealed a possible vacuolar
transporter of Zn in plants (Yang and Chu,
2011). The CDF family members have been
identified and characterized in plants such as
barley (Podar et al., 2012) and Arabidopsis
(Bloß et al., 2002; Kobae et al., 2004; Tanaka et
al., 2013), and Populus spp. (Blaudez et al.,
2003).
CAX
Members of CAX family have been identified in
animals, plants, fungi, and bacteria (Shigaki et
al., 2006). The CAX transporters are divalent
cation/H+ antiporters and are located on the
vacuoles, which are involved in cation transport
in plants (Jain et al., 2009). It contains 10-14
TM domains (Hanikenne et al., 2005). The CAX
family members were primarily found to
transport Ca2+; further studies revealed their
ability to transport a wide range of ions
including Zn (Socha and Guerinot, 2014). Mg2 +/
H+ exchanger (MHX) is also a member of CAX
family, which is an H+ antiporter, localized in the
vacuolar membrane and involved in transport of
Mg and Zn across the tonoplast in Arabidopsis
(Shaul et al., 1999). CAX family of transporters
is crucial for the redistribution of cations
including Zn in Arabidopsis (Socha and
Guerinot, 2014). These transporters were
identified and characterized in Arabidopsis
(Shigaki et al., 2006; Shaul et al., 1999) and rice
(Kamiya et al., 2005).
P-type ATPase
The P-type ATPase family of transporters was
identified in both prokaryotes and eukaryotes
(Hussain et al., 2004; Rastgoo et al., 2011;
Wang et al., 2014). Heavy metals including Zn
are transported across the membrane against
their electrochemical gradient by the energy of
ATP hydrolysis (Møller et al., 1996). The
structures of P-type ATPases contain eight TMs
and a CPx/SPC signature motif is found in sixth
TM, which has a key role in metal binding and
translocation (Williams and Mills, 2005). The
function of P-type ATPases has been proved in
transition metal transport and homeostasis in
Arabidopsis (Williams and Mills, 2005). Heavy
metal ATPase (HMAs) is one of the members of
the P-type ATPase (Hussain et al., 2004) which
is involved in transport of Cu, Zn, Cd, lead (Pb),
and Co (Rensing et al., 1999). Hussain et al.
(2004) reported that HMA2 and HMA4 were
involved in Zn transport and played an essential
role for increasing the Zn content in roots, stems,
and leaves of Arabidopsis. Xylem-loading is an
important step for the translocation of Zn from
root to rest of the plant. The HMA2 and HMA4
family of transporters is involved in xylem
loading of Zn in xylem parenchymatous cells
(Hussain et al., 2004; Hanikenne et al., 2008;
Sinclair and Krämer, 2012). The role of
AtHMA1 gene was tested in 3 different hma1
knock out Arabidopsis mutants (hma1-1, hma1-
2 and hma1-3) in Zn toxicity (Kim et al., 2009).
This study confirmed Zn detoxification function
of AtHMA1 and it has been found to be localized
on chloroplast envelope. The Zn transporting
activity of AtHMA1 was also tested in Zn
sensitive zrc1 yeast mutant and it confirmed that
expression of AtHMA1 exacerbated the
sensitivity of zrc1 in the excess Zn (Kim et al.
2009). Many P-type ATPase family members
have been identified and characterized in plants
viz. Arabidopsis (Hussain et al., 2004), rice
(Sasaki et al., 2014) and barley (Mills et al.,
2012).

Ajeesh Krishna et al. (2017)
219
PCR
PCR transporter is the largest gene family and
the members of these metal transporters are
found in fungi, algae, plants and animals (Song
et al., 2011). These transporters act as secondary
transporters mainly in epidermal cells and in the
xylem of new roots. In Arabidopsis, PCR2 gene
was found to be expressed both in roots and
shoots and is involved in plant survival in excess
and deficient conditions of Zn. PCR2 is also
important for redistribution and detoxification of
Zn in plants like Arabidopsis (Song et al., 2010).
The main role of PCR2 is the transporting of Zn
and maintaining of optimal Zn concentration in
roots of Arabidopsis.
Agronomic aspects of managing low Zn in soil
Many agronomic aspects are to be taken into
consideration while managing the soils with low
Zn that affect both the growth and yield of crop
plants. The effects of warmer temperatures, dry
soils, soil microbial activity and root-induced
chemicals help largely in the Zn uptake from the
soil by plants. Improvements of agronomic
factors may influence chemical and physical
processes in soils that influence the nutrient
availability (Pilbeam, 2015). When soil
temperature is low, mineralization of soil
organic matter slows down resulting in less
amount of Zn release in the soil. The application
of green leaf manuring is one of the important
practices for increasing organic matter and Zn
content in the soil. For example, the application
of 1 t/ha of Gliricidia (Gliricidia sepium) leaf
manure provides 85g Zn in soil (Srinivasara and
Rani 2011). Also, the incorporation of whole
parts of horse gram (Macrotyloma uniflorum)
into soils showed increase in the availability of
Zn in soil (Venkateswarlu et al., 2007). Manzeke
et al. (2012) reported that the supply of quality
organic nutrient resources apparently had a
strong influence on available Zn in soil.
Remarkably, the application of farmyard manure
showed relatively higher status of existing Zn
content in soil (Srinivasarao et al., 2013).
Mixed cropping and intercropping
systems are common and most important for the
nutritional improvement of crops grown in
nutrient-poor soils or low-input agro-ecosystems
(Li et al., 2004; Zuo and Zhang, 2008). These
agro-systems may have numerous advantages in
terms of increasing availability of Zn (Zuo and
Zhang, 2008). The crucial role played by the
interspecific root contacts in nutrient acquisition
in mixed stands of plants was also reported (Li
et al., 2001). Manzeke et al. (2012) suggested
that the legume-cereal intercropping system
proved a possible avenue for improving plant
available soil Zn. This is due to the high capacity
of legumes to scavenge nutrients from the soil
and release it back to the soil through falling off
(Zuo and Zhang, 2008). In field study,
intercropping system had higher Zn
concentration in shoot and seed of wheat and
chickpea compared to monocropping system (Li
et al., 2001).
In farming, the greatest management
practices and the best external alternative are to
supply Zn to low Zn soil. The application of
inorganic Zn containing fertilizers maintains or
restores Zn content in soil (Rengel 2002). With
wide range of soil types, the addition of Zn
fertilizer varies and it ranges from 0.5 to 1.5
kg/ha (Takkar et al., 1989). Zn containing
fertilizers like Zn sulphate heptahydrate (21%),
Zn sulphate monohydrate (33%), Zn-EDTA
(12%), zincated urea (2%) and zincated
phosphate (17.6%) are widely used to address
Zn deficiency problem all over the world
(Cakmak et al., 2010; Das et al., 2013). Zn
fertilizers provide an immediate and effective
remedy to increase Zn concentration under soil
with severe Zn stress. Similarly, the foliar
application of Zn is also a quick solution to
plants growing in low Zn soil. Supplying Zn
through seed soaking and seed coating are also
other agro-practices that result in increased crop
yields in low Zn soils (Rengel, 2002). When
compared, the seed treatment gave higher ZUE
than soil application of Zn sulphate at the rate of
5.5 kg Zn ha-1 (Singh et al., 2003).
Crop varieties respond differently to
varying systems of fertility management and the
mechanisms for the uptake of different nutrients
from soil also differ (Valizadeh et al., 2002).
ZUE genotypes contribute not only to reduce the
costs of fertilizer inputs but also to overcome the
problems related to subsoil Zn deficiency (Torun
et al., 2000). A genotype with high nutritional-
use efficiency gives high yields in infertile soil.

SABRAO J. Breed. Genet. 49 (3) 211-230
220
In many crop plants genetic variation with
respect to ZUE has been validated (Graham and
Rengel 1993; Erenoglu et al., 2000; Fageria
2001; Lonergan et al., 2009; Yamunarani et al.,
2016). The Zn deficiency tolerant genotypes are
designed to manage low Zn in soil.
Efforts made to improve ZUE
Efforts have been made by researchers to
improve ZUE of plants using various approaches
including, conventional breeding, transgenic
modification and marker assisted breeding. The
details of conventional breeding, transgenic and
marker assisted breeding (MAB) approaches
made to improve the ZUE are presented below.
Conventional breeding approach
Conventional breeding has been used to improve
agricultural production for thousands of years in
several biotic and abiotic stress conditions.
Hybridization is the most common method of
creating genetic variation with improved
characteristics. Plant breeding strategies hold
great promise for making a significant low-cost
and sustainable contribution to improve Zn
uptake. The ZUE may be improved through
conventional plant breeding by selecting
genotypes on the basis of genetic variability.
These genotypes are used to develop ZUE lines.
Germplasm screening can be used to raise ZUE
in plants. Many Zn deficiency tolerant crop
plants in Zn deficient condition were
characterized and reported (Rengel and
Römheld, 2000; McDonald et al., 2008).
ZUE plants have been successfully
developed in few crops. For example, the dry
land rice cultivar was developed from crossing
IR74 (intolerant) and Jalmagna (tolerant)
(Wissuwa et al., 2006). This dry land rice
cultivar exhibited significant genotypic variation
in tolerance to low soil Zn (Gao et al., 2006).
Similarly, the sorghum hybrid variety CSH-7
(36-A × 168) showed greater capacity to absorb
Zn in soil (Ramani and Kannan 1985). The
doubled haploid barley population was
developed from crossing two genotypes, Clipper
(low Zn accumulator) and Sahara 3771 (high Zn
accumulator). While screening this population,
some of the lines showed higher Zn uptake
efficiency than parental lines (Lonergan et al.,
2009). The hexaploid wheat line derived from
the crossing between Miracle wheat (Triticum
turgidum) and Einkorn wheat (Triticum
monococcum) had a significant increase in the
total amount of Zn in shoot with increased shoot
and root growth under Zn deficient conditions
(Cakmak et al., 1999). Similarly, the synthetic
hexaploid wheat lines (Triticum durum ×
Aegilops tauschii) had higher Zn content in its
grains than other cultivated wheats (Calderini
and Ortiz-Monasterio 2003). The ZUE
contributed to higher Zn uptake efficiency which
was responsible for the higher grain Zn
concentration in the hexaploid wheat lines.
Many more efforts are needed to develop ZUE
plants through conventional breeding. It will
ensure to improve growth and yield of important
crop plants in low Zn soil without relying much
on synthetic Zn fertilizers.
Transgenic approach
Plant genetic engineering has been considered as
a straight forward approach for imparting
specific trait in crop plants. Genes of many Zn
transporters have been overexpressed in various
plants through transgenic modification to
improve the ZUE. Overexpression of these
transporters increased the Zn uptake in Zn
deficiency. For example, overexpression of
AtZIP1 increased plant growth and Zn transport
rates in Arabidopsis in Zn deficiency condition
(Ramesh et al., 2004). Similarly, overexpression
of Arabidopsis AtMTP1 and AtZIP1 genes in
cassava improved the accumulation of Zn in the
edible portion of the storage root compared to
control plants in Zn deficiency (Gaitán et al.,
2015). Also, several other Zn transporters
(OsZIP4, AtZIP1, AtMHX1, AtMPT1, AhHMA4,
ZmIRT1, ZmZIP3 and NcTZN1) were
overexpressed in rice, Arabidopsis and
Nicotiana tabacum (tobacco) to improve Zn
acquisition during Zn deficiency (Table 3).
Molecular marker approach
Molecular marker assisted selection (MAS) and
breeding are also utilized for the improvement of
crop plants for many agronomically important
traits such as grain yield, quality, disease

Ajeesh Krishna et al. (2017)
221
resistance, nutritional quality, etc (Genc et al.,
2009; Hash et al., 2002; Srinives et al., 2010).
The identification of quantitative trait loci (QTL)
provides the basis for devising plant breeding
strategies to improve ZUE through marker
assisted selection. These approaches can be used
to develop new genotypes with improved ZUE
in various crop plants. However, only little effort
has been made so far in utilizing molecular
markers to improve ZUE in crop plants. Nagesh
et al. (2013) reported that QTL SC129 was
associated with ZUE in rice. This QTL is located
on chromosome number 3. In barley,
microsatellite-anchored fragment length
Table 3. The details of Zn transporter genes overexpressed in various plants through transgenic
modification.
Transformed
species
Name of the gene
Source of the
gene
Function
References
Oryza sativa
OsZIP4
Oryza sativa
Zn transport and
distribution
Ishimaru et al. (2007)
Hordeum vulgare
AtZIP1
Arabidopsis
Zn uptake
Ramesh et al. (2004)
Manihot esculenta
AtMTP1 and AtZIP1
Arabidopsis
Zn accumulation
Gaitán-Solís et al. (2015)
Arabidopsis
ZmIRT1 and ZmZIP3
Zea mays
Zn accumulation
Li et al. (2015)
Nicotiana tabacum
AtMHX1
Arabidopsis
Zn transport
Shaul et al. (1999)
Nicotiana tabacum
NcTZN1
Neurospora
crassa
Zn accumulation
Dixit et al. (2010)
Nicotiana tabacum
AhHMA4
Arabidopsis
Zn accumulation
Barabasz et al. (2010)
polymorphism (MFLP) marker SZnR1 has been
found to be associated with Zn concentration
and content (Sadeghzadeh et al., 2010).
Similarly, Nawaz et al. (2015) reported that
QTL RM237, RM3562, RM6863 and RM105
were associated with grain Zn concentration of
brown rice. These markers are located on
chromosome numbers 1, 3, 8, and 9 respectively.
Several other reports are also available for the
identification of QTL for Zn content of rice
grains. Four candidate genes (OsNAC, OsZIP8a,
OsZIP8c and OsZIP4b) were also found to be
connected with the Zn content of rice grain
(Gande et al., 2015). Similarly, markers for
candidate genes OsARD2, OsIRT1, OsNAS1 and
OsNAS2 were found to be associated with the
grain Zn content of rice (Anuradha et al., 2012).
QTL were also identified for leaf bronzing
(Zbz1a, Zbz1b, Zbz4, Zbz7 and Zbz12), plant
mortality (Zmt1, Zmt2, Zmt7 and Zmt12) and
dry matter (Zdm7, Zdm3, Zmt2, and Zmt12) in
Zn deficient conditions in rice (Wissuwa et al.,
2006). Most of these marker studies so far
focused on rice only; only a few studies have
been performed on other plants to find the
marker for ZUE. Gelin et al. (2007) found QTL
associated with Zn content of the seed in
recombinant inbred lines (RILs) of Phaseolus
vulgaris (navy bean). Roshanzamir et al. (2013)
identified the QTL associated with Zn
concentration of bread wheat grain using
composite interval mapping (CIM). Similarly,
QTL are also identified using CIM for Zn
concentration/content in wheat, rice,
Arabidopsis, barley etc. (Table 4). These QTL
could be used for marker assisted selection and
breeding for developing new genotypes with
improved ZUE in the same or other plants. The
candidate genes and molecular markers
established through basic research in model
plants like Arabidopsis could be extended to
other crops to improve their ZUE.
CONCLUSION
In conclusion, Zn deficiency is a major problem
for crop production worldwide. It has caused
yield losses in major crops such as rice, maize
and wheat. Currently, the non-conventional or
advanced molecular plant breeding techniques
help improve ZUE in crop plants. MAB and
transgenic are two widely used plant breeding
techniques to produce plants with favorable

SABRAO J. Breed. Genet. 49 (3) 211-230
222
Table 4. Details of quantitative trait loci (QTL) for grain Zn content detected by composite interval
mapping (CIM) in various plants.
Name of the plant
Traits identified
Chromosome
position
Detail of markers
Reference
Triticum aestivum
Grain Zn content and
concentration
1A
P3156.2-WMC59
Shi et al. (2008)
2D
P3470.3-P3176.1
3A
Xgwm391-P8422
Grain Zn content
4A
P3446-205-CWM145
4D
WMC331-Xgwm624
5A
WMC74-Xgwm291
7A
WMC488-P2071-180
Triticum aestivum
Grain Zn concentration
1A
Xgwm3094-Xgwm164
Roshanzamir et al.
(2013)
4A
Xgwm4026-Xgwm1081
Oryza sativa
Grain Zn concentration
1
RM34-RM237
Stangoulis et al. 2007)
12
RM235-RM17
Oryza sativa
Grain Zn concentration
4
CT206-G177
Zhang et al. (2011)
6
RZ516-G30
Arabidopsis
Grain Zn content
1
AXR1
Vreugdenhil et al.
(2004)
2
FD150
3
FD111
5
HH480
Hordeum vulgare
Grain Zn concentration
2HS
bcd175 - psr108
Sadeghzadeh et al.
(2015)
2HL
vrs1 - ksuF15
3HL
wg178 - HVM60
4HS
cdo358 - awbma29
Grain Zn content
2HS
bcd175 - psr108
2HL
vrs1 - ksuF15
Zea mays
Grain Zn concentration
4-08
ZM136
Simic et al. (2012)
3-05
bnlg1456
Triticum aestivum
Shoot Zn content
3A
Xmwg30
Balint et al. (2007)
7A
Xcdo545b
Oryza sativa
Grain Zn content
2
RM521-RM29
Kumar et al. (2014)
10
RM473-RM184
10
RM496-RM591
Triticum aestivum
Grain Zn content
7A
Xcfd31 – Xcfa2079
Singh et al. (2008)
Triticum aestivum
Grain Zn concentration
2D
wPt-730057-wPt-671700
Pu et al. (2014)
3D
wPt-6191-wPt-8658
4D
wPt-671648-wPt-667352
5B
wPt-7237-wPt-0708
Triticum aestivum
Grain Zn concentration
3D
Gdm136-gwm3
Genc et al. (2009)
4B
Wms149-gmw113
6B
Barc146a-p41/M48-76
7B
Gwm282-gwm63

Ajeesh Krishna et al. (2017)
223
traits such as Zn-use efficiency. The plants
produced by MAB have already been accepted
worldwide unlike the transgenic plants that face
the challenge with regard to safety on human
health. Zn transporters play important role to
maintain Zn uptake during Zn deficiency
condition. Only a little information is available
on the mechanism of Zn homeostasis in crop
plants. Many types of Zn transporters are
involved in uptake and translocation of Zn in
plants. Further characterization of these
transporters is crucial to understand the
mechanism behind the acquisition and transport
of Zn in crop plants. QTL have been identified
using molecular markers. Most of the molecular
marker studies are focused on rice only. The
QTL needs to be identified for ZUE in other
major cereals such as maize, wheat and millets.
Conventional and non-conventional
plant breeding techniques play an important role
in crop improvement. A collective effort coupled
with research is needed especially in the
developing world to improve the crop plants for
ZUE using MAB.
ACKNOWLEDGEMENTS
We sincerely thank Loyola College-Times of India grant
(7LCTOI14ERI001) for providing the financial support.
We also acknowledge the Vice Deanship of Scientific
Research Chairs at King Saud University, Riyadh, Saudi
Arabia for its funding through Research Group Project no.
RGP-213.
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