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Critical Reviews in Plant Sciences
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Finger Millet (Eleusine coracana (L.) Gaertn):
Nutritional Importance and Nutrient Transporters
Theivanayagam Maharajan, Stanislaus Antony Ceasar & Thumadath
Palayullaparambil Ajeesh Krishna
To cite this article: Theivanayagam Maharajan, Stanislaus Antony Ceasar & Thumadath
Palayullaparambil Ajeesh Krishna (2022): Finger Millet (Eleusine�coracana (L.) Gaertn):
Nutritional Importance and Nutrient Transporters, Critical Reviews in Plant Sciences, DOI:
10.1080/07352689.2022.2037834
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Finger Millet (Eleusine coracana (L.) Gaertn): Nutritional Importance and
Nutrient Transporters
Theivanayagam Maharajan, Stanislaus Antony Ceasar, and
Thumadath Palayullaparambil Ajeesh Krishna
Department of Biosciences, Rajagiri College of Social Sciences, Kochi, India
ABSTRACT
Finger millet is a nutri-rich cereal crop of poor people living in the developing countries of
Asia and Africa. Finger millet grains contain high amounts of potassium, phosphorus, mag-
nesium, calcium, manganese, copper, zinc, and iron. Its calcium content is 10-fold higher
than all other cereals and even three times higher than milk. Finger millet seeds are also
rich in cystine, methionine, tryptophan, and total aromatic amino acids as compared to
other cereals. Genome sequence of finger millet gives us the opportunity to study the nutri-
ent transporters. No attempt has been made to analyze and improve the nutrient transport
in finger millet based on the genome sequence. In this review, we discuss the nutritional
importance of finger millet and report the details on key nutrient transporters for the first
time. We have performed a genome-wide identification of various mineral nutrient trans-
porters (nitrogen, ammonia, phosphorous, sulfur, potassium, and micronutrients) of finger
millet and analyzed their protein sequences with those of various model cereals by various
computational tools. Phylogenetic relationship of each nutrient transporter with those of
other plants was analyzed and reviewed. The conserved and functional residues of nutrient
transporters are analyzed through homology modeling and multiple sequence alignment
using transporters with available crystal structures as templates and those from key cereals.
This review may provide a foundation for further studies on these nutrient transporters and
would help improve the nutrient transport in finger millet and other cereals to conserve
food and nutrient security in the developing countries of Asia and Africa.
Abbreviations: AMF: arbuscular mycorrhizal fungi; AMTs: ammonium transporters; COPT:
copper transporter; HAK: high-affinity potassium transporter; HKT: high-affinity potassium
uptake transporter; MCs: main clusters; MSA: multiple sequence alignment; NRTs: nitrate
transporters; PHTs: phosphate transporters; SULTR: sulfate transporter; TPK: tandem-pore K
channels; ZIP: zinc-regulated, iron-regulated transporter-like proteins
KEYWORDS
Functional residues;
genome sequence;
homology modeling;
human health;
nutrients; phylogeny
I. Introduction
Finger millet (Eleusine coracana (L.) Gaertn) repre-
sents the millet of the Poaceae. It is a highly self-polli-
nated and allotetraploid (2n¼4x¼36) crop (Ceasar
et al.,2018; Hatakeyama et al.,2018; Hittalmani et al.,
2017). Finger millet is a major crop in the arid and
semi-arid regions of developing countries of Asia and
Africa. It is also cultivated in America, Oceania, and
Europe (Dosad and Chawla, 2016; Krishna et al.,
2021). Finger millet ranks fourth on a global scale of
small grains production next to sorghum (Sorghum
bicolor), pearl millet (Cenchrus americanus), and fox-
tail millet (Setaria italica) (Upadhyaya et al.,2007;
Krishna et al.,2020a). Finger millet grains are used as
flour in the preparation of cakes, bread, and other
pastry products, and also malted and used as a benefi-
cial food for infants (Ceasar and Ignacimuthu, 2011).
The germinated seeds are also nutritious and easily
digestible. Finger millet grains contain a higher con-
tent of minerals, such as calcium (Ca), phosphorus
(P), iron (Fe), and manganese (Mn), compared to
other major cereals (Ceasar et al.,2018). Notably, it
has 10-fold higher Ca in seeds compared to other
major cereals and can be a panacea for Ca bio-fortifi-
cation (Kumar et al.,2016; Maharajan et al.,2021a).
The increase in population leads to industrializa-
tion throughout the world particularly in semi-arid
CONTACT Stanislaus Antony Ceasar antony_sm2003@yahoo.co.in Department of Biosciences, Rajagiri College of Social Sciences, Kochi 683104,
Kerala, India.
These authors contributed equally to this work.
Supplemental data for this article can be accessed at publisher’s website.
ß2022 Taylor & Francis Group, LLC
CRITICAL REVIEWS IN PLANT SCIENCES
https://doi.org/10.1080/07352689.2022.2037834
tropical areas where the availability of agricultural
land is declining quickly (Gupta et al.,2017). The
population of the world is expected to reach around
9.8 billion in 2050. World’s food demand is projected
to rise to 14,886 million tons in 2050 (Islam, 2019).
To overcome such a situation, there will be a demand
in the requirement of cereals, which includes finger
millet, to increase its production from 2.5 to 4.5 t
ha
1
(Saxena et al.,2018). An increase in finger millet
production with high nutritional value will benefit the
agriculture systems of Asia and Africa where famine
and malnutrition are widespread. Despite possessing
an excellent nutrient profile, finger millet suffers due
to less research emphasis, lack of improved varieties,
non-adoption of technologies, diseases like blast, lodg-
ing, and moisture stress, and threshing and milling
limitations (Wambi et al.,2021). All these reduce the
yield and utilization of finger millet considerably
throughout the world.
Mineral nutrients are required for plant growth,
development, and crop yields. Nitrogen (N), P, potas-
sium (K), Ca, magnesium (Mg), and sulfur (S) are
needed in large amounts for plant growth. Chloride
(Cl), copper (Cu), Mn, Fe, zinc (Zn), cobalt (Co),
molybdenum (Mo), and nickel (Ni) are required in
very small amounts for plant growth (White and
Brown, 2010; Imran and Gurmani, 2011). Plants
obtain all these nutrients primarily from the soil by
their root systems, translocated to the shoots via the
xylem, and then distributed into various tissues
depending on their demands. N is an important com-
ponent of proteins, chlorophyll, nucleic acids, cell
walls, phospholipids, amino acids, nucleic acids, vita-
mins, hormones, enzymes, and alkaloids (O’Brien
et al.,2016; Wang et al.,2018b). Nitrate (NO
3
)isan
essential source of N for most of the cultivated crops
(Forde, 2000; Noguero and Lacombe, 2016). NO
3
is
absorbed from the soil by NO
3
transporters (NRTs).
Ammonium (NH
4
þ
) is also the common N source for
higher plants, especially for the plants grown under
flooded or acidic soils where NH
4
þ
is dominant
(Sonoda et al.,2003; Hao et al.,2020b). Acquisition of
NH
4
þ
in plants is mediated by the NH
4
þ
transporters
(AMTs) (Ludewig et al.,2007; Pantoja, 2012). P is one
of the three indispensable macronutrients involved in
several important functions in plants (Baker et al.,
2015; Maharajan et al.,2018,2021b; Roch et al.,
2019). Plants acquire P from the soil in the form of
inorganic phosphate (Pi). Pi acquisition, translocation,
remobilization, and distribution depend on transport
processes mediated by the membrane-bound
Phosphate transporters (PHTs) (Roch et al.,2019;
Ceasar, 2020). K plays a crucial role in many bio-
chemical and physiological processes (Hasanuzzaman
et al.,2018). Plants require K ranging between 100
and 200 mM (Schroeder et al.,1994). Numerous K
channels and transporters are involved in the uptake
and distribution of K in plants. S is necessary for
plant growth and it is involved in the biosynthesis of
proteins, amino acids coenzymes, vitamins, prosthetic
groups, and secondary metabolites (Buchner et al.,
2004; Takahashi, 2019). S is accessible to plants pre-
dominantly in the form of anionic sulfate (SO
42
)
which is obtained via SO
42
transporters (SULTR).
Micro-nutrients also play important role in carbo-
hydrate metabolism, maintenance of the integrity of
cellular membranes, protein synthesis, regulation of
auxin synthesis, pollen formation, stabilization of ribo-
somal fractions and synthesis of cytochrome, and
regulation of gene expression (Hafeez et al.,2013;
Krishna et al.,2020b). Zn is acquired and transported
in plants primarily as a divalent cation (Zn
2þ
) by spe-
cialized transporters like Zn-regulated, iron-regulated
transporter-like proteins (ZIP). A minimal amount of
Cu is required to ensure cellular functions in plants.
Cu functions as a structural element in regulatory
proteins in addition to being a cofactor of many
enzymes (Sancen
on et al.,2004; Printz et al.,2016).
Cu is transported from the soil by Cu transporter
(COPT) (Pilon, 2011).
Many of these nutrient transporters are well char-
acterized both at the genomic and proteomic levels in
many plants. However, these are not yet identified
and characterized in finger millet although it possesses
high levels of key nutrients. Only a very few prelimin-
ary studies were conducted using partial gene
sequence of the finger millet to analyze their expres-
sion by RT-PCR (reviewed in Ceasar et al.,2018).
In this review, we highlight the nutritional import-
ance of finger millet offering various health benefits.
We further report various nutrient transporters from
the genome sequence of finger millet and provide
information on the structure, functions, and phylo-
genetic relationship of these nutrient transporters.
This review would form a foundation for further stud-
ies in finger millet and other millets to understand
and improve nutrient transport.
II. Nutritional importance of finger millet
Finger millet is often referred to as a nutraceutical
crop due to its nutrient-dense seeds offering health
benefits (Devi et al.,2014; Kumar et al.,2016). Finger
millet grains are rich in Ca, P, K, Mg, Mn, Na, dietary
2 T. MAHARAJAN ET AL.
fiber, polyphenols, and proteins (Izadi et al., 2012;
Amadou et al., 2013; Srikanth and Chen, 2016; Annor
et al.,2017). Compared to the other millets, finger
millet grain contains higher amounts of K
(430–490 mg/100 gm) as well as Ca (398.0 mg/100 gm)
(Roopa and Premavalli, 2008; Manjula et al.,2015).
The high amount of potassium in the finger millet
helps for the proper functioning of the kidneys and
brains and allows the brain and muscles to work
smoothly. Ca which is rich in finger millet, plays an
important role in growing children, pregnant women
as well as people suffering from obesity, diabetes, and
malnutrition (Jideani, 2012; Chappalwar et al.,2013;
Maharajan et al.,2021a). Both young and elderly peo-
ple can mitigate Ca deficiency by consuming finger
millet-based foods in their daily life (Towo et al.,
2006). The phosphorus content of finger millet is up
to 283.0 mg/100 g, which contributes to the develop-
ment of body tissue and energy metabolism
(Vanithasri et al.,2012; Ramashia et al.,2018). The
concentration of Mg in finger millet grains ranges
from 78 to 201 mg/100 g and can help to reduce the
risk of high blood pressure, asthma, migraine head-
aches, and heart attack (Saleh et al.,2013; Verma and
Patel, 2013; Prashanth and Muralikrishna, 2014).
Finger millet is also an excellent source of vitamins,
which play beneficial roles for brain function and
healthy cell division. Finger millet grains contain both
fat and water-soluble vitamins and are rich in vita-
mins A [retinol (6.0 mg/100 g)] and B [thiamine
(0.2–0.48 mg/100 g) and riboflavin (0.12 mg/100 g)]
(Chappalwar et al.,2013; Devi et al.,2014). The seeds
of finger millet contain more than 40% essential
amino acids, such as lysine, isoleucine, cysteine,
methionine, tryptophan, leucine, phenylalanine, and
threonine (Singh and Raghuvanshi, 2012; Sood et al.,
2017; Ramashia et al.,2018) compared to other cere-
als. In particular, methionine content (194 mg/100 gm)
is higher in finger millet compared to other millets
and cereals (Singh et al.,2012; Prashanth and
Muralikrishna, 2014). The presence of essential amino
acids helps to lower cholesterol levels and reduces the
risk of cancer and obesity in humans. The grains of
finger millet also contain essential fatty acids (lino-
lenic and palmitic acids) which are necessary for the
development of the brain and neural tissue (Kunyanga
et al.,2013; Muthamilarasan et al.,2016). The fat con-
tent is very low (1–2%) in finger millet grains which
helps to prevent the risk of obesity (Singh et al. 2012;
Verma and Patel, 2013; Gunashree et al.,2014). In
contrast, other cereal grains contain higher amounts
of fat ranging from 3.5 to 5.2% (Shahidi and
Chandrasekara, 2013). So finger millet-based food
products are considered a valuable food source for
obese people. Polyphenols (phenolic acids and tan-
nins) are present in finger millet grains which help to
maintain the body’s immune system (Siwela et al.,
2007; Devi et al.,2014; Okwudili et al.,2017). The
outer layer of finger millet seeds contains tannins,
which play an important role in the biological func-
tion of humans (Devi et al.,2014). Apart from their
excellent nutritional profile, finger millet grains are
non-glutinous food that is easily digestible and nonal-
lergenic (Saxena et al.,2018). It also has many health
benefits to prevent cancer and heart diseases, reduces
the tumor risk, cholesterol, fat absorption, and blood
pressure, delayed gastric emptying, and supply of
gastrointestinal bulk (Truswell, 2002; Gupta et al.,
2012). Finger millet’s phenolic compounds like syrin-
gic, ferulic, p-hydroxy benzoic, protocatechuic, p-cou-
maric, vanillic, trans-cinnamic acids, gallic, and
quercetin exhibit major antidiabetic and antioxidant
properties and also inhibit cataract effectively (Saleh
et al.,2013). The dietary fiber and polyphenols of fin-
ger millet contain various health benefits like antidia-
betic, antioxidant, hypocholesterolemic, antimicrobial
effects, increase in the fecal bulk, reduce the blood lip-
ids and protects from diet-associated chronic diseases
(Devi et al.,2014). Globally, most countries are facing
a high and increasing rate of cardiovascular diseases
due to smoking, unhealthy diet, obesity, and lack of
physical activity. Finger millet reduces plasma trigly-
cerides in hyperlipidemic rats and prevents cardiovas-
cular disease (Lee et al.,2010). In India, nearly every
third child is undernourished, with underweight
(35.7%), stunted (38.4%), and wasted (21%) among
the children under 5 years, whereas every second child
is anemic (58.4%) (Durairaj et al.,2019). Finger millet
with high nutritional and medicinal properties can
ensure, apart from food, the nutritional security of the
people in developing countries.
III. Nutrient transporters of finger millet
Plants acquire essential mineral nutrients through
roots from the soil for use in plant growth and devel-
opment. The uptake of mineral nutrients is mediated
by different transporters belonging to various trans-
porter families. Several families of membrane-bound
nutrient transporters were identified to be involved in
the acquisition of specific nutrients from the soil,
transport from root to shoot and redistribution within
the plant (Saski et al., 2016). Each nutrient has a spe-
cific family of a membrane transporter for the
CRITICAL REVIEWS IN PLANT SCIENCES 3
acquisition and re-distribution of nutrients in plants.
In finger millet, these nutrient transporters are poorly
studied and details on gene expression, transport
property, structure, and phylogenetic relationship of
these nutrient transporters have not yet been reported.
So we review the past works on nutrient transporters
in finger millet and analyze the phylogenetic relation-
ship and functional residues of key nutrients trans-
porters in this review.
A. Current status of understanding of finger millet
nutrient transporters
Nutrient transporters of finger millet are poorly studied
and only a few candidate genes have been identified
related to nutrient transport at the gene expression
level. For example, expression levels of the Ca trans-
porter genes, such as Ca exchanger (CAX1), two pore
channel1 (TPC1), calmodulin stimulated type IIB,Ca2
þ
ATPase,andcalmodulin dependent protein kinase
(CaMK1 and CaMK2) were analyzed in finger millet
(Mirza et al.,2014). Similarly, Ca sensor and
Calciuneurin B-like protein interacting protein kinases
24 (CIPk24) genes were identified in the developing
spikes of finger millet (Singh et al.,2014; Chinchole
et al.,2017). Expression of transcription factor prola-
min-binding factor DNA binding with one finger only
(PBF Dof) was analyzed in root, stem, leaf, and devel-
oping spikes of finger millet (Gupta et al.,2011).
Likewise, NRT1;2,NO
3
reductase (NR), glutamine syn-
thetase (GS), glutamine oxoglutarate aminotransferase
(GOGAT), and DNA binding with one finger (Dof1 and
Dof2) genes were analyzed in finger millet (Gupta
et al.,2013,2014). Four PHT1 (EcPHT1;1 to
EcPHT1;4) family transporters were analyzed in root
and leaf of finger millet (Pudake et al.,2017).
Notably, all of these studies were carried out before
the release of the genome sequence of finger millet
using partial sequence or primers of other cereals. The
genome sequence of two different finger millet geno-
types (ML-365 and PR-202) was released from 2017
to 2018 (Hittalmani et al.,2017; Hatakeyama et al.,
2018). Around 80–82% of the finger millet genome is
covered by these two works which are sufficiently
high due to the presence of repetitive elements in
grass genomes. Many researchers predicted that the
availability of genome sequence will help to identify
the candidate genes for agronomically and nutrition-
ally important traits (Ceasar et al.,2018; Puranik
et al.,2020). Hittalmani et al. (2017) identified and
validated some candidate genes related to drought,
disease resistance, Ca transport, and C4
photosynthetic pathway. Therefore, the new genomic
resources produced by Hittalmani et al. (2017) would
help to conduct more comprehensive functional gen-
omics studies on nutrient transport in finger millet.
IV. Finger millet genome and nutrient
transporter analysis
The genome sequence of finger millet gives us an
opportunity to analyze the nutrient transporters.
Several nutrient transports of other major cereal crops
were identified and analyzed based on their genome
sequences including rice (Sasaki et al.,2016), maize
(Zhang et al.,2012; Mondal et al.,2014; Wang et al.,
2018a) and wheat (Shukla et al.,2016;Liet al.,2017;
Cheng et al.,2018). To the best of our knowledge, no
candidate genes and proteins associated with biotic
and abiotic stresses have yet been identified and ana-
lyzed based on the genome sequence of finger millet.
We have performed genome-wide identification of
various nutrient transporters (N, P, K, NH
4
þ
, S, Cu,
and Zn) for finger millet from its genome sequence
(NCBI Accession ID: LXGH00000000, BioSample:
SAMN04849255, BioProject: PRJNA318349)
(Hittalmani et al.,2017). Specifically, we have pre-
dicted and analyzed the gene and protein sequences of
NRTs, PHTs, high affinity K transporters (HAKs), K
uptake (HKTs), tandem pore K channels (TPKs),
AMTs, SULTs, COPTs, and ZIP family members in
finger millet for the first time.
The steps used for the identification and analysis of
finger millet nutrient transporters are illustrated in
Supplemental Figure 1. The Arabidopsis (Arabidopsis
thaliana), rice and foxtail millet protein sequences of
macro- and micronutrient transporters were used for
Tblastn search with finger millet genome assembly
(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE¼
BlastSearch&PROG_DEF=blatn&BLAST_SPEC=Assem
bly&ASSEMBLY_NAME=GCA_002180455.1) at NCBI
website (Accession ID: LXGH00000000). The phylo-
genetic relationships of each family with those of
other plants were analyzed by molecular evolutionary
genetics analysis (MEGA) v6 software tool (Tamura
et al.,2013), multiple sequence alignment by
ClustalW, and homology modeling using Modeler
v9.22 (Eswar et al.,2007).
A. Nitrate transporters
The NRT1 family belongs to the Major Facilitator
Superfamily (MFS) of secondary active transporters
that use the proton electrochemical gradient to drive
substrate uptake into the cell (Reddy et al.,2012).
4 T. MAHARAJAN ET AL.
Plant NRT family members were first identified and
functionally characterized long ago (Tsay et al.,1993).
They are encoded by four gene families, NRT1 (Leran
et al., 2014), NRT2 (Orsel et al.,2002; Krapp et al.,
2014), chloride channel (CLC) (Bi et al.,2007), and
slow anion channel-associated 2 (SLAC2)/SLAC homo-
logs (SLAH) (Negi et al.,2008). Among the four fami-
lies, NRT1 and NRT2 are involved in uptake,
transport, and remobilization of NO
3
from root to
shoot (Wang et al.,2012; Chen et al.,2016; Fan et al.,
2017). The NO
3–
uptake system of higher plants con-
sists of two NO
3
absorption systems viz. low-affinity
transport system (LAS) and high-affinity system
(HAS) which are facilitated by NRT1 and NRT2 fam-
ily transporters, respectively (Wang et al.,2012). The
first NRT1 family transporter was identified in
Arabidopsis by screening chlorate resistance with T-
DNA insertion mutants (Tsay et al.,1993). Since then
NRT1 family transporters have been identified in
many plant species, such as Arabidopsis (Tsay et al.,
1993; Kotur et al.,2012), rice (Feng et al.,2011;Hu
et al.,2015; Yang et al.,2020), wheat (Yin et al.,2007;
Duan et al.,2015,2016), maize (Plett et al.,2010;
Garnett et al., 2013), barley (Vidmar et al.,2000;
Tong et al.,2005; Guo et al.,2020), and foxtail millet
(Nadeem et al.,2018). Both NRT1 and NRT2 family
members perform proton-coupled transport of NO
3–
and have 12 putative TMDs (Chen et al.,2008). We
have identified 19 NRT family genes in finger millet;
12 belong to NRT1 (EcNRT1;1to 1;12) family and the
remaining seven belong to NRT2 (EcNRT2;1to 2;7)
family. The NRT family transporters identified could
serve as the basis for future investigations on NO
3–
transporter functions in finger millet and eventually
could provide useful information for optimam N
management in finger millet.
1. Phylogenetic relationship
A phylogenetic tree was constructed with EcNRT fam-
ily proteins and those of other plants (Figure 1).
EcNRT2 members formed an independent cluster
(group II) from the EcNRT1 members (group 1), sug-
gesting that NRT1 and NRT2 subfamily members may
share different ancestors (Figure 1). For NRT1 sub-
family (group I, 107 plant NRT1 members comprised
of 11 main clusters (MCs) (Figure 1). In MC10,
EcNRT1;3 is closely clustered with AtNRT1;3 and
EcNRT1;4 distantly clustered with NRT1 family mem-
bers of foxtail millet, green foxtail, sorghum, and
maize (Figure 1). Interestingly, EcNRT1;5 and
EcNRT1;8 clustered together (MC9) and distantly
related with transporters of other cereals like barley
(HvNRT1;5) sorghum (SbNRT1;5), foxtail millet
(SiNRT1;5), and green foxtail (SvNRT1;5) (Figure 1).
NRT1;5 has not yet been identified and analyzed in
sorghum, foxtail millet, and green foxtail. Functional
analysis of AtNRT1;5 in Xenopus laevis oocytes con-
firmed it as a low affinity, pH-dependent bidirectional
nitrate transporter (transport nitrate in both directions
and export nitrate out of pericycle cells into the
xylem) (Lin et al., 2008). Functional characterization
of EcNRT1;5 and 1;8 transporters in finger millet
using X. laevis oocytes will help to identify the role of
these transporters. EcNRT1;2 (MC8) closely clustered
with TaNRT1;2 and distantly clustered with three
transporters of cereals. Similarly, EcNRT1;1 (MC7) is
distantly clustered with SbNRT1;1, SvNRT1;1, and
SiNRT1;1. Most of the EcNRT1 family transporters
are closely and distantly associated with those of fox-
tail millet, green foxtail, sorghum, and maize. NRT1
family transporters have not yet been identified and
functionally characterized in all these cereals.
However, NRT1 family transporters have been identi-
fied and functionally characterized in rice (Islam,
2019; Yang et al.,2020). Overexpression of
OsNRT1;1a and 1;1 b increased plant N accumulation
in rice under low N conditions (Fan et al.,2016).
OsNRT1;6 is also involved in the uptake of N under
low N conditions (Xia et al.,2015). As like rice, fur-
ther expression analysis and functional characteriza-
tion studies may help to identify the role of each
NRT1 family transporter in finger millet and other
cereals which may help to improve the growth and
yield of small millets and other cereals.
The 50 NRT2 family members of plants clustered
into seven MCs according to their evolutionary rela-
tionships. NRT2s family has at least seven members in
Arabidopsis (Miller et al.,2007) and five in rice (Feng
et al.,2011). NRT2 family plays an important role in
NO
3
acquisition from the soil under low N condi-
tions (Wang et al.,2012; Fan et al.,2016;O’Brien
et al.,2016). EcNRT2;3 is distantly associated with
high-affinity transporter of SbNRT2;3, ZmNRT2;2,
SvNRT2;6, and SiNRT2;4 (Figure 1). In general, NRT2
family transporters, such as NRT2;2, 2;3, and 2;4 are
considered as high-affinity transporters in rice (Feng
et al.,2011; Yan et al.,2011) and Arabidopsis (Li
et al.,2007), because both transporters are involved in
uptake and translocation of N under low N condition.
Unfortunately, the NRT2 family members have not
yet been identified and functionally characterized in
other cereals including sorghum, foxtail millet, green
foxtail, and finger millet. The EcNRT2;3 is distantly
associated with SbNRT2;3, ZmNRT2;2, SvNRT2;6, and
CRITICAL REVIEWS IN PLANT SCIENCES 5
SiNRT2;4 in MC5. The SbNRT2;3 improved uptake
efficiency of both organic and inorganic forms of N
(Gelli et al.,2014). This result revealed that EcNRT2;3
transporter may be involved in the NO
3
uptake
under N limitation condition. EcNRT2;4 and 2;7
clustered together and associated with the SvNRT2;7
of green foxtail. Future studies like analysis of expres-
sion dynamics and functional characterization of NRT
family transporters under differential N supply may
help to understand the roles of EcNRT family
Figure 1. Phylogenetic analysis of finger millet (Eleusine coracana) nitrate transporter 1 (EcNRT1) and EcNRT2 family proteins with
those of other plants. The protein sequences finger millet NRT transporters were obtained from genome assembly of finger millet
at NCBI website (Accession ID: LXGH00000000) as per the details mentioned (Supplemental Figure 1). The protein sequences of
rice, maize, wheat, barley, sorghum, foxtail millet, and green foxtail and model plant Arabidopsis were collected from phytozome
website (https://phytozome-next.jgi.doe.gov/). The expression profile and functions of rice (Feng et al.,2011; Tang et al.,2012;
Yang et al.,2020), maize (Garnett et al., 2013; Ibrahim et al.,2017), wheat (Bajgain et al.,2018; Wang et al.,2020), barley (Guo
et al.,2020), sorghum, foxtail millet (Nadeem et al.,2018), and green foxtail and model plant Arabidopsis (Okamoto et al.,2003;
Kotur et al.,2012) were inferred from respective previous reports and utilized for the comparison and analysis. The tree was con-
structed using MEGA version 6 software using the maximum likelihood method based on the Jones-Taylor-Thornton matrix-based
model with 1000 bootstrap replicates. The phylogenetic was visualized and edited using FigtreeV1.4.2 tool. Then Microsoft PPT
was used for labeling groups and main clusters (MCs) for NRT family members. The phylogenetic tree was divided into two groups
which are highlighted in aqua (group I; NRT1 family) and pink (group II; NRT2 family). The EcNRT family proteins of finger millet
are indicated in red.
6 T. MAHARAJAN ET AL.
transporters. The NRT family transporters are respon-
sible for N assimilation in plants through the uptake
of peptides (Parker and Newstead, 2014).
2. Functional residues in finger millet NRT1
transporter
Crystal structure of NRT1;1 is available for
Arabidopsis (AtNRT1;1) that helps to model and
study other plant NRT1 transporters. The structure of
AtNRT1;1 transporter contains 12 transmembrane
domains, consisting of amino-terminal (TM1 to TM6)
and carboxyl-terminal (TM7 to TM12) (Sun et al.,
2014; Parker and Newstead, 2014). It also consists of
three unique and conserved structural elements, such
as well-structured N-terminal cytoplasmic segment,
disulfide bond-stabilized extracellular loop, and par-
tially ordered central linker sequence (Sun et al.,
2014). The crystal structure of AtNRTI;1 has been
compared with bacterial peptide transporter from
Streptococcus thermophiles, thereby identifying the role
of residues involved in N uptake (Sun et al.,2014;
Parker and Newstead, 2014). Crystal structure of
NRT1 family homolog transporter of S. thermophilus
(StPepT) showed that Tyr29 and Tyr68 are important
functional residues and involved in peptide binding of
NRT1 transporter (Solcan et al.,2012). Two residues,
Tyr29 and Tyr68 involved in peptide binding of
StPepT transporter are well conserved in EcNRT1;1
(Supplemental Figure 2). Several residues involved in
proton binding of StPepT, such as Glu22, Glu25,
Arg26, Tyr30, and Lys126 are conserved in finger mil-
let, Arabidopsis, rice, and foxtail millet NRT1s
(Supplemental Figure 2). Glu22, Glu25, Arg26, Tyr30,
and Lys126 residues play a vital role in proton bind-
ing (Solcan et al.,2012). Similarly, four residues
(Arg33, Glu299, Asn328, and Glu400) have important
roles in peptide transport (Solcan et al.,2012). Among
these, Glu400 is conserved in NRT1s of plants includ-
ing EcNRT1;1 of finger millet. Glu299 was poorly
conserved and often replaced by Tyr334 in EcNRT1;1,
Tyr346 in AtNRT1;1 and OsNRT1;1 and Tyr356 in
SiNRT1;1. Glu300 plays an important role in the pro-
ton coupling (proton binding and transport) mechan-
ism. The Glu300 is appeared in GkPOT transporters
but not conserved in EcNRT1;1 and other plant
NRT1s. Arabidopsis AtNRT1.1 is a dual affinity NO
3
transporter that can uptake NO
3
over a wide range
of concentrations (Liu and Tsay, 2003;Hoet al.,2009;
Gojon et al.,2011; Bouguyon et al.,2015; Islam,
2019). The NRT1;1 also acts as a sensor for nitrate
(Ho et al.,2009). It will be very interesting to study
the functions of EcNRT1;1 transporter and analyze
their roles in the future especially under the influence
of other nutrients.
B. Phosphate transporters
One of the important responses in plants under low
Pi conditions is the induction of the PHT family of
genes. The plant PHTs are grouped into five families’
viz. PHT1, PHT2, PHT3, PHT4, and PHT5 (Roch
et al.,2019). Among these, plasma membrane located
PHT1 family transporters are involved in the acquisi-
tion of Pi from the soil and redistribution within
plants (Paszkowski et al.,2002; Ceasar et al.,2014;
Baker et al.,2015; Walder et al.,2015; Roch et al.,
2019), whereas Pi distribution within the cell, such as
translocations into chloroplasts, mitochondria, golgi,
and vacuole are mediated by PHT2, PHT3, PHT4,
and PHT5 family members, respectively (Guo et al.,
2008; Guo et al.,2013; Jia et al.,2015; Mlodzinska
and Zboinska, 2016; Shukla et al.,2016; Ceasar, 2020).
PHT1 family was first identified and characterized in
Arabidopsis, they are the primary transporters
involved in the acquisition of Pi from soil and redis-
tribution within plants and increase the Pi-influx cap-
acity by several folds in low Pi condition (Nussaume
et al.,2011; Plaxton and Tran, 2011; Baker et al.,
2015; Roch et al.,2019). PHT1 family genes have
been identified in several plants including rice
(Paszkowski et al.,2002; Guimil et al., 2005; Glassop
et al.,2007), wheat (Glassop et al.,2005;
Sisaphaithong et al.,2012; Maharajan et al.,2021c),
maize (Nagy et al.,2006; Liu et al.,2016), barley
(Glassop et al.,2005), foxtail millet (Ceasar et al.,
2014), finger millet (Pudake et al.,2017) and sorghum
(Sisaphaithong et al.,2012; Walder et al.,2015;
Maharajan et al.,2021d), to be involved in both direct
and indirect (arbuscular mycorrhizal fungi (AMF)-
mediated) Pi-uptake. In finger millet, a total of 23
putative EcPHT genes, including 12 EcPHT1, one
EcPHT2, four EcPHT3 and three each EcPHT4 and
EcPHT5 family members were identified from the
genome sequence of finger millet.
1. Phylogenetic relationship
Phylogenetic analysis of 12 members of finger millet
PHT1 (EcPHT1;1 to 1;12) proteins with those of other
monocots revealed their phylogenetic relationships.
The predicted EcPHT1 family protein sequences were
also clustered with those previously identified in
Poaceae family, such as from wheat, barley, maize,
foxtail millet, green foxtail, rice, sorghum, and maize
(Figure 2). More PHT1 transporters are clustered in
CRITICAL REVIEWS IN PLANT SCIENCES 7
MC5 (39 PHT1s) and this is followed by MC4 and
MC2 (23 and 21 PHT1s, respectively). EcPHT1;3 and
EcPHT1;11 transporters are clustered together and
distantly clustered with the SiPHT1;2, SvPHT1;2,
SbPHT1;7, ZmPHT1;1, ZmPHT1;2, and ZmPHT1;4 of
other monocots (Figure 2). SiPHT1;2was expressed in
shoot, root, and leaves of all millets at all stages of
growth under low and high Pi conditions (Ceasar
et al.,2014; Maharajan et al.,2019; Roch et al.,2020).
Down-regulation of SiPHT1;2severely reduced the
total P and Pi contents in shoot and root tissues of
foxtail millet (Ceasar et al.,2017). Therefore, expres-
sion analysis and functional characterization of
EcPHT1;3 and 1;11 would help us to understand the
further relationship in these two homologs. EcPHT1;8
and EcPHT1;10 (MC5) closely clustered with the
Figure 2. Phylogenetic analysis of finger millet phosphate transporter 1 (EcPHT1) family members with those of other plants. The
protein sequences finger millet PHT1 family transporters were obtained from genome assembly of finger millet at NCBI website
(Accession ID: LXGH00000000) as per the details mentioned (Supplemental Figure 1). The protein sequences of rice, maize, wheat,
barley, sorghum, foxtail millet, and green foxtail and model plant Arabidopsis were collected from phytozome website (https://
phytozome-next.jgi.doe.gov/). The tree was constructed using 106 potential PHT1 family proteins from nine plant species including
12 EcPHT1 of finger millet. The expression profile and functions of rice (Paszkowski et al.,2002; Glassop et al.,2005;Aiet al.,
2009), maize (Nagy et al.,2006; Liu et al.,2018), wheat (Teng et al.,2017), barley (Schunmann et al.,2004; Huang et al.,2011), sor-
ghum (Walder et al.,2015; Wang et al.,2019), foxtail millet (Ceasar et al.,2014), and green foxtail (Ceasar, 2019) and model plant
Arabidopsis (Muchhal et al.,1996; Mudge et al.,2002) were inferred from respective previous reports and utilized for the compari-
son and analysis. Methods used were as per the Figure 1 legend. The phylogenetic tree was divided into five MCs which are high-
lighted in pink (MC1), orange (MC2), light green (MC3), aqua (MC4), and blue (MC5). The EcPHT1 family proteins of finger millet
are indicated in red.
8 T. MAHARAJAN ET AL.
SvPHT1;6 and SiPHT1;6 also distantly with the
SbPHT1;3 ZmPHT1;7, OsPHT1;7, and HvPHT1;7.
ZmPHT1;7 induced 3-fold in roots of maize colonized
by AMF (Glomus etunicatum and Funneliformis mos-
seae) compared to un-colonized plants (Liu et al.,
2016; Sawers et al., 2017). SbPHT1;3 expression level
was also higher in AMF colonized roots of sorghum.
EcPHT1;8 and 1;10 might be AMF-specific transport-
ers induced in AMF-colonized roots of finger millet
which needs further studies. EcPHT1;1 is closely clus-
tered with SbPHT1;4, OsPHT1;3, and SvPHT1;5. In
rice, OsPHT1;3 increases Pi concentration in shoots
and roots and can uptake Pi under low Pi conditions
(Chang et al.,2019). SbPHT1;4 is expressed in roots
of sorghum under low Pi conditions (Walder et al.,
2015). So, EcPHT1;1 might be involved in Pi uptake
under low Pi conditions. EcPHT1;2 is distantly corre-
lated with the PHT1 family transporters of cereals
including SiPHT1;4 of foxtail millet. SiPHT1;4 was
considered as a root-specific transporter in the small
millets (Ceasar et al.,2014; Maharajan et al.,2019;
Roch et al.,2020). Therefore, EcPHT1;2 may be
involved in Pi transport in roots of finger millet under
low Pi conditions. EcPHT1;6 is closely clustered with
OcPHT1;12. EcPHT1;7 and 1;9 are closely linked to
HvPHT1;6 and TaPHT1;5. HvPHT1;6 is confirmed to
be a low-affinity transporter in sorghum with a Km of
385 mM (Rae et al. 2003). Other EcPHT1 proteins are
distantly related to PHT1s of cereals (Figure 2). Four
EcPHT1 (EcPHT1;1 to 1;4) family transporters were
analyzed for their expression in roots and shoots of
finger millet under both low and high Pi conditions
by Pudake et al. (2017). This study was performed
before the release of the genome sequence of finger
millet using PHT1 family gene-specific primers of
rice. Fortunately, the recently released genome
sequence of finger millet allowed the identification of
the entire PHT1 family transporters. Further charac-
terization of these genes with specific primers will
shed more light on their expression dynamics.
A separate phylogenetic tree was constructed for
PHT2-PHT5 family members of finger millet and of
other Poaceae consisting of 118 PHT2-PHT5 protein
sequences (Figure 3). The phylogenetic tree was div-
ided into four groups (group I-IV) each group con-
sisting of a separate family member (PHT2 to PHT5).
In group I, EcPHT2;1 is distantly clustered with
AtPHT2;1, SbPHT2;1, SvPHT2;1, ZmPHT2;1, and
SiPHT2;1 (Figure 3). Transporters of AtPHT2;1 and
SbPHT2;1 were predominantly expressed in leaves of
Arabidopsis and sorghum under low Pi conditions
(Daram, 1999; Wang et al.,2019). All EcPHT3
members are distantly clustered with other cereal
members of the same family in group II. PHT3 family
transporters are involved in Pi exchange between the
cytoplasm and the mitochondrial matrix (Baker et al.,
2015; Roch et al.,2019). PHT3 has three members in
Arabidopsis (Rausch and Bucher, 2002) and six mem-
bers in rice (Liu et al.,2011) and sorghum (Wang
et al.,2019). In wheat, TaPHT3;1and 3;2were
expressed in embryo and aleurone layer, which are
reported to be involved in grain development (Shukla
et al.,2016). AtPHT3;2 and 3;3 in Arabidopsis are
reported to be involved in Pi transport into mitochon-
dria (Hamel et al.,2004). SbPHT3;2and 3;6were
upregulated >2-fold in leaves of sorghum under low
P conditions (Wang et al.,2019). The application of
such studies will help to identify the accurate role of
EcPHT3 family members in finger millet. In group
III, EcPHT4;2 (MC4) is closely clustered with
ZmPHT4;2 and distantly clustered with OsPHT4;2,
TaPHT4;2, HvPHT4;2, and TaPHT4;3 (Figure 3). In
wheat, the expression level of TaPHT4;2 was higher in
the endosperm during grain development and
involved in Pi allocation within the seed (Shukla
et al.,2016). In MC3, EcPHT4;3 (group III member)
is distantly clustered with the SiPHT4;4 and
ZmPHT4;4. EcPHT5;1 has a distant relationship with
SiPHT5;2 and SvPHT5;2. EcPHT5;2 (group IV mem-
ber) is distantly related to SbPHT5;1, ZmPHT5;1,
TaPHT5;2, and OsPHT5;1 (Figure 3). As such, further
study is needed to investigate the expression analysis
on EcPHT2-5 family genes in finger millet.
2. Functional residues in finger millet PHT1 family
transporters
The crystal structure of the eukaryotic Pi-transporter
was deciphered from a high-affinity fungal Pi trans-
porter PiPT with bound Pi (Pedersen et al.,2013). It
provides the opportunity to analyze the structure and
functional residues of plant phosphate transporters
(Ceasar et al.,2016;Guet al.,2016; Liao et al.,2019;
Roch et al.,2019). It has 12 transmembrane helices
(M1-M12) divided into two homologous N-and C-
domains which are common for all major facilitator
superfamily (MFS) transporters. An MSA was per-
formed for all 12 EcPHT1s, yeast PHO84, and PiPT
transporters to analyze the conservation of functional
residues involved in Pi binding and transport
(Supplemental Figure 3). Totally seven residues,
Tyr150, Gln177, Trp 320, Asp324, Tyr328, Asn431,
and Lys459 are involved in Pi binding and transport
in PiPT (Pedersen et al.,2013). Among these, Tyr150,
Gln177, Trp 320, Asp324, Tyr328, and Asn431 are
CRITICAL REVIEWS IN PLANT SCIENCES 9
well conserved in all 12 finger millet PHT1s
(Supplemental Figure 3). The Lys459 is conserved for
all the EcPHT1s except for EcPHT1;12. The length of
EcPHT1;12 is short when compared to the other
EcPHT1 family transporter; it has only 429 amino
acids. Lack of complete sequence may be the reason
for variation in conserved residues with EcPHT1;12.
Asp324 is involved in the binding and transport of
H
þ
and Pi besides Gln177 by moving the N-domain
toward the binding site (Pedersen et al.,2013; Ceasar
et al.,2016). Lys459 is responsible for increasing the
affinity of Pi in PiPT (Pedersen et al.,2013; Ceasar
et al.,2016). Asp45, Asp48, Glu108 Asp149, and
Glu440 pull H
þ
from the binding site and this creates
electrostatic repulsion between Pi and the carboxy-
lated oxygen at the binding site (Pedersen et al.,
2013). Among these five residues, Asp48 and Asp149
are well conserved in all 12 EcPHT1 transporters
Figure 3. Phylogenetic analysis of finger millet phosphate transporter 2 (PHT2), PHT3, PHT4, and PHT5 family members with
those of other plants. The protein sequences finger millet PHT family transporters were obtained from genome assembly of finger
millet at NCBI website (Accession ID: LXGH00000000) as per the details mentioned (Supplemental Figure 1). The protein sequen-
ces of rice, maize, wheat, barley, sorghum, foxtail millet, and green foxtail and model plant Arabidopsis were collected from phy-
tozome website (https://phytozome-next.jgi.doe.gov/). Phylogeny tree was constructed using 90 potential PHT1 family proteins
from nine plant species including 10 EcPHT family members (1 EcPHT2, 4 EcPHT3, 3 EcPHT4, and 2 EcPHT5) of finger millet. The
expression profile and functions of rice (Liu et al.,2011;Shiet al.,2013; Ruili et al.,2020), maize (Takabatake et al.,1999), wheat
(Guo et al.,2013;Azizet al.,2014; Shukla et al.,2016), barley, sorghum (Wang et al.,2019), foxtail millet, and green foxtail and
model plant Arabidopsis (Rausch and Bucher, 2002; Hamel et al.,2004; Guo et al.,2008) were inferred from respective previous
reports and utilized for the comparison and analysis. Methods used were as per the Figure 1 legend. The phylogenetic tree was
divided into four groups which are highlighted in yellow (group I; PHT2 family) and bright green (group II; PHT3 family), pink
(group III; PHT4 family), and blue (group IV; PHT5 family). The EcPHT family proteins of finger millet are indicated in red.
10 T. MAHARAJAN ET AL.
(Supplemental Figure 3). Both these two residues are
also well conserved in rice (OsPHT1;2, 1;6, 1;11, and
1;13), foxtail millet (SiPHT1;1 to 1;12) and
Arabidopsis (AtPHT1;1, 1;2, and 1;5) transporters
(Ceasar et al.,2016). Asp45 is conserved in all
EcPHT1s except EcPHT1;4. Glu108 was not conserved
in all the 12 PHT1 family transporters. This residue is
also conserved in all PHT1 family transporters of rice,
foxtail millet, maize, and Arabidopsis (Ceasar et al.,
2016). Glu440 is conserved in all the EcPHT1s except
for EcPHT1;12. The functions of key residues in
Arabidopsis’AtPHT1 transporter were studied by site-
directed mutagenesis and yeast complementation
assays (Fontenot et al.,2015; Liao et al.,2019). Four
residues (Asp35, Asp 38, Arg134, and Asp144) are
implicated in H
þ
transfer and two residues (Tyr312
and Asn421) are involved in the initial interaction
and translocation of Pi (Liao et al.,2019). These
results revealed that all of these residues are essential
for Pi transport activity. Such studies with finger mil-
let PHT1 family transporters may help to shed more
light on the mechanism of Pi transport.
Functional residues were also analyzed by hom-
ology models for all the 12 EcPHT1 (EcPHT1;1 to
1;12) transporters with bound Pi as a ligand using
PiPT as a template. Key functional resides involved in
Pi binding are well conserved for most of the EcPHT1
models (EcPHT1;1 to 1;12) as in PiPT except for
EcPHT1;12 (Supplemental Figure 4). In the structure
of PiPT, Pi binding is mediated by Tyr150, Gln177,
Trp320, Asp324, Tyr328, and Asn425 (Pedersen et al.,
2013) and these residues are well conserved in most
of the EcPHT1 models except for EcPHT1;12 and
only Tyr150 is missing in EcPHT1;1. In EcPHT1;12
only two residues, Tyr150 and Gln177 are exposed to
the Pi binding site. Pi binding residue of Tyr150 is
well conserved in all PHT1 family transporters of rice,
maize, foxtail millet, and Arabidopsis (Ceasar et al.,
2016). However, in ClustalW alignment (Supplemental
Figure 3) the key functional residues present in all 12
EcPHT1s including EcPHT1;12 as in PiPT (Pedersen
et al.,2013). Many plant PHT1 family transporters
were characterized in yeast and xenopus for uptake
assays (Ai et al.,2009; Ceasar et al.,2017; Wang et al.,
2014; Teng et al.,2017). Similar studies with finger
millet PHT1 family transporters would shed more
light on the transport function.
C. Potassium transporter
Numerous K channels and transporters are involved
in the uptake and distribution of K in plant cells. K
transporters can be classified into four families; HAK,
HKT, TPK, K efflux anti-porter (KEA), and cation/
hydrogen exchanger (CHX) (Maser et al., 2001; Gupta
et al.,2008; Ragel et al.,2019; Hasanuzzaman et al.,
2018). Among these, HAK is the largest K transporter
family; it plays a different role in K uptake and trans-
port, regulation of plant growth and development, salt
tolerance, and regulation of osmotic potential (Cheng
et al.,2018;Liet al., 2018). In the past decade, several
HAK genes were identified and their functions charac-
terized in many plant species (Gierth and Maser,
2007; Very et al., 2014; Ragel et al.,2019) including
Arabidopsis (Quintero and Blatt, 1997; Nieves
Cordones et al.,2016), rice (Amrutha et al.,2007;
Gupta et al.,2008; Yang et al.,2009), wheat (Cheng
et al.,2018), maize (Zhang et al.,2012, Cao et al.,
2019), and barley (Santa-Mar
ıaet al., 1997; Coskun
et al.,2013). For example, a total of 13 and 27 HAK
family members have been identified in the
Arabidopsis (M€
aser et al., 2001; Very and Sentenac,
2003) and rice (Amrutha et al.,2007; Yang et al.,
2009), respectively. However, a genome-wide analysis
of the K transporter family genes has not yet been
performed in finger millet. We have identified a total
of 37 putative K transporter genes from the finger
millet genome, such as 23 EcHAK family genes, seven
genes encoding EcTPK family, and four each for
EcHKT1 and EcHKT2 families. Together with previ-
ously reported sequences, 37 assumed genes might
code transporters involved with K
þ
transport in fin-
ger millet.
1. Phylogenetic relationship
A phylogenetic tree was constructed with 309 poten-
tial K
þ
transporter sequences identified from nine
plant species including those from finger millet. The
tree is divided into three groups (group I to III)
(Figure 4). Group I contained the members of HAK1
family while group II and group III included the HKT
and TPK members, respectively. Group I consisted of
10 MCs. Among the 10 MCs, EcHAK16, and
EcHAK17 are found in MC7 and are closely related to
SiHAK19 and SvHAK19 (Figure 4). HAK family
transporters have not yet been identified and func-
tionally characterized in millets. In barley, the tran-
scriptomic analysis revealed that HvHAK19 was
slightly expressed in developing grain and inflores-
cence (Cai et al.,2021). Similar transcriptome analysis
revealed that TaHAK19a and TaHAK19b were slightly
expressed in the grain of wheat (Cheng et al.,2018).
Based on the previous transcriptomic reports, we
assume that HAK19 transporters may be involved in
CRITICAL REVIEWS IN PLANT SCIENCES 11
Figure 4. Phylogenetic analysis of potassium transporter family members [high-affinity K transporters (EcHAKs), high-affinity K
uptake transporter (EcHKT), and tandem-pore K channel (EcTPK)] of finger millet. The protein sequences finger millet HAK, HKT,
and TPK family transporters were obtained from genome assembly of finger millet at NCBI website (Accession ID: LXGH00000000)
as per the details mentioned (Supplemental Figure 1). The protein sequences of rice, maize, wheat, barley, sorghum, foxtail millet,
and green foxtail and model plant Arabidopsis were collected from phytozome website (https://phytozome-next.jgi.doe.gov/). The
tree was constructed using 309 potential potassium family proteins from nine plant species including 38 finger millet potassium
family transporters. The expression profile and functions of rice (Horie et al.,2011; Isayenkov et al.,2011; Wang et al.,2015; Okada
et al.,2018), maize (Zhang et al.,2012; Jiang et al.,2018), wheat (Cheng et al.,2018;Xuet al.,2020), barley (Hazzouri et al.,2018;
Cai et al.,2021), sorghum (Wang et al.,2014), foxtail millet (Zhang et al.,2018), and green foxtail and model plant Arabidopsis
(Quintero and Blatt, 1997; Baxter et al.,2010; Isayenkov and Maathuis, 2013) were inferred from respective previous reports and
utilized for the comparison and analysis. Methods used were as per the Figure 1 legend with bootstrap values of 500 replicates
instead of 1000. The phylogenetic tree was divided into three groups which are highlighted in green (group I; HAK family mem-
ber), red (group II; HKT family member), and pink (group III; TPK family member). The potassium transporter family proteins of fin-
ger millet are indicated in red.
12 T. MAHARAJAN ET AL.
the translocation of K
þ
for grain development under
low K
þ
conditions.
EcHAK4 is distantly related to four HAKs of cere-
als. EcHAK5 is distantly clustered with the HAK fam-
ily transporters of plants including ZmHAK5 of maize
(Figure 4). ZmHAK5 played a key role in the uptake
and translocation of K in maize under low K condi-
tions (Qin et al.,2019). EcHAK6 is found in the MC1
and is distantly clustered with HvHAK20 and
HvHAK21. The recent transcriptomic study suggested
that HvHAK21 was slightly expressed in root and
developing grains of barley (Cai et al.,2021).
HvHAK20 was expressed in various tissues, such as
shoot, root, embryo, senescing leaves, developing till-
ers, inflorescences, epidermal strips, and etiolated
seedling of barley through the same transcriptomic
analysis (Cai et al.,2021). These results clearly indi-
cate that HAK20 may be involved in uptake, trans-
location, and redistribution of K
þ
under low K
þ
conditions. EcHAK1 and EcAHK18 are clustered
together in MC7 and they are distantly related to
OsHKA1. OsHAK1 is highly upregulated in rice roots
under low K conditions and overexpression of
OsHAK1 in rice increased the K
þ
uptake as well as K/
Na
þ
ratio (Banuelos et al.,2002). EcHAK2 (MC3) is
distantly clustered with ZmHAK2, SbHAK3, SvHAK2,
and SiHAK2 (Figure 4). ZmHAK2 was expressed in
embryo, tassel, ear, and seed of maize under low K
þ
conditions. This study suggested that HAK2 may be
involved in the regulatory and developmental proc-
esses of seed (Zhang et al.,2012). EcHAK10 and
EcHAK11 are linked in the same cluster (MC4) and
distantly associated with SiHAK11, SvHAK11,
SbHAK18, and ZmHAK10. The ZmHAK10 was
expressed in root, tassel, ear, seed, embryo, and meri-
stem under low K conditions in maize (Zhang et al.,
2012). Other EcHAK family proteins are distantly
clustered with the HAK family proteins of cereals
(Figure 4). EcHAK3 and EcHAK7 clustered together
and are distantly assembled with the OsHAK7,
HvHAK7, and TaHAK7. TaHAK7 protein functions
in maintaining normal growth of wheat and mediated
K
þ
absorption under K deficiency (Cheng et al.,
2018). In rice, OsHAK7 transporter plays an import-
ant role in mediating K influx or efflux, depending on
the conditions (Banuelos et al.,2002). Further charac-
terization of multiple roles of EcHAKs in finger millet
is essential to understand and improve the K
þ
trans-
port in other cereals since finger millet has a rich
source of K.
HKT family proteins are primarily involved in
mediating salt tolerance in plants (Horie et al.,2009;
Ragel et al.,2019). For example, HKT family trans-
porters are expressed in xylem parenchyma cells and
protect leaves from salinity stress by removing Na
2þ
from the xylem sap (Ren et al.,2005; Sunarpi Horie
et al., 2005). In phylogenetic analysis, finger millet
HKT family members clustered separately under
group II (Figure 4). Two HKT1 family proteins
(EcHKT1;1 and 1;2) of finger millet were identified in
MC3 and they are closely clustered with SbHKT1;1
and SbHKT1;3 (Figure 4). In general, HKT1 family
gene (HKT1;1) has not yet been identified in many
cereals including sorghum. However, the role of
HKT1;1 was identified in Arabidopsis (Berthomieu
et al.,2003). They have reported that AtHKT1;1 is
involved in Na
þ
reticulation from shoot to root and
this process plays an important role in plant tolerance
to salinity.
EcHKT1;4 is found on MC1 and distantly corre-
lated with the other HKT1 family members of cereals.
EcHKT2;1, EcHKT2;3, and EcHKT2;4 are clustered
together and distinctly linked with the proteins of
other cereal HKT2 family transporters including
OsHKT2;1 of rice (Figure 4). OsHKT2;1 is involved in
Na
þ
uptake and supports plant growth under limiting
K
þ
supply (Horie et al.,2007; Haro et al.,2010).
EcHKT2;2 (MC4) is distantly associated with
HvHKT2;1, TaHKT2;1 and TaHKT2;2. TaHKT2;1,
when expressed in X. oocyte and yeast systems, espe-
cially mediate K
þ
uptake when external Na
þ
is at
sub-millimolar levels but facilitates Na
þ
influx when
external Na
þ
are in excess of K
þ
(Gassmann et al.,
1996). HvHKT2;1 was expressed in roots of barley
and involved in mediating Na
þ
uptake under low K
þ
conditions (Haro et al.,2005). The phylogenetic inves-
tigation was conducted with the inclusion of TPKs of
finger miller and other plants. EcTPK1, EcTPK5,
EcTPK6 and EcTPK7 found in MC1 (Figure 4).
EcTPK1 and EcTPK7 clustered together and are dis-
tantly linked with SbTPK1, ZmTPK1, and SiTPK1. In
Arabidopsis, TPK1 transporter has been reported to
be involved in vacuolar K
þ
release during stomatal
closure, seed germination, and radicle growth (Gobert
et al.,2007). Based on this report, we assume that
TPK1 transporters may participate in vacuolar K
þ
release during stomatal closure, seed germination, and
radicle growth of all cereals. EcTPK5 and EcTPK6
also clustered together and distantly connected with
the ZmTPK5, SbTPK4, SiTPK5, and SiTPK4. AtTPK5
was functionally characterized in Escherichia coli and
has been reported to be involved in K
þ
transport
under low K
þ
conditions (Isayenkov and Maathuis,
2013). TPK family members have not yet been
CRITICAL REVIEWS IN PLANT SCIENCES 13
identified in sorghum, foxtail millet, and maize.
Expression analysis and functional characterization of
EcTPK family transporters may help to identify the
role of each TPK family in finger millet and other
cereals. Altogether, it provides information for future
functional analyses on K transporters and improve the
K-utilization efficiency in finger millet and
other cereals.
D. Ammonium transporters
Acquisition of NH
4
þ
in plants is mediated by the
AMTs (Ludewig et al.,2007; Pantoja, 2012). There are
four families of AMTs, such as AMT1, AMT2, AMT3,
and AMT4. AMT families participate in many physio-
logical processes, such as uptake of NH
4
þ
from the
soil solution (AMT1) (Kaiser et al.,2002; Loque et al.,
2006), transport of NH
4
þ
from root to shoot (AMT1
and AMT2) (Sohlenkamp et al.,2002; Yuan et al.,
2007; Giehl et al.,2017), relocation of NH
4
þ
in leaves
and reproductive organs (AMT1, AMT2, and AMT3)
(Lee and Tegeder, 2004; Koegel et al.,2013) and
transfer of NH
4
þ
from symbiotic fungi to host plants
(AMT3 and 4) (Behie and Bidochka, 2014; Chen
et al.,2018). AMT family transporter has been identi-
fied in several plant species, such as Arabidopsis
(Ninnemann et al. 1994; Yuan et al.,2007;Wuet al.
2019), rice (Li et al.,2009; Sumei et al., 2012; Yang
et al.,2015), maize (Gu et al.,2013; Dechorgnat et al.,
2019), sorghum (Koegel et al.,2013) and wheat (Li
et al.,2017; Jiang et al.,2019). A total of nine AMT
family genes were identified in the finger millet gen-
ome, including 4 AMT1, 2 each AMT2 and AMT2
members and one AMT4.
1. Phylogenetic relationship
To understand the phylogenetic relationship between
AMT family proteins in finger millet and other plants,
an un-rooted phylogenetic tree of AMT family mem-
bers was constructed. A total of 62 AMT plant pro-
teins, including 28 AMT1, 12 AMT2, 15 AMT3, and 7
AMT4 family members were used to construct the
phylogenetic tree (Figure 5). EcAMT1;1 is closely clus-
tered with OsAMT1;1 and EcAMT1;2 is distantly clus-
tered with AMT1s of sorghum, maize, green foxtail,
and foxtail millet in MC3. Overexpression of
OsAMT1;1helps both for uptake and translocation of
NH
4
þ
under low NH
4
þ
conditions (Hoque et al.,
2006; Kumar et al.,2006). This result suggested that
EcAMT1;1 may be involved in both uptake and trans-
location of NH
4
þ
under the conditions of limiting
NH
4
þ
supply. EcAMT1;3 is clustered with the
HvAMT1;1. EcAMT1;4 is distantly clustered with
TaAMT1;2 and TaAMT1;4. The expression level of
TaAMT1;2was higher in the leaf and root tissues of
wheat under low NH
4
þ
conditions (Li et al.,2017).
EcAMT2;1 is found in the MC2 of group II and is
distantly linked to the SiAMT2;1 (Figure 5).
EcAMT2;2 is closely clustered with the SbAMT2;2. In
sorghum, SbAMT2;1 and SbAMT2;2 were expressed
in root, shoot, stem, pistil, and stamen tissues under
low NH
4
þ
conditions (Koegel et al.,2013). EcAMT2;2
of finger millet may play a role in NH
4
þ
transfer to
the reproductive organs of finger millet. The
EcAMT3;1 and 3;2 clustered together and they are
closely associated with the SiAMT3;1 and SvAMT3;2
(Figure 5). AMT3 family transporters have not yet
been identified and functionally characterized in many
cereals including small millets. AtAMT3;1was
expressed in senescing leaves of Arabidopsis under
low NH
4
þ
condition (Couturier et al., 2007).
OsAMT3;1was expressed in roots of rice under low
NH
4
þ
conditions. These two results suggest that
EcAMT3;1 transporter may be involved in both
uptake and translocation of NH
4
þ
under low NH
4
þ
conditions (Suenaga et al.,2003).
2. Functional residues in finger millet AMT1
transporters
The bacterial EcAMTb is the first AMT protein to
have its crystal structure solved (Khademi et al.,2004;
Zheng et al.,2004). The EcAMT1 family protein
sequences were analyzed with the sequences of plant
AMT transporters, such as Arabidopsis (5), rice (3),
and foxtail millet (3). EcAMTb was used to identify
the conserved residues of EcAMT1s (Supplemental
Figure 5). The structure of EcAMTb showed that two
His residues, such as His168 and His318 are essential
for the de-protonation process (Khademi et al.,2004).
Very interestingly, these two residues (His168 and
His318) are strongly conserved with EcAMT1s of fin-
ger millet (Supplemental Figure 5). Phe107, Trp148,
and Ser219 play a major role at the NH
4
þ
binding site
in EcAMTb (Khademi et al.,2004; Zheng et al.,2004;
Javelle et al.,2006). All these residues are well con-
served with the AMT1s of finger millet and other
plants (Supplemental Figure 5). Ile110, Leu114,
Leu208, and Trp212 are the pore-lining residues
(Khademi et al.,2004). Among these, Leu208 and
Trp212 residues are strongly conserved in EcAMT1s.
The remaining two residues (Ile110 and Leu114) are
replaced by Ala and Ile, respectively at the ligand-
binding site of all 4 EcAMT1s. Further expression
analysis and functional characterization of EcAMTs
14 T. MAHARAJAN ET AL.
may be helpful to understand their specific function
in the future.
Homology models were produced for all four finger
millet EcAMT1s using E. coli EcAMTb as a template.
In EcAMTb, the ligands are coordinated by His168,
His318, Trp212, Ile110, Leu114, and Leu208 (Khademi
et al.,2004). Many variations were seen for the resi-
dues at the ligand-binding site of finger millet
EcAMT1s and only His and Trp are similar in all 4
EcAMT1s and Leu (Leu114) is found only in
EcAMT1;4 transporter (Supplemental Figure 6). Other
ligand site residues showed much variation in all 4
EcAMT1s (Supplemental Figure 6). The second His
residue (His 318) is not found in all 4 EcAMT1 mod-
els and both Ile110 and Leu114 are replaced by Phe
and Val at the ligand-binding site of all 4 EcAMT1s.
Figure 5. Phylogenetic analysis of finger millet ammonium transporter 1 (EcAMT1) to ECAMT4 family members. The protein
sequences finger millet AMT family transporters were obtained from genome assembly of finger millet at NCBI website (Accession
ID: LXGH00000000) as per the details mentioned (Supplemental Figure 1). The protein sequences of rice, maize, wheat, barley, sor-
ghum, foxtail millet, and green foxtail and model plant Arabidopsis were collected from phytozome website (https://phytozome-
next.jgi.doe.gov/). The expression profile and functions of rice (Sonoda et al.,2003; Sumei et al.,2012; Bao et al.,2015), maize (Gu
et al.,2013; Hao et al.,2020a), wheat (Li et al.,2017; Jiang et al.,2019), barley, sorghum (Koegel et al.,2013), foxtail millet, and
green foxtail and model plant Arabidopsis (Shelden et al.,2001; Sohlenkamp et al.,2002; Yuan et al.,2009) were inferred from
respective previous reports and utilized for the comparison and analysis. Methods used were as per the Figure 1 legend. The
phylogenetic tree was divided into four groups which are indicated in blue (group I), aqua (group II), green (group III), and pink
(group IV). The EcAMT family proteins of finger millet are indicated in red.
CRITICAL REVIEWS IN PLANT SCIENCES 15
This demands further studies with mutagenesis of key
resides of plant AMTs and test their transport func-
tion in yeast and in planta to confirm their function.
E. Sulfate transporter
S is accessible to plants predominantly in the form of
anionic sulfate (SO
42
) which is obtained by plants
via SO
42
transporter (SULTR). Generally, SULTR
can be classified into four groups, such as SULTR
1–4. We identified SULTR3 family genes in finger
millet. We could not find the sequences of SULTR1,
2, and 4 family members in the genome sequence of
finger millet.
1. Phylogenetic relationship
A phylogenetic tree was constructed with five identi-
fied EcSULTR3 family members of finger millet and
37 previously characterized SULTR3 proteins sequen-
ces of other plants (Figure 6). In MC2, the
EcSULTR3;2 and EcSULT3;5 are distantly clustered
with SvSULTR3;2 and SiSULTR3;2 (Figure 6). In
MC4, two EcSULTR3 family proteins (EcSULTR 3;1
and 3;3) are distantly clustered with monocot
SULTR3 family transporters. SULTR3 family members
play an important role in the transport and distribu-
tion of SO
42
during seed development (Takahashi,
2019). The role of SULTR3 transporter has not been
reported in wheat, maize, barley, sorghum, and small
millets. However, their function has been reported in
rice and Arabidopsis (Cao et al.,2013; Zhao et al.,
2016). For example, AtSULTR3;1 has been reported to
be involved in the transport of SO
42
across the
chloroplast envelope in Arabidopsis (Cao et al.,2013).
OsSULTR3;3was expressed in vascular bundles of
shoots, leaves, flowers, and seeds of rice (Zhao et al.,
2016). In the same study, functional analysis revealed
that OsSULTR3;3 play a major role in SO
42
trans-
port and homeostasis.
F. Copper transporter
Cu is transported from soil to plant by CTR family
proteins (Pilon, 2011). The CTR-like transporters are
called Cu transporter (COPT) in plants and up to six
members of the COPT family (COPT1 to COPT6)
have been reported in plants (Kampfenkel et al.,1995;
Penarrubia et al., 2010; Sancenon et al.,2003).
Members of COPT family transporters contain three
TMDs with N-and C-terminal regions in extracellular
space and cytosol, respectively (Puig, 2014; Yuvan
et al., 2011). Among these six members, COPT1 plays
a vital role in the acquisition of Cu from the soil solu-
tion (Printz et al.,2016) and specifically, COPT6
mediates Cu transport, remobilization, and redistribu-
tion of Cu from senescing to sink organs in all plants
(Jung et al.,2012; Garcia Molina et al.,2014). COPT5
is localized to the tonoplast and involved in intracellu-
lar Cu homeostasis. COPT proteins have been identi-
fied including AtCOPT1-6 in Arabidopsis (Sancenon
et al.,2003; Sancen
on et al.,2004) OsCOPT1-6 in rice
(Yuan et al.,2011), ZmCOPT1-3 in maize
(Wang et al.,2018a), and TaCOPT1 in wheat (Li
et al.,2014). To date, Cu transporters have not
been reported in finger millet. We have identified 6
Cu transporter family genes in the finger mil-
let genome.
1. Phylogenetic relationship
A Phylogenetic tree was constructed using 47 potential
COPT family proteins from nine plant species includ-
ing six finger millet COPT proteins (Figure 7). The
COPT family transporter sequences are clustered into
six MCs (MC1 to 6). MC2 contained 11 transporters
MCs 5 and 6 consists of 10 proteins each from various
Poaceae members including finger millet (Figure 7).
EcCOPT1 is distantly clustered with ZmCOPT1 and
TaCOPT2. Among COPT transporters, COPT1 is con-
sidered to be the best-characterized member. It was
first identified in Arabidopsis and its role was function-
ally characterized by expressing in S.cerevisiae mutant
(ctr1D)(Kampfenkelet al.,1995). They have reported
that the COPT1 transporter allows the entrance of Cu
into cells from the exterior to the cytoplasm, therefore
it was considered as a high-affinity transporter
(Kampfenkel et al.,1995;Sancenonet al.,2003). The
high-affinity transporter ZmCOPT1 was strongly upre-
gulated in shoots of maize under Cu deficiency condi-
tions and involved in Cu transport (Wang et al.,
2018a). EcCOPT6 is closely clustered with HvCOPT1.
EcCOPT5 is distantly associated with SiCOPT5,
SvCOPT5, SbCOPT4, SbCOPT2, and ZmCOPT4
(Figure 7). Expression analysis of COPT2,4,and5
have not yet been studied in foxtail millet, maize, sor-
ghum, and green foxtail. AtCOPT2 was expressed in
shoot and root tissues of Arabidopsis under Cu defi-
cient condition (Sancenon et al.,2002).OsCOPT2 and
5were also expressed in both shoot and root tissues of
rice under Cu deficient conditions (Yuvan et al.,2011).
In the same study, OsCOPT4 was found to be
expressed in stem, leaf, root, and panicle of rice under
Cu deficient conditions (Yuvan et al.,2011).
These results suggest that COPT2, 4, and 5 transporters
of finger millet may be involved in uptake, transport,
16 T. MAHARAJAN ET AL.
and remobilization of Cu under low Cu conditions. Both
EcCOPT3 and EcCOPT4 are closely clustered with
TaCOPT1 and HvCOPT2 (Figure 7). Functional analysis
of TaCOPT1 transporter revealed that it is involved in
Cu uptake and homeostasis in wheat under low Cu con-
ditions (Li et al.,2014). EcCOPT1 is distantly clustered
with the ZmCOPT1 and TaCOPT. The TaCOPT1 is
highly induced in both root and leaf tissues under Cu-
deficient conditions in wheat (Li et al.,2014). TaCOPT1
is localized in golgi apparatus and regulated Cu homeo-
stasis in common wheat and improves tolerance to low
Cu stress to maintain normal growth and development
(Li et al.,2014). EcCOPT2 is distantly connected with
the SvCOPT2 and SiCOPT1.
Figure 6. Phylogenetic analysis of finger millet sulfate transporter (SULTR3;1 to 3;5) family members. The protein sequences finger
millet SLUTR family transporters were obtained from genome assembly of finger millet at NCBI website (Accession ID:
LXGH00000000) as per the details mentioned (Supplemental Figure 1). The protein sequences of rice, maize, wheat, barley, sor-
ghum, foxtail millet, and green foxtail and model plant Arabidopsis were collected from phytozome website (https://phytozome-
next.jgi.doe.gov/). The tree was constructed using 37 potential SULTR3 family transporter proteins from nine plant species including
five finger millet EcSULTR proteins. The expression profile and functions of rice (Ye et al.,2011; Zhao et al.,2016; Yuan et al.,
2021), maize (Huang et al.,2018), wheat (Buchner et al.,2010), barley, sorghum (Akbudak et al.,2018), foxtail millet, and green fox-
tail and model plant Arabidopsis (Kataoka et al.,2004;Chenet al.,2019) were inferred from respective previous reports and utilized
for the comparison and analysis. Methods used were as per the Figure 1 legend. The phylogenetic tree was divided into seven
MCs and which are highlighted in pink (MC1), blue (MC2), sky blue (MC3), green (MC4), gold (MC5), light green (MC6), and orange
(MC7). The EcSULTR family proteins of finger millet are indicated in red.
CRITICAL REVIEWS IN PLANT SCIENCES 17
2. Functional residues in finger millet COPT1
transporter
Structural characterization of Cu transporters in plant
species is still limited. However, Ren et al. (2019) pro-
vided a molecular understanding of Cu transport by
COPT1 family transporter through X-ray crystal struc-
tures of an engineered Atlantic salmon (Salmo salar)
COPT1 (SsCOPT1) transporter. Protein sequences of
S. salar COPT1 (SsCOPT1), EcCOPTs of finger millet,
AtCOPTs of Arabidopsis, OsCOPTs of rice, and
SiCOPTs of foxtail millet were used for alignment to
identify the conserved residues involved in Cu binding
and transport (Supplemental Figure 7). Previously, X-
ray crystal structure of COPT1 from S. salar revealed
that two methionine residues (Met146 and Met150)
are essential for Cu binding and transport (Ren et al.,
2019). Met146 and Met150 are well conserved in all
EcCOPTs analyzed (Supplemental Figure 7). Two resi-
dues namely, Glu80 and His135 are found on the Zn
binding site in SsCOPT1 (Ren et al.,2019). Glu80 and
Figure 7. Phylogenetic analysis of finger millet copper transporter (EcCOPT1-EcCOPT6) family members. The protein sequences fin-
ger millet COPT family transporters were obtained from genome assembly of finger millet at NCBI website (Accession ID:
LXGH00000000) as per the details mentioned (Supplemental Figure 1). The protein sequences of rice, maize, wheat, barley, sor-
ghum, foxtail millet, and green foxtail and model plant Arabidopsis were collected from phytozome website (https://phytozome-
next.jgi.doe.gov/). Phylogeny tree was constructed using 47 potential COPT family transporter proteins from nine plant species
including six finger millet EcCOPT proteins. The expression profile and functions of rice (Yuan et al.,2011), maize (Wang et al.,
2018a), wheat (Li et al.,2014), barley, sorghum, foxtail millet, and green foxtail and model plant Arabidopsis (Kampfenkel et al.,
1995; Sancenon et al.,2003; Penarrubia et al.,2010) were inferred from respective previous reports and utilized for the comparison
and analysis. Methods used were as per the Figure 1 legend. The phylogenetic tree was divided into six MCs and which are high-
lighted in white (MC1), gold (MC2), light green (MC3), pink (MC4), light blue (MC5), and light gray (MC6). The EcCOPT family pro-
teins of finger millet are indicated in red color.
18 T. MAHARAJAN ET AL.
His135 are predicted to play a key role in increased
Cu uptake. Glu80 is well conserved in all six
EcCOPTs analyzed except for EcCOPT5 wherein
Glu80 is replaced by Gln42 (Supplemental Figure 7).
Similarly, the His135 residue found in all six
EcCOPTs, except for EcCOPT5 which, has Phe98 in
place of His135. This might be a valuable basis for
future studies to characterize these transporters in fin-
ger millet and other millets.
G. Zinc transporter
Zn is one of the essential micro-nutrients for crop
production. The ZIP family transporters help to carry
Zn
2þ
ions across cellular membranes into the cyto-
plasm (Eide et al.,1996; Conte and Walker, 2011).
ZIP transporters are mainly involved in the uptake,
transport, and distribution of Zn in plants (Gupta
et al.,2016; Krishna et al.,2020b;Liet al.,2013). ZIP
transporters are essential for Zn uptake and transport.
We have identified four putative EcZIPs (EcZIP1-
EcZIP4) from the genome of the finger millet. For
example, 12 ZIP family members were identified in
Arabidopsis (Grotz et al.,1998), 16 in rice (Ramesh
et al.,2003; Chen et al.,2008), 12 in maize (Li et al.,
2013; Mondal et al.,2014), 14 in wheat (Durmaz
et al.,2011; Evens et al.,2017), 13 in barley (Pedas
et al.,2009; Tiong et al.,2015), and seven in foxtail
millet (Alagarasan et al.,2017). Plant ZIP family pro-
teins consist of 300–476 amino acid residues with six
to eight TMDs (Guerinot, 2000; Vatansever et al.,
2016). Lengths of the EcZIP family transporter pro-
teins ranged from 213 to 296 and contained
2–7 TMDs.
1. Phylogenetic relationship
A phylogenetic tree was constructed with 76 plant ZIP
protein sequences including 4 EcZIPs of finger millet
(Figure 8). The finger millet EcZIP1 is distantly clus-
tered with SvZIP1 and SiZIP1 in the MC2 (Figure 8).
SiZIP1 gene has been found to be expressed in root,
shoot, and leaf of foxtail millet under drought condi-
tions (Alagarasan et al.,2017). So EcZIP1 transporter
might be active at various tissues for efficient Zn
transport in finger millet. EcZIP2 is closely clustered
with the ZmZIP2.ZmZIP2 was highly expressed in
flag leaf and kernel of maize under Zn deficient con-
dition (Mondal et al.,2014) and EcZIP2 might be
induced in flag leaf and developing spike of finger
millet by Zn deficiency. Therefore, further expression
and functional analysis are required to understand the
specific role of EcZIP2 transporter in finger millet.
The EcZIP3 and EcZIP4 are found in MC4 and MC5,
respectively, and are distantly clustered with monocot
ZIPs (Figure 8). OsZIP1 was expressed in root tissues
of rice and reported to be responsible for Zn uptake
from the soil (Ramesh et al.,2003). The phylogenetic
analysis revealed that EcZIP4 is distantly clustered
with the ZIP family proteins of cereals including
OsZIP1 of rice. This finding will serve as a basis to
understand the role of EcZIP4 in finger millet in the
future. This analysis gives information on the possible
functions of EcZIP genes and lays foundation for fur-
ther studies.
2. Functional residues in finger millet ZIP
transporter
No information is available till now on structural fea-
tures and mechanism of Zn uptake of plant ZIP trans-
porters. The high-resolution crystal structure of BbZIP
is available now and its functional residues are well
characterized (Zhang et al.,2017). More than eight
residues, such as Asn178, Pro180, Glu181, Gly182,
Gln207, Asp208, Pro210, Glu211, Gly212, and Glu240
are involved in Cd/Zn binding and transport in
BbZIP (Zhang et al.,2017). Five hydrophobic residues
(Met99, Ala102, Leu200, Ile204, and Met269) are
involved in the blocking of metals at the extracellular
surface in BbZIP. Three residues (Asp144, His275,
and Glu276) are involved in the metal release into the
cytoplasm (Zhang et al.,2017). Krishna et al. (2020b)
predicted the functional residues of plant ZIP proteins
using BbZIP. They have reported that only a few
functional residues are conserved with plant ZIPs
compared to BbZIP (Zhang et al.,2017). We have
aligned four finger millet ZIPs (EcZIP1-EcZIP4) with
other plant ZIP proteins (using the BbZIP as a tem-
plate (Supplemental Figure 8). The MSA alignment
shows that only a few finger millet amino acid resi-
dues are conserved with the BbZIP as in the previous
report (Krishna et al. 2020b). Most of the functional
residues are not conserved in the finger millet and
other plant ZIPs compared to that of BbZIP
(Supplemental Figure 8). Asp144 is involved in metal
release in BbZIP (Zhang et al.,2017) and it is con-
served in all EcZIPs of finger millet. Moreover, the
EcZIP3 and EcZIP4 protein sequences have conserved
His177 residue as in BbZIP; His177 is involved in the
metal release from the metal-binding site (Zhang
et al.,2017). It is replaced by Asp in EcZIP1 and by
Lys in EcZIP2 (Supplemental Figure 8). The His177
residue is conserved in ZIP protein sequences of rice
(expect OsZIP16), Arabidopsis (expect AtZIP10), and
maize (Krishna et al.,2020b). His177 is replaced by
CRITICAL REVIEWS IN PLANT SCIENCES 19
Gln in AtZIP10 (Gln222) and OsZIP13 (Gln140)
(Krishna et al.,2020b). It is interesting to note that
EcZIP3 and EcZIP4 protein sequences have conserved
Gly182 residue which is replaced by Asp in EcZIP1
and EcZIP2 (Supplemental Figure 8). Gly182 is
involved in the metal release from the metal-binding
site (Zhang et al.,2017). Other functional residues,
such as Leu200, Glu211, and Gly212 are essential for
metal-binding and these are conserved only in EcZIP3
(Zhang et al.,2017). Glu181 residue is conserved only
in EcZIP4. The Ile204 residue is conserved in EcZIP2
and EcZIP4. Notably, some of the key residues are
absent in the EcZIP. It may be due to the lack of
complete annotation of the genome sequence of fin-
ger millet.
To deduce further the functional residues involved
in Zn
2þ
binding and transport of finger millet ZIP
transporters, homology models were produced using
Figure 8. Phylogenetic analysis of finger millet zinc-regulated, iron-regulated transporter-like proteins (EcZIP1–4) family members.
The protein sequences finger millet ZIP family transporters were obtained from genome assembly of finger millet at NCBI website
(Accession ID: LXGH00000000) as per the details mentioned (Supplemental Figure 1). The protein sequences of rice, maize, wheat,
barley, sorghum, foxtail millet, and green foxtail and model plant Arabidopsis were collected from phytozome website (https://phy-
tozome-next.jgi.doe.gov/). The tree was constructed using 76 potential ZIP family transporter proteins from nine plant species includ-
ing four finger millet EcZIP proteins. The expression profile and functions of rice (Ramesh et al.,2003; Ishimaru et al.,2005; Chen
et al.,2008), maize (Li et al.,2013; Mondal et al.,2014), wheat (Evens et al.,2017; Deshpande et al.,2018), barley (Tiong et al.,2015),
sorghum, foxtail millet (Alagarasan et al.,2017), and green foxtail and model plant Arabidopsis (Grotz et al.,1998; Inaba et al.,2015;
Lilay et al., 2018) were inferred from respective previous reports and utilized for the comparison and analysis. Methods used were as
per the Figure 1 legend. The phylogenetic tree was divided into six MCs and which are highlighted in pink (MC1), gold (MC2), aqua
(MC3), blue (MC4), yellow (MC5), and green (MC6). The EcZIP family proteins of finger millet are indicated in red.
20 T. MAHARAJAN ET AL.
BbZIP as a template. In BbZIP, the Zn
2þ
binding is
coordinated by Glu181, Gln207, Glu187, Glu276, and
His177, based on the models created by us (Krishna
et al.,2020b). However, the finger millet ZIP proteins
showed greater variation for residues involved in
Zn
2þ
binding. Even variation was seen for Zn
2þ
bind-
ing residues among 4 EcZIP proteins (Supplemental
Figure 9). One of the Zn
2þ
binding residues, Glu, is
seen only in EcZIP3 (Glu149) and EcZIP4 (Glu142)
proteins. The Zn
2þ
binding residues of Glu were
found in ZIP family protein sequences of maize
(ZmZIP6 and 11), rice (OsZIP6 and 13), and
Arabidopsis (AtZIP1 and 2) (Krishna et al.,2020b).
His residue is also found only in EcZIP1 and EcZIP4
proteins. His is conserved in OsZIP6 and ZmZIP6;
shifting in the positioning of His is seen in AtZIP1,
AtZIP2, OsZIP13, and ZmZIP11 (Krishna et al.,
2020b). The other Zn
2þ
binding residues seem to be
entirely different from those seen in BbZIP. In
EcZIP1, the Zn
2þ
binding is coordinated by His119,
Lys96, Asn92, Arg93, and Ile33. Similar variation is
seen for EcZIP2 (Asp129, Arg35, Asp99, and Asn39),
EcZIP3 (Leu65, Glu149, Asp191, and Ala61), and
EcZIP4 (Glu142, Leu64, His165, and Leu60) transport-
ers. Further transport assay with mutagenesis of these
residues may shed more light on the transport func-
tion of EcZIP proteins.
V. Conclusion and future prospects
Finger millet is a valuable crop in the semi-arid
tropics due to its climate-resilient and nutrient-rich
properties. Although this crop was not given due
attention in the first green revolution and following
modern genetic studies, most of the recent studies
focused on crops like finger millet considering its cli-
mate-hardy behaviors. The genome sequence of two
finger millet genotypes was released recently which
provides an opportunity for modern genomic studies.
Nutrient transporters of finger millet were poorly
studied when compared to other plants despite being
a crop with dense mineral nutrients. The recently
released genome sequence provides an opportunity to
identify and characterize the genes and proteins
involved in nutrient transport. Many nutrient trans-
porters of other plants like rice, wheat, maize, and
barley were extensively studied including the transport
assay in yeast and xenopus. Such studies could be
undertaken with the mining of genes and proteins in
the genome sequence of finger millet. Many gene and
protein sequences of finger millet nutrient transport-
ers were identified and analyzed in this review which
may form the basis for further functional studies in
the future. Further, the modern genome editing tool
CRISPR/Cas could be utilized for the efficient charac-
terization of nutrient transporters with precise muta-
genesis studies which would help to study and
improve the nutrient use-efficiency in finger millet
and other millets. Overall, improving the nutrient use
efficiency of finger millet will help strengthen the
food and nutrient security amid changing climate.
Acknowledgments
We thank Rajagiri College of Social Sciences for all the sup-
port and help for the research.
Disclosure statement
No potential conflict of interest was reported by
the authors.
Funding
The research works in our lab is funded by the Department
of Biotechnology, Govt. of India under grant (No: BT/
PR21321/GET/119/76/2016).
Author contributions
SAC conceived and designed the experiments. TM and SAC
retrieved nucleotide sequences of various nutrient transport-
ers from the genome sequence of finger millet. TM, SAC,
and TPAK predicted and validated the protein sequences of
various nutrient transporters. TM, SAC, and TPAK con-
structed the phylogenetic tree of each nutrient transporter
and ClustalW alignment. SAC performed the homology
modeling. TM, SAC, and TPAK wrote the manuscript.
ORCID
Theivanayagam Maharajan http://orcid.org/0000-0002-
7343-2854
Stanislaus Antony Ceasar http://orcid.org/0000-0003-
4106-1531
Thumadath Palayullaparambil Ajeesh Krishna http://
orcid.org/0000-0002-7788-4436
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