Chiang Mai J. Sci.

2020; 47(6) : 1158-1171

http://epg.science.cmu.ac.th/ejournal/
Contributed Paper (Short Communication)

Mining Late Embryogenesis Abundant (LEA) Family Genes

in Amaranthus hypochondriacus
Phi Bang Cao
Faculty of Natural Sciences, Hung Vuong University, Vietnam.
*Author for correspondence; e-mail: phibang.cao@hvu.edu.vn
Received: 24 December 2019
Revised: 6 May 2020
Accepted: 15 July 2020
A BSTRACT
Late Embryogenesis Abundant (LEA) was first described to accumulate during plant seed
dehydration, absolutely at the late stages of embryogenesis. Since then they have also been found in
plant vegetative tissues under water limitation, thereby, associated with the acquisition of desiccation
tolerance. This work provides furnishes the first comprehensive study about the LEA gene family
in Amaranthus hypochondriacus, which is that the important grain crops and widely grown around the
world. A total of 46 genes encoding LEAs (AhLEAs) were identified and classified into eight groups
(LEA1, LEA2, LEA3, LEA4, LEA5, LEA6, DHN, and SMP) depending on basis of their predicted
amino acid sequences and further on their phylogenetic relationships with the Arabidopsis thaliana LEA
proteins (AtLEAs). A linking gene structure and motif architecture were observed within each LEA
group. Analysis of their chromosomal localizations revealed that the AhLEAs were non-randomly
distributed across all 16 chromosomes and that 43% of all AhLEAs are tandemly or whole-genome
duplicated genes. In silico expression analysis using RNA-seq data revealed that the AhLEA genes
were widely expressed in various vegetative and reproductive tissues especially including mature seeds.
The tissue-specific expression was observed for most of AhLEA genes. Additionally, most of AhLEA
genes were up-regulated by water deficit stress. These results bring about hand over a useful reference
to further investigate the AhLEAs functions and to apply on crop improvement.

Keywords: Amaranthus hypochondriacus, Late Embryogenesis Abundant (LEA), gene expression analysis,
gene ontology analysis, phylogeny analysis

1. I NTRODUCTION
Late embryogenesis abundant (LEA) protein
was first identified from cotton seeds in the late
stages of embryo development [1]. However,
these proteins are not restricted to seeds. Many
LEA proteins have been also found in vegetative
tissues[2] Moreover, LEA homologs have been
found in non-viridiplantae species such as bacteria
[3], mosses [4], fungi [5] and animals [6].
As a result of their particular amino acid
composition, most LEA proteins displayed a high
positive hydrophilicity and heat stabilization in
solution [7]. However, some of them can exhibit
particular three-dimensional structures under
desiccation or extreme temperature conditions
[8]. In plants, LEA proteins play an important
physiological role in development and stress
Chiang Mai J. Sci. 2020; 47(6) 1159
tolerance via many proposed mechanisms, such as
protection of proteins and organelles, interactions
with membranes, stabilization of sugar glasses,
hydration buffers, and ion sequestration [9]. Many
LEA proteins may function as chaperones and
protect enzyme activities under in vitro conditions
[10].
The LEA proteins were classified into different
groups according to various authors [11, 12]. Firstly,
Dure (1994) divided these proteins into 8 groups,
D-7, D-11, D-19, D-29, D-34, D-73, D-95, and
D-113, depending on their molecular weight [12].
Recently, the availability of many plant genomes
allowed annotating and classifying LEA superfamily.
In Arabidopsis thaliana, 51 LEA proteins were classified
into nine groups using the Pfam nomenclature [11].
Deduced amino acid sequences of LEA proteins
allowed the identification of eight groups using the
Pfam nomenclature in cotton (Sorghum bicolor) [2]
and upland cotton [13] and sweet orange (Citrus
sinensis) [8]. Indeed, 29 potato LEA proteins were
divided into nine distinct groups [14]. Surprisingly,
a total of 242, 136, and 142 LEA genes were
identified in Gossypium hirsutum, G. arboreum, and
G. raimondii respectively [13]. Most recently, 68
LEA genes were identified in Sorghum bicolor [2].
The expression of LEA genes was correlated
with abiotic stress tolerance in many plants [2,
14]. LEA gene expression can be involved in
ABA-dependent or ABA-independent signaling
pathways. Many LEA genes are expressed in
response to ABA, drought, and salt stress. ABA-
responsive element (ABRE) or/and dehydration
responsive element (DRE/CRT) were found in
their gene promoter regions [2, 8]. Overexpression
of a LEA gene confers tolerance to water deficit
in the transgenic plants including rice [15].
Amaranthus hypochondriacus belongs to the
Amaranthus genus which has gained much attention,
particularly for its high economic and nutritional
values [16]. Previous studies on A. cruentus have
displayed a novel late embryogenesis abundant
protein that belongs to the LEA5 group in seeds
[17]. The other LEA was identified in seed and
many plant tissues of different amaranth wild
and domesticated species. This protein could
play an important function during the seed drying
process. Experimental analysis indicated that
this LEA was an intrinsically disordered protein
involved in protection against desiccation, oxidant
conditions, and osmotic stress [17]. Recently,
differential accumulation of late embryogenesis
abundant as storage proteins in seeds of wild
and cultivated amaranth species was reported
[18]. However, there has been little knowledge
available about the LEA family in Amaranthus.
In this work, we first identified the LEA genes
in A. hypochondriacus genome and then conducted
detailed characterization and expression analyses
by using a bioinformatic approach.
2. M ATERIALS AND METHODS
2.1 Identification of LEA from A. hypochondriacus
Genomic Sequences and Construction of the
Phylogenetic Tree
BLAST searches and sequence analyses were
performed by BLASTp on the A. hypochondriacus
genome v2.1 (https://phytozome.jgi.doe.gov/pz/
portal.html#!info?alias=Org_Ahypochondriacus_er)
using known A. thaliana [11] as queries. The selected
Amaranthus genes were used for a second TBLASTN
round on the A. hypochondriacus genome; this additional
step allowed discovering A. hypochondriacus paralogs
that had been excluded by their dissimilarity to the
Arabidopsis orthologs.
All the identified A. hypochondriacus candidates
were analyzed using the Pfam server (https://
pfam.xfam.org/) [19] to confirm the presence
of  LEA conserved domains in their protein
structure. Proteins without any appropriate motif
were manually re-predicted and corrected by
Fgenesh software (http://linux1.softberry.com/
berry.phtml) using dicotyledon plant models.
Joint phylogenetic analysis with all Arabidopsis
LEAs allowed validating sequences without
LEA conserved domains. Proteins grouped with
Arabidopsis LEAs were considered to belong to the
LEA family. The multiple sequence alignment of
1160 Chiang Mai J. Sci. 2020; 47(6)
the A. hypochondriacus and Arabidopsis LEA proteins
was performed using MAFFT (http://mafft.cbrc.
jp/alignment/) [20] with default parameters and
Iterative refinement methods. The phylogenetic
tree was conducted using Mega X [21].
2.2. Sequence Analysis
MEME suite tool (http://meme.sdsc.edu/
meme) was used to analyze protein motif with
the following parameters: distribution of motifs:
Anny number of repetitions; maximum number of
motifs: 5; minimum number of sites: 2; maximum
number of sites: 5; minimum motif width: 6 and
maximum motif width: 50.
The chromosomal localization of AhLEA genes
was retrieved from phytozome (https://phytozome.
jgi.doe.gov/pz/portal.html#!info?alias=Org_
Ahypochondriacus_er).
The characteristics of AhLEA proteins
were calculated using the ProtParam online
tool (http://www.expasy.org/tools/protparam.
html) [22], including the number of amino acids,
molecular weight, theoretical isoelectric point (pI),
grand average of hydropathicity (GRAVY). The
prediction of the LEA subcellular location was
performed using the Yloc program (http://abi.inf.
uni-tuebingen.de/Services/YLoc/webloc.cgi) [23].
2.3. Gene Ontology (GO) and In Silico
Expression Analysis
Functional analysis of LEA proteins was taken
using Omicsbox program, version 1.1.164 [24] to
predict biological functions, cellular content, and
molecular functions. Amino acid sequences of
predicted LEA proteins were loaded to OmicsBox.
Functional analysis was done by Blast2GO program
with three steps (i) matching was conducted with
the loaded sequences in the program (BLASTp)
(ii) mapping associated with BLAST results was
completed (Mapping) (iii) dump file related to
sequences was created (Annotation).
The gene expression Fragments Per Kilobase
of transcript per Million fragments mapped (FPKM)
values were obtained from RNA-seq libraries,
including Green Cotyledon (SRX722062), Root tissue
(SRX722060), Leaf tissue (SRX722059), Stem tissue
(SRX722057), Floral tissue (SRX722058), Immature
seeds (SRX722056), Mature seeds (SRX722063),
Water stressed tissue sample (SRX722061) [25].
The results showed the log-transformed (base
10) values of FPKM counts.
3. R ESULTS
3.1. Identification of A. hypochondriacus
LEA Genes
The members of LEA gene family in
A. hypochondriacus genome were identified by a series
of basic local alignment search tool (BLAST) using
LEA proteins of Arabidopsis as query sequences
and then a second round of TBLASTN against
the A. hypochondriacus genome. Sequences that
excluded start or stop codons or exon sequences
in comparison to Arabidopsis orthologs were
re-predicted using the Fgenesh software (http://
linux1.softberry.com/berry.phtml).
In total, 46 full-length encoding putative
LEA proteins were selected (Table 1). Domain
analysis using Pfam allowed to valid 42 of the 46
candidate genes. The remaining four genes which
did not contain LEA-conserved domains were
further validated by joint phylogenetic analysis
with all Arabidopsis orthologs (Figure 1).
3.2. Phylogenetic Analysis and Classification
of AhLEAs
The phylogenetic tree was constructed
with LEA sequences from A. hypochondriacus
and A. thaliana by Mega X [21] software using
the Maximum Likelihood methods with 1,000
bootstrap replicates (Figure 1). Phylogenetic analysis
revealed that AhLEA proteins can be divided into
eight major groups (Figure 1): LEA1-6, SMP, and
DHN corresponding to the Pfam nomenclature.
With 14 members, the LEA4 group was the
largest. Next, LEA2 had 11 members. SMP group
was made up of seven genes. DHN contained
five members. LEA1 and LEA3 comprised three
genes in each group and LEA6 contained only one
Chiang Mai J. Sci. 2020; 47(6) 1161

Table 1. Identification and characteristics of the LEA genes in A. hypochondriacus.

Gene name Subgroup Locus ID PL (aa) MW (kDa) pI GRAVY IN SCL
AhDHN-1 DHN Ah001781 208 21.96 5.89 -0.96 1 Cyt
AhDHN-2 DHN Ah002326 174 19.58 5.84 -1.51 1 N
AhDHN-3 DHN Ah003804 265 27.67 6.69 -1.05 1 Cyt
AhDHN-4 DHN Ah005786 147 16.01 5.96 -1.17 2 Cyt
AhDHN-5 DHN Ah023683 225 25.48 5.62 -1.50 3 M
AhLEA1-1 LEA1 Ah009310 80 9.14 9.13 -1.16 1 N
AhLEA1-2 LEA1 Ah011491 159 16.86 9.05 -0.86 1 Cyt
AhLEA1-3 LEA1 Ah020326 83 9.06 8.93 -1.31 1 N
AhLEA2-1 LEA2 Ah002467 231 25.02 9.10 0.23 1 Cyt
AhLEA2-2 LEA2 Ah007830 258 28.80 10.04 -0.20 1 N
AhLEA2-3 LEA2 Ah009482 325 35.75 9.24 -0.53 0 Cyt
AhLEA2-4 LEA2 Ah009631 159 17.62 4.91 -0.23 0 Cyt
AhLEA2-5 LEA2 Ah010338 215 23.26 9.75 0.20 0 N
AhLEA2-6 LEA2 Ah011754 317 35.04 4.87 -0.33 0 Cyt
AhLEA2-7 LEA2 Ah013240 186 20.55 6.19 0.03 1 M
AhLEA2-8 LEA2 Ah016081 276 30.06 9.88 -0.23 1 M
AhLEA2-9 LEA2 Ah021219 229 26.09 9.51 -0.22 1 N
AhLEA2-10 LEA2 Ah021764 208 23.34 9.40 0.11 0 N
AhLEA2-11 LEA2 Ah022417 169 18.51 9.80 -0.13 1 Cyt
AhLEA3-1 LEA3 Ah012945 96 10.49 10.09 -0.27 0 M
AhLEA3-2 LEA3 Ah013427 122 14.11 9.08 -0.91 2 Cyt
AhLEA3-3 LEA3 Ah022093 88 9.77 5.43 -0.53 0 Cyt
AhLEA4-1 LEA4 Ah000279 648 68.44 9.00 -0.88 0 N
AhLEA4-2 LEA4 Ah002005  149 15.89 8.92 -1.20 1 Cyt
AhLEA4-3 LEA4 Ah012685 295 32.36 6.19 -1.07 2 Cyt
AhLEA4-4 LEA4 Ah012686 231 25.59 8.55 -1.15 1 Cyt
AhLEA4-5 LEA4 Ah015080  390 41.85 6.22 -0.90 0 Chp
AhLEA4-6 LEA4 Ah015243  175 19.87 9.44 -1.30 1 M
AhLEA4-7 LEA4 Ah016434 67 6.92 5.15 -0.80 1 ER
AhLEA4-8 LEA4 Ah016488 65 6.98 4.96 -1.10 3 M
AhLEA4-9 LEA4 Ah016489 72 7.52 4.76 -0.89  2  N
AhLEA4-10 LEA4 Ah021631  439 47.02 6.11 -0.99 1 N
AhLEA4-11 LEA4 Ah021661  283 31.92 6.70 -1.24 2 N
AhLEA4-12 LEA4 Ah021865  420 45.88 6.23 -1.18 1 N
AhLEA4-13 LEA4 Ah022926 70 7.48 4.81 -1.19 1 N
PL: Protein full length, MW: Molecular weight, IN: Introns number, SCL: Sub cellular Location, Cyt: Cytoplasm, Chp:
Chloroplast, ER: Endoplasmic reticulum M: Mitochondrion, N: Nucleus.
1162 Chiang Mai J. Sci. 2020; 47(6)

Table 1. (Continued)
Gene name Subgroup Locus ID PL (aa) MW (kDa) pI GRAVY IN SCL
AhLEA4-14 LEA4 Ah022928 65 6.80 5.22 -1.02 1 N
AhLEA5-1 LEA5 Ah011466 81 8.53 5.85 -1.40 0 Cyt
AhLEA5-2 LEA5 Ah018817 86 9.67 5.88 -1.59 2 N
AhLEA6-1 LEA6 Ah006187 86 9.29 5.49 -1.37 1 Cyt
AhSMP-1 SMP Ah002544  144 14.85 4.44 -0.19 1 Cyt
AhSMP-2 SMP Ah007095 239 24.53 5.03 -0.23 0 N
AhSMP-3 SMP Ah010846  188 20.11 9.01 -0.53 0 Cyt
AhSMP-4 SMP Ah017845 272 28.66 4.98 -0.40 2 N
AhSMP-5 SMP Ah017846 240 25.12 4.70 -0.48 2 N
AhSMP-6 SMP Ah017848 105 10.69 4.31 -0.11 1 N
AhSMP-7 SMP Ah017849  243 25.38 4.36 -0.22 1 Cyt
PL: Protein full length, MW: Molecular weight, IN: Introns number, SCL: Sub cellular Location, Cyt: Cytoplasm, Chp:
Chloroplast, ER: Endoplasmic reticulum M: Mitochondrion, N: Nucleus.

Figure 1. Phylogenetic tree of the LEA family from A. hypochondriacus (Ah) and A. thaliana (At). The
tree was generated using Mega X program by Neighbor-Joining method. Bootstrap values are indicated
at each branch.
Chiang Mai J. Sci. 2020; 47(6) 1163
member. The species-specific Arabidopsis group
(AtM) of the LEA gene family was absent in the
A. hypochondriacus genome.
3.3. Characteristics of Predicted LEA Genes
in A. hypochondriacus
In silico analysis allowed determining the
characteristics of predicted LEA genes in
A. hypochondriacus. The AhLEA genes included a
few introns in which only two of them contained
three introns, seven had two introns while most
(24/46) of them consisted only one intron and
12 had no intron. Most AhLEA deduced proteins
(37/46) were smaller than 30 kDa (Table 1).
Eleven of them were very small LEA proteins
(<10 kDa). The AhLEA4-1 was the unique
protein that showed high molecular weights
(~68 kDa). The theoretical pI value of AhLEA
proteins ranged from 4.31 to 10.09. Most of
AhLEA (42/46) were hydrophilic. The GRAVY
scores of all A. hypochondriacus LEA proteins
were negative, except for four AhLEA2 proteins
named AhLEA2-1, AhLEA2-5, AhLEA2-7, and
AhLEA2-10 (Table 1).
LEA1 group
The LEA1 group of A. hypochondriacus was
composed of three genes, AhLEA1-1, AhLEA1-2,
and AhLEA1-3. Each of them exhibited one
intron. The molecular weights of these proteins
were 9.14, 16.86, and 9.06 kDa, respectively. The
theoretical pI values of these proteins were 9.13,
9.05, and 8.93, respectively, showing that they
were alkaline. All three AhLEA1 proteins were
hydrophilic with GRAVY values ranging from
-1.31 to -0.86. AhLEA1-1 and AhLEA1-3 were
predicted to be located in the nucleus whereas
AhLEA1-2 was in the cytoplasm (Table 1). MEME
analysis could detect only one consensus motif
containing 13 amino acids (MQ[AS][AV]K[DQ]
K[IV][KS][DE][IM][AS]S) in three AhLEA1
proteins. Additionally, three other motifs, 2, 3,
and 4, were only presented in two out of three
LEA1 members (Table 2).
LEA2 group
The LEA2 group contained 11 genes (AhLEA2-1
to AhLEA2-11), indicating that this group was the
second largest group in A. hypochondriacus LEA family.
Six of them (AhLEA2-1, AhLEA2-2, AhLEA2-7,
AhLEA2-8, AhLEA2-9, and AhLEA2-11) had
only one intron while five others were intronless.
Their deduced full-length protein sequences ranged
from 159 to 325 amino acids while the molecular
weights of these proteins varied from 17.62 to
35.75 kDa. The theoretical pI values of the eleven
AhLEA2 proteins fluctuated from 4.78 to 10.04,
with three acidic and eight alkaline proteins. Four
proteins, AhLEA2-1, AhLEA2-5, AhLEA2-7,
and AhLEA2-10, were hydrophobic with positive
GRAVY values. In contrast, the GRAVY values
of seven other proteins were negative, indicating
that they were hydrophilic. Subcellular localization
prediction of AhLEA2 proteins showed that they
were in ambiguous localized subcellular, four in
the nucleus, four in the cytoplasm, and two in the
mitochondrion (Table 1). MEME analysis revealed
that the two most common motifs (motif 5 and 6)
were displayed in nine out of 11 AhLEA2s except
for AhLEA2-4 and AhLEA2-6. Besides, motif 7
was exhibited in seven out of 11 AhLEA2s. Four
genes, AhLEA2-2, AhLEA2-5, AhLEA2-8 and
AhLEA2-9, shared consensus motif [NK]R[NS]
C[CS]CR[AKV][CIL]CC in N-terminal (Table 2).
LEA3 group
The LEA3 group of A. hypochondriacus was
made up of three members. The AhLEA3-2
gene exhibited two introns whereas AhLEA3-1
and AhLEA3-3 lacked introns. The theoretical pI
values of AhLEA3-1 and AhLEA3-2 were 10.09
and 9.08, which supposed that they were alkaline.
However, the AhLEA3-3 protein was acidic (pI
5.43). The GRAVY values of all AhLEA3 proteins
were negative (ranging from -0.91 to -0.27),
indicating that they were hydrophilic. Subcellular
localization prediction showed that AhLEA3-1
was in the mitochondrion, whereas, AhLEA3-2
and AhLEA3-3 were presented in the cytoplasm
(Table 1). Motif 9 was discovered in all three
1164 Chiang Mai J. Sci. 2020; 47(6)

Table 2. Conserved motifs in different groups of A. hypochondriacus LEA proteins.

Group Motif Sites E-value Consensus sequence
1 3 3.50E+02 MQ[AS][AV]K[DQ]K[IV][KS][DE][IM][AS]S
2 2 9.90E-01 [NY][ET]TGY[PR]A[AP][GH][NY][IN]
LEA1
3 2 5.00E+01 IAKEVR
4 2 7.70E+01 [GQ]H[DL]Q[HV]D
5 9 1.60E-10 [FP][PV]SL[DS][AS]X[FL]QLT[IV][TH][AV][RK]NPN[KI][KV]I[GI]IYY
6 9 1.10E-05 [LS][AIL]L[LIV][LGV][LAV][IT][GAL][AIL][AT][IAT][LT][IV][FIL][FLWY]
L[LVA][FAY][KRH]P[KRS][KV]P[HKT][FY][ST][LV][DIQ]S
LEA2
7 7 3.00E-13 [LMF][ST][IVA][YMW]Y[KD][GDN][IST][PLQR]L[GC][QSR][GA]
[HRSVY][LVF][PE][ADGK][FG][YS]Q[PG][APH][RHLN][SN][CTV]
8 4 5.60E-10 [NK]R[NS]C[CS]CR[AKV][CIL]CC
9 3 2.90E-15 [SF]W[IM][PK]DP[VE]TG[YN][YW][RI]P[AE][NT][RH][AF][AG]
E[IV]D[VI][AV]ELR[ER][IKM][LF][LI]

LEA3 10 3 5.90E+00 [FL]A[AM]ASQ

11 3 2.70E+01 CN[RW][IQ][DT][FY]
12 2 1.70E+02 [QR]KIRSQ
13 32 7.70E-98 [DN]KA[KS][EQ][AI]KDX[TA][VM]EK[AT]GEAKD[YK][TVA]
LEA4 14 31 9.90E-37 E[AK]ASK[MA]KEKA[KS][DG][AT]A[ED]S[VA]K[ED][AS]T
15 7 3.70E-07 [ECQ]N[AY]S[YI][QA][AS]G[QG][AGV][KR][AG][TAE]T
16 3 5.00E-01 QTR[KR]EQ[LM]G[TH]EGY[QK][EQ][LM]
17 2 4.40E+01 MA[ST][GR]Q[EQ][NS][KR][EK]ELD
LEA5 18 2 3.90E+01 [AI][EK][IQ][DG]E[ST][KV][FV][PR]
19 2 1.10E+03 G[LR][ST]T[GM]D
20 2 1.10E+03 [DL]EA[GQ][AQ][EH][DL][AV]
LEA6 21 1 1.20E-04 T[DE]APT
22 9 3.70E-15 [HKQ][ED]KKG[IF][ML][DE]KIK[ED]K[LI]PG[TFG][HG]

DHN 23 5 9.70E-10 [LI][HQ][RH][SDT][GNS][SY]S[ST]S[SG]S[SDK]E

[LH][RK][DE][EDKS][YEHL][GAE][AEKNQ][PAK][VL][HRE]
24 6 3.00E-05
[QEHL][TDN][DH][EDH][FLNTY][GN][NRV][PH][VHI][QEK]
25 17 1.70E-78 [LV][EF]A[VT]AKLA[GA]DK[PA][VI][TD][PR][SR]DA[AE]A[IVM]
QAAEMR[AN]T[GP]
26 16 1.40E-23 PGG[VLP][AG][AES]X[AM]Q[AS]AA[TR]IN
SMP 27 7 6.30E-01 VTIGEA
28 4 2.10E-06 [HR][LR][MI]YD[EQ]DKT[KT]I[SA]EI[LI]
29 4 3.00E-04 [PA]IKYGD
Chiang Mai J. Sci. 2020; 47(6) 1165
AhLEA3 proteins by MEME tool. In addition,
three motifs 10, 11, and 12 were found in two out
of three AhLEA3 members (Table 2). Variation
in motifs in this group suggested functional
divergence.
LEA4 group
The LEA4 was the largest LEA group of
A. hypochondriacus with the presence of 14 members.
Most of them (12/14) exhibited one to three
introns. However, AhLEA4-1 and AhLEA4-5
lacked intron. Their protein lengths varied from
65 to 648 amino acids corresponding to molecular
weights ranging from 6.80 to 47.02 kDa. The
theoretical pI values of the LEA4 proteins were
variable, from 4.76 to 9.44 with four alkaline and
ten acidic proteins. All AhLEA4 proteins were
hydrophilic due to negative GRAVY scores,
ranging between -1.24 and -0.80 (Table 1). The
results of subcellular prediction revealed that
the AhLEA4 group was widely distributed in
most of the subcellular compartments, including
endoplasmic reticulum (AhLEA4-7), chloroplast
(AhLEA4-5), mitochondrion (AhLEA4-6 and
AhLEA4-8), cytoplasm (AhLEA4-2, AhLEA4-3,
and AhLEA4-4) and nucleus (seven remaining
members) (Table 1). MEME analysis showed
three most common motifs in this group including
motif 14, E[AK]ASK[MA]KEKA[KS][DG][AT]
A[ED]S[VA]K[ED][AS]T, which was shared by
all AhLEA4 proteins and repeated several times
in some genes. Next, motif 13 was found in eight
members with several repetitions in some genes.
Motif 15 was exhibited in the N-terminal of seven
AhLEA4 proteins (Table 2).
LEA5 group
The LEA5 group of A. hypochondriacus contained
two members, AhLEA5-1 and AhLEA5-2,
respectively. The former lacked intron whereas
the latter exhibited two introns. The protein
sequences were from 81 to 86 amino acids in length
corresponding to molecular weights ranging from
8.53 to 9.67 kDa. These two proteins were acidic
since their theoretical pI values ranged from 5.85
to 5.88. Both AhLEA5 proteins were hydrophilic
due to their negative GRAVY scores, ranging from
-1.59 to -1.40. The results of subcellular prediction
showed that the AhLEA5-1 was in the cytoplasm
whereas AhLEA5-2 was in the nucleus (Table 1).
MEME analysis indicated that two AhLEA5
proteins shared four common motifs, 16, 17, 18,
and 19. In which, motif 16 was repeated twice in
AhLEA5-2. Additionally, motif 20 was repeated
twice only in AhLEA5-1 (Table 2).
LEA6 group
The LEA6 group of A. hypochondriacus contained
only one gene, AhLEA6-1, which exhibited one
intron. The protein sequence possessed 86 amino
acids corresponding to their molecular weight
of 9.29 kDa. The theoretical pI value was 5.49.
AhLEA6-1 protein was hydrophilic due to its
negative GRAVY score (-1.37). AhLEA6-1 was
predicted to locate in cytoplasmic (Table 1). No
motif was detected in LEA6 by using MEME
analysis since this group contained the unique gene.
Dehydrin (DHN) group
DHN was the fourth largest group in the
A. hypochondriacus LEA gene family with five genes,
AhDHN-1 to AhDHN-5, respectively. Introns were
found in each of them. The three first contained
only one intron whereas the AhDHN-4 exhibited
two introns and three introns were observed in the
AhDHN-5. Their protein lengths ranged from 147
to 265 amino acids corresponding to molecular
weights ranging from 16.01 to 27.67 kDa. Their
theoretical pI values ranged from 5.62 to 6.69,
suggesting that these DHNs were weak acidic.
All of five AhDHN proteins were hydrophilic
due to their negative GRAVY values, ranging
from -1.51 to -0.96. The results of subcellular
prediction showed that AhDHN-1, AhDHN-3,
and AhDHN-4 were in the cytoplasm whereas
AhDHN2 in the nucleus and AhDHN5 in the
mitochondrion (Table 1). By using MEME, two
motifs, 22 and 23, were discovered in all five
members of the DHN group. In detail, Motif 22
([ER][KEH][EK]KKG[FLI][LM][DE]KIK[ED]
KLPG[GT][HGK][HK]) was repeated two or
three times. Additionally, motif 24 ([ADN][FHR]
1166 Chiang Mai J. Sci. 2020; 47(6)
LRD[ES][YH]G[KNQ]PV[RE][QL]TDE[FLY]
G[NR]PV[QK][HL][TK][GD][TE][MH]G[AQ]
[YP]) was detected in only three genes, AhDHN-1,
AhDHN-3 and AhDHN-4 respectively.
Seed Maturation Protein (SMP) group
SMP was the third-largest group within the
A. hypochondriacus LEA gene family. This group
contained seven genes. Within them, two genes,
AhSMP-2 and AhSMP-3 lacked intron while the
AhSMP-1, AhSMP-6, and AhSMP-7 exhibited one
intron and therefore two other, AhSMP-4 and
AhSMP-5, contained two introns. The polypeptide
full length of those SMPs ranged from 105 to 272
amino acids like their molecular weights starting
from 10.69 to 28.66 kDa. The pI value of AhSMP-3
was 9.01, suggesting that it was alkaline. The six
other proteins were acidic with pI values ranging
from 4.31 to 5.03. The negative GRAVY values
indicated that each one of seven AhSMPs were
hydrophilic. Three members, AhSMP-1, AhSMP-3,
and AhSMP-7, located in cytoplasmic and four
others were in the nucleus. (Table 1). MEME
analysis revealed three common motifs, 25, 26,
and 27, which were shared by all seven AhSMP
genes. Motif 25 and 26 were repeated twice or
thrice in six out of seven SMP members, apart
from AhSMP-6. Additionally, two other motifs,
28 and 29, were discovered in four out of seven
AhSMPs (Table 2).
3.3. Gene Ontology (GO) Analysis
Gene ontology (GO) analysis of AhLEA
genes was carried out by using OmicsBox
v1.1.164 (Figure 2). The biological processes,
molecular functions, and cellular components of
A. hypochondriacus LEA genes were investigated
by GO database. The results indicated that 15 out
of 46 AhLEAs were putatively implicated in a
variety (during a short) of biological processes.
Four AhLEA genes (AhLEA2-4, AhLEA2-6,
AhLEA2-7, AhLEA2-11) were predicted to
function in the response to desiccation, followed
by response to water deprivation and response to
abscisic acid (three genes each). Cold acclimation,
protein stabilization, embryo development ending
in seed dormancy, and response to water were
associated with two genes each. Prediction of
molecular function showed that AhDHN-3 was
involved in protein-containing complex binding,
AhDHN-4 was linked to binding and AhLEA2-1
was associated with translation initiation factor
activity. Eighteen out of 46 AhLEA genes
were putatively linked to cellular components.
Prediction analysis showed that nine AhLEAs
involved in integral component of membrane,
seven related in the cytosol, and two linked to the
mitochondrion. Three other cellular components
including membrane, plasma membrane, nucleus,
and chromosome were be involved by one gene
each.
In all the LEA groups of A. hypochondriacus,
molecular functions, biological process, and cellular
components were recorded except in LEA1 in
which only two GO functions, biological process,
and cellular components were observed.
3.4. Expression Profiling of LEA Genes
The expression profile of the 46 AhLEA genes
was analyzed via the bioinformatic analyses from
an open data bank archive (SRA, Sequence Read
Archive) libraries (Figure 3). Expression analysis
was conducted on different tissues including
green cotyledons (GC), roots (RT), leaves (LT),
stems (ST), floral tissues (FT), immatured seeds
(IMS), matured seeds (MS) and water-stressed
tissue sample (mixed- root, stem, and leaf) [25].
Heat map indicated that all of the AhLEA genes
were expressed at least in green cotyledons, floral
tissues, and seeds (Figure 3). All AhDHNs were
expressed in all analyzed tissues. The AhDHN-1
and AhDHN-3 were strongest expressed in the
green cotyledon. These two genes were also
highly expressed in floral tissues and mature
seeds. Especially, AhDHN-5 had high expression
levels in all analyzed tissues. All DHN genes were
higher expressed in a water-stressed tissue sample
(mixed- root, stem, and leaf) than in separated
tissues under normal conditions.
Chiang Mai J. Sci. 2020; 47(6) 1167

Figure 2. GO analysis of LEA genes in A. hypochondriacus. (A) Biological process (BP) results of GO
functional enrichment analysis, (B) Molecular function (MF) results of GO functional enrichment
analysis, (C) Cellular component results of GO functional enrichment analysis.

LEA2 and LEA6 were two groups containing
similar expression profiles as DHN group. Expression
of all LEA2 and LEA6 genes was observed in
all tissues with various levels. Two out of three
AhLEA1 genes, AhLEA1-2 and AhLEA1-3,
were expressed in all eight tissues whereas
expression of AhLEA1-1 was not determined in
leaves. Similarly, expression of two out of three
AhLEA3 genes, AhLEA3-1 and AhLEA3-3,
was found in all tissues whereas the transcript of
AhLEA3-2 was not detected in the stem. Two
LEA5 genes were exhibited expression in most
1168 Chiang Mai J. Sci. 2020; 47(6)

Figure 3. Heatmap showing expression level of AhLEAs in eight tissues. Colour scale represents RPKM
normalized log10 transformed counts. The green scale indicates low expression and red indicates high expres-
sion. GC: Green Cotyledon (no perisperm, no seed coat), RT: Root tissue, LT: Leaf tissue, ST: Stem tissue,
FT: Floral tissue (tepals from both male and female flower), IMS: Immature seeds, MS: Mature seeds, WS:
Water stressed tissue sample (mixed- root, stem, and leaf).

of the analyzed tissues, except an expression of green cotyledons in comparison to vegetative

AhLEA5-2 was not found in the stem. Seven out
of 14 LEA4 genes and three out of seven SMP
genes displayed expression in all tissues. However,
other members of these two groups were not
discovered in one or two vegetative tissues. Higher
expression levels in the floral tissues, seeds and
tissues were observed for 46 LEA genes of A.
hypochondriacus Except for four LEA4 (AhLEA4-8,
AhLEA4-9, AhLEA4-13, and AhLEA4-14) and
one SMP (AhSMP-3) genes, most of AhLEAs
expressed stronger in water stress tissue sample
than in separated vegetative tissues.
Chiang Mai J. Sci. 2020; 47(6) 1169
4. D ISCUSSION
Genome identification of LEA superfamily
proteins was done for many species such as
A. thaliana [11], potato [14], and upland cottons
(e.g. Gossypium hirsutum, G. arboreum and G. raimondii)
[13]. A. hypochondriacus is a C4 dicotyledonous
seed-producing crop. Investigation of proteins
encoded by LEA family genes in this C4 plant
should benefit crop improvement. According to
literature, the gene number of LEA family of A.
hypochondriacus was higher than the numbers of
LEA genes previously reported in the genomes of
potato (29 genes) [14] but smaller than in A. thaliana
(51 genes) [11], and upland cottons (with 242, 136
and 142 in Gossypium hirsutum, G. arboreum and
G. raimondii, respectively) [13]. It is confusing
to note that the copy-number of LEA genes is
abundant and variable between different taxa. The
abundance perhaps suggests their conservative role
under abiotic stress conditions as well as during
growth and development. Most of AhLEA shared
common characteristics including small molecular
weights, richness in hydrophilic amino acids, and
containing few introns [13]. Indeed, only two LEA
genes, AhDHN5 and AhLEA4-8, contained three
introns. Remaining AhLEA genes (96%) exhibited
one, two, or no intron. It has been indicated that
genes involving in the stress response usually
contain few introns [26]. A proposed hypothesis
to explicate such observation is that introns may
have a hurtful effect on gene expression since
they can delay transcript production, likewise the
further energetic cost caused by the increase of
transcript length [26]. The variation in biochemical
characteristics among the 46 AhLEA proteins
suggested functional divergence.
Phylogenetic analysis categorized the AhLEA
proteins into eight different groups, e.g., LEA_1
to LEA_6, SMP, and DHN, respectively. Theses
eight majors groups of LEA were reported in
many plants, such as cotton and upland cotton
[2, 13], sweet orange [8]. Differently, there were
nine groups in Arabidopsis [11], and potato [14].
Phylogenetic analysis further allowed detecting
duplication events. A tandem duplication event
was detected in chromosome 11, originating four
SMP genes. Five whole-genome duplication events
allowed explicating the expansion of LEA4 group
in A. hypochondriacus genome. In addition, two
paralogs, AhDHN-3 and AhDHN-4, were duplicates
made up of whole-genome duplication. Similarly,
AhLEA3-1 and AhLEA3-3 were whole-genome
duplicated paralogs, too. Segmental duplication,
tandem duplication, and transposition events were
the main drivers of LEA gene family expansion
which were observed in the genome of sweet
orange [8], cotton and upland cotton [2, 13].
Motif analysis of the A. hypochondriacus
LEA proteins by using MEME tool showed that
members of each LEA group possessed several
group-specific conserved motifs (Table 2), except
for LEA6. Similar characteristics have been
reported for LEA proteins in Potato [14], and sweet
orange [8]. For example, MEME analysis revealed
three motifs (motifs 22–24) in DHN group. All
AhDHN shared K-segment and the S-segment,
two known typical motif of DHNs. Three proteins,
AhDHN-1, AhDHN-3, and AhDHN-4, contained
two Y-segments whereas two others lacked this
type of motif. (Figure 3). Conserved motifs in the
DHN group were the 11-amino acid repeated motif,
EKKGIMDKIKEKLPG (K-segment, richness
in lysine residues). In this study, consensus motif
[ER][KH][EK]KKG[FLI][LM][DE]KIK[ED]
KLPG[GT][HK][HK] was identified among the
DHN group. Additionally, the commonly known
motif [LI][HQ][RH][SDT][GNS][SY]S[ST]S[SG]
(S-segment) was also observed. The Y-segment
was not detected by MEME analysis. Instead,
motif 24 ([ADN][FHR]LRD[ES][YH]G[KNQ]
PV[RE][QL]TDE[FLY]G[NR]PV[QK][HL][TK]
[GD][TE][MH]G[AQ][YP]) was discovered in this
study. It can be found that this motif includes
Y-segment with two repetitions. K-segment was
rich in the DHN group, indicating that the LEA
proteins evolved from the gene expansion within
their specific gene groups. In addition, LEA4
genes were found to possess a repetition of
1170 Chiang Mai J. Sci. 2020; 47(6)

conserved motif 14. In some proteins, the time deficit. This gene is orthologs of At1G76180,
of repetition was seven. The same characteristic ERD14 gene, which was induced early on in
was also noted in harboring repeat motifs, and response to dehydration stress [28].
so was in motif 14 [8].
5. Conclusions
Expression analysis supports new insights 5. C ONCLUSIONS
into the function of AhLEA genes. RNA-seq data In this work, the whole repertoire of LEA
In this work, the whole repertoire of LEA encoding genes in the A. hypochondriacus
from the databases show different expressions of encoding genes in the A. hypochondriacus genome
AhLEA genes in different tissues. This indicates has been identified and characterized for the first
genome has been identified and characterized for the first time. The results indicated that LEA
that they have a different role during growth and time. The results indicated that LEA constitutes
development
constitutes [2]. Generally,
a multigene all AhLEA
family includinggenes a multigene
eight groups family including
in A. hypochondriacus, eight groups
displaying a in
exhibit higher expression levels in mature seeds A. hypochondriacus, displaying a diversity of
anddiversity
cotyledons than in other
of sequences, tissues. Thegene
motif composition, highstructure,
sequences, motif and
gene ontology, composition, gene structure,
expression patterns.
expression level of AhLEA genes in cotyledons, gene ontology, and expression patterns. It appears
lateItstage
appearsof that
embryogenesis.
segmental and Gene expression
whole-genome thatplays
duplication segmental and whole-genome
an important role in the expansionduplication
in the mature seed is similar to LEA of Pisum plays an important role in the expansion of some
of some
sativum, groups. Further,
supporting the role the diversified
of genes duringandloss
tissue-specific
groups.expression
Further, the profiles provide
diversified andfurther
tissue-specific
and re-establishment of desiccation tolerance expression profiles provide further insight into
[27].insight into theexpression
Abundant possible functional
of LEA1, divergence
LEA4,in the the
AhLEA gene functional
possible family. Thedivergence
future attempts
in thetoAhLEA
LEA5, SMP, and DHN groups was observed in gene family. The future attempts to clear up their
clear up their functional role in A. hypochondriacus
flower tissue, suggesting their roles in reproductive should greatlyrole
functional gain
in from this comprehensive
A. hypochondriacus should greatly
development [27]. The majority of the AhLEAs gain from this comprehensive analysis.
analysis.
were expressed in leaf tissues, consistent with
the observations in Sorghum bicolor L. [2] and in A CKNOWLEDGEMENT 
Acknowledgement
sweet orange [8]. The paralogous genes of LEA4 The author is grateful to Msc. Ngô Thi Thanh
group exhibited
The author similar expression
is grateful to Msc.features in
Ngô Thị Thanh Huy ề nn for
Huy for checking
checking the theEnglish
Englishlanguage
language in the
different tissues, indicating distinct divergence manuscript.
andinevolution
the manuscript.
of duplicated genes for different
functions during plant growth and development. R EFERENCES
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