Plant Gene 5 (2016) 109117

Contents lists available at ScienceDirect

Plant Gene

journal homepage: www.elsevier.com/locate/plantgene

Molecular cloning and characterization of a novel basic helixloop

helix-144 (bHLH144)-like transcription factor from Morus alba (L.)
R.S. Sajeevan, Karaba N. Nataraja
Department of Crop Physiology, University of Agricultural Sciences (UAS), GKVK, Bangalore, Karnataka 560 065, India

a r t i c l e i n f o a b s t r a c t

Article history: Basic helixloophelix (bHLH) transcription factors (TFs) are associated with many biological processes, including
Received 28 June 2015 abiotic stress response in plants. With an objective to prospect novel drought stress responsive bHLH gene/s from
Received in revised form 12 December 2015 mulberry (Morus alba L.), a woody perennial tree, we isolated 1512 bp of bHLH-like gene, and its transcript variant
Accepted 29 January 2016
of 1419 bp by PCR-based approach using cDNA generated from leaf tissue. Since, the cloned sequences showed iden-
Available online 3 February 2016
tity with Prunus mume bHLH144-like transcript, as per the BLASTn analysis, the variants are designated as
Keywords:
MabHLH144a-like and MabHLH144b-like genes. The full-length MabHLH144a- and b-like cDNAs have 726 bp open
bHLH144 reading frames and the gene is predicted to encode a protein of 241 amino acids. The genomic clone of
Morus alba MabHLH144-like has 2304 bp consisting of a single 786 bp intron at the 5 UTR region. Fold-Index analysis predicted
miRNA that MabHLH144-like is highly disordered protein, a characteristic feature of TFs, and the expression analysis indicat-
Abiotic stress ed that the transcript variants are differentially expressed in diverse tissue types and also under different abiotic
stresses. Analysis of the possible miRNA binding sites revealed the absence of a target site in one the transcript var-
iants, which would have contributed for the observed differential expression. As per our knowledge, this is the rst
report on cloning, and characterization of bHLH144-like gene and its transcript variant from mulberry.
2016 The Authors. Published by Elsevier Inc. All rights reserved. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction 1018 amino acids, located at the N-terminus, has the ability to bind
to DNA (Atchley et al., 1999; Toledo-Ortiz et al., 2003; Li et al., 2006).
Basic helixloophelix (bHLH) proteins are the second largest family The specic amino acids at the basic region of the bHLH protein are es-
of transcription factors (TFs) in plant genomes that are present in all sential to recognize the E-Box (5-CANNTG-3) and/or G-Box (5-CACG
eukaryotic organisms (Castilhos et al., 2014; Jin et al., 2014). These pro- TG-3) cis elements in the promoter region of target genes, which
teins are characterized by the presence of a typical 5060 amino acid were identied in a large group of plant genes (Carretero-Paulet et al.,
bHLH domain, with two functionally distinctive segments, the basic 2010). The HLH region contains two alpha helices separated by a loop
and helixloophelix (HLH) regions. The basic region, which is about of variable length that functions as a dimerization domain (Toledo-
Ortiz et al., 2003).
The bHLH genes are identied as regulators of various plant physio-
logical and developmental processes. These proteins have been shown
Abbreviations: bHLH, Basic helixloophelix; TF, Transcription factor; NCBI, National to be involved in chloroplast development (Monte et al., 2004); devel-
Center for Biotechnology Information; cDNA, DNA complementary to RNA; RNA,
Ribonucleic acid; RACE, Rapid amplication of cDNA ends; Ma, Morus alba; gDNA,
opment of trichome or root hairs (Karas et al., 2009); stomata
Genomic DNA; ORF, Open reading frame; BLAST, Basic Local Alignment Search Tool; NJ, (Kanaoka et al., 2008); owering (Ito et al., 2012); axillary meristem
Neighbor joining; MEGA, Molecular Evolutionary Genetics Analysis; ExPASy, Expert formation (Yang et al., 2012); nodule vascular patterning (Godiard
Protein Analysis System; I-TASSER, Iterative Threading ASSEmbly Renement; SIB, Swiss et al., 2011); biosynthesis of avonoid, anthocyanin, and isoquinoline
Institute of Bioinformatics; 3D, Three dimensional; ProSA, Protein Structure Analysis; PP,
alkaloids (Ohno et al., 2011); and hormone signaling (Yin et al., 2005).
PredictProtein; PONDR, Predictor Of Naturally Disordered Regions; miRNA, microRNA;
ELF1, Elongation factor 1 alpha; RT-PCR, Reverse transcription polymerase chain These proteins also participate in light signaling (Ni et al., 1998;
reaction; qRT-PCR, Quantitative reverse transcription polymerase chain reaction; Ct, Fairchild et al., 2000) and are responsive to different abiotic stresses,
Threshold cycle; FC, Field capacities; UTR, Untranslated region(s); kDa, Kilodalton(s); such as drought (Dong et al., 2014), low temperature (Chinnusamy
mRNA, Messenger ribonucleic acid; PDB, Protein Data Bank; TM, Template modeling; Cd, et al., 2003; Feng et al., 2012), salinity (Zhou et al., 2009; Li et al.,
Cadmium; Hg, Mercury; Al, Aluminum.
Corresponding author.
2010), osmotic (Liu et al., 2013), wound stresses (Kiribuchi et al.,
E-mail addresses: nataraja_karaba@yahoo.com, nnkaraba@uasbagalore.edu.in 2004). There are also reports on the role of bHLH in iron acquisition
(K.N. Nataraja). and heavy metal detoxication (Yuan et al., 2008; Wu et al., 2012).

http://dx.doi.org/10.1016/j.plgene.2016.01.004
2352-4073/ 2016 The Authors. Published by Elsevier Inc. All rights reserved. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
110 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117

Table 1 rst time, cloning and characterization of a novel bHLH144-like gene

List of primers used for the isolation and expression analysis of MabHLH144-like gene from (bHLH144a-like) and its transcript variant (bHLH144b-like) from peren-
mulberry.
nial tree, mulberry. The transcript variants reported in this study are
Name Sequence 5 3 Application differentially expressed in diverse tissue types and are induced under
MabHLH144-F1 GGGTAACAATTATATGCATAYCW Initial PCR different abiotic stresses.
MabHLH144-F2 GGGTAACAATTATATGCATAYCWC Initial PCR
MabHLH144-R1 CAAGTTCATGACTTGTCGAAGCVG Initial PCR
MabHLH144-R2 CCTATGAATTGAAGAAGATGATCTGTTC 5 RACE (1st PCR)
2. Materials and methods
MabHLH144-R3 CAAGATTCAGGTGACGAGCTTCCG 5 RACE (Nested PCR)
MabHLH144-F3 CCCTGTTGCACCTGCTTATGGTGCAG 3 RACE (1st PCR)
MabHLH144-F4 CGCAGTCAGATCATGTTCCACCCTGC 3 RACE (Nested PCR) 2.1. Total RNA isolation and cDNA synthesis
MabHLH144-F5 GCTGGTTGGAGGAGTGAGAATGTTC Full-length amplication
MabHLH144-R4 CAACGGTGATGAGAGCTGATGATAGATG Full-length amplication Total RNA was extracted from 100 mg young mulberry (Morus alba,
MabHLH144a-F6 GTGGTGAATGGTAATTGCTATCGAACTC Expression of variant-a
MabHLH144b-F7 GGGGTATACCAATTTGAGACTGCATC Expression of variant-b
genotype Dudia white) leaves exposed to drought stress (as mentioned
ELF1 Forward CCCAAATATTCGAAGGCTAGGTACGAT Internal control in Section 2.8) using modied lithium chloride precipitation method by
ELF1 Reverse CAAGTTGGTGGACCTCTCAATCATG Internal control Sajeevan et al. (2014). The rst strand cDNA was synthesized from total
RNA (5 g) using the RevertAid First Strand cDNA Synthesis Kit follow-
ing the manufacturer's protocol (Thermo Fisher Scientic, USA) and
used as the template for PCR reactions.
Recent whole genome analysis in different plant species added up
the number of bHLH genes in the databases. In model systems
Arabidopsis and Oryza, 225 and 211 bHLH genes, respectively, were 2.2. RT-PCR and cloning of bHLH-like gene
identied (Jin et al., 2014), and the number is much high in Brassica
rapa (230), Musa acuminata (252), Glycine max, and Zea mays (289) Degenerate primers MabHLH144-F1, MabHLH144-F2, and
(Song et al., 2014). There are many reports on functional roles of bHLH MabHLH144-R1 (Table 1) were designed based on the nucleic acid se-
in plant growth and survival under stressful conditions (Zheng et al., quences of bHLH-like genes from other plant species reported in NCBI
2010; Dong et al., 2014; Babitha et al., 2015). Overexpression of nger (http://www.ncbi.nlm.gov/) to clone the gene from mulberry. PCR
millet bHLH57 in tobacco signicantly increased tolerance to salinity was performed in a 20 l nal reaction volume using cDNA as template
and drought stress with improved root growth (Babitha et al., 2015). under standardized conditions in a PCR machine (Eppendorf,
The majority of the bHLH genes reported in NCBI are identied and an- Germany). The PCR products were cloned into T/A cloning vector
notated through computational gene prediction methods, and their bi- using InsT/A clone PCR product cloning kit (MBI, Fermentas Life
ological functions remain unknown. In this study, we report, for the Sciences), and sequenced (ABI 3730Xl sequencer).

Fig. 1. Cloning of MabHLH144-like gene from cDNA (a) and genomic DNA (b) of mulberry leaf tissue. The genomic organization of the MabHLH144-like gene (c). White boxes with number
inside indicate the intron, and black boxes with number below are exons with their respective size. Lengths of intron and exons are indicated with the number of bases. The arrows indicate
the CDS region. bp, base pair; scale showing the size of 1 kb marker (MBI, Fermentas Life Sciences).
 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117 111

2.3. Cloning of 3 and 5 cDNA regions of bHLH-like gene annealing temperature 64 C. The PCR products were cloned and se-
quenced as mentioned in Section 2.2.
The 3 and 5 ends of the gene were cloned by RNA ligase-mediated
rapid amplication of cDNA ends (RACE) approach using GeneRacer Kit 2.4. Amplication of full length bHLH-like gene from cDNA and genomic
(http://www.invitrogen.com) following the manufacturer's instruc- DNA
tions. For 3 RACE, PCR was performed with MabHLH144-F3 (Table 1)
and GeneRacer 3 primer. The product of the primary PCR was diluted To amplify the full-length bHLH-like gene from cDNA and geno-
(20-fold) and used as template in the second PCR with gene-specic mic DNA (gDNA), gene-specic forward (MabHLH144-F5, Table 1)
MabHLH144-F4 nested primer (Table 1) and GeneRacer 3 nested prim- and reverse (MabHLH144-R4, Table 1) primers were designed using
er at annealing temperature 65 C. Similarly, 5 RACE was carried out sequence information generated from the 5 and 3 RACE. Using
with GeneRacer 5 and MabHLH144-R2 (Table 1) primer following the cDNA as template, PCR was carried out in a 20 l reaction volume
manufacturer's PCR conditions. The primary PCR product was diluted containing Phusion DNA polymerase (Finnzymes, Thermo Fisher Sci-
(15-fold) and used as template in the secondary PCR with GeneRacer entic, USA) at an annealing temperature of 60 C. Genomic DNA was
5 and gene-specic MabHLH144-R3 (Table 1) nested primer at isolated from leaves using the DNeasy Plant Mini Kit (QIAGEN)

Fig. 2. Full-length cDNA sequence of MabHLH144a-like gene. The amino acid sequence is displayed in a one-letter code under the coding sequence, with the underlined amino acids
representing the bHLH domain. The sequences highlighted in red bold italics is the E-box specicity site and the sequences in black bold italics is absent in MabHLH144b-like (transcript
variant). The translation start codon is framed. The asterisk denotes the stop codon.
112 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117
 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117 113

Fig. 4. Phylogenetic relationship between MabHLH144-like and related bHLH144-like proteins from other species. Protein sequences were aligned using the ClustalX and the phylogenetic
tree was constructed in MEGA 4.1 program. MabHLH144-like protein is shown in a full triangle. The appended bHLH144-like proteins are as follows: Hevea brasiliensis (ACZ04445.1), Vitis
vinifera (XP 002275393.1), Glycine max (XP 003524798.1), Medicago truncatula (XP 003608491.1), Arabidopsis thaliana (AEE31153.1), Brachypodium distachyon (XP 003569396.1), Zea
mays (NP 001145247.1), Solanum lycopersicum (XP 004233098.1), Aegilops tauschii (EMT06805.1), Setaria italica (XP 004952349.1), Oryza sativa (ABF97395.1), Cicer arietinum (XP
004504689.1), Cucumis sativus (XP 004134628.1), and Prunus mume (XP 008245935.1).

according to the manufacturer's protocol and PCR was performed protein was carried out with the help of ExPASy and I-TASSER software
using 100 ng of gDNA as template under standardized annealing (http://zhanglab.ccmb.med.umich.edu/I-TASSER/; Ambrish et al.,
temperature as described above, with an initial denaturation of 2010), respectively. The modeled structures were also validated using
6 min. The amplied PCR products were cloned and sequenced as de- the program ProSA (Wiederstein and Sippl, 2007). In silico localization
scribed in Section 2.2. of bHLH144-like gene was predicted using the software PredictProtein
(PP, https://www.predictprotein.org) and intrinsic disorder regions of
2.5. Bioinformatics analysis the cloned bHLH144-like TF gene was predicated using the software Pre-
dictors of Natural Disordered Regions with default parameters (PONDR,
2.5.1. Sequence analysis http://www.pondr.com/research.html; Romero et al., 1997).
The nucleotide and deduced amino acid sequence of the cloned gene
was used for BLAST search on the NCBI database (http://www.ncbi.nlm. 2.6. MicroRNA target prediction
gov/) using the BLASTn and BLASTx tools. The basic information on ORF
and translation of the nucleotide sequence, prediction of isoelectric The microRNA (miRNA) targets of MabHLH144a- and MabHLH144b-
point (pI), and determining the molecular weight (MW), etc., were ac- like genes were predicted as described by Enright et al. (2003). The mi-
complished using tools at ExPASy website (http://www.expasy.org) Randa scanning algorithm, which utilizes dynamic programming align-
and DNA Star software. ment and thermodynamics to predict miRNA targets, was employed in a
stand-alone version 1.9 (http://www.microrna.org/miranda new.html).
2.5.2. Phylogeny, structure, localization, and intrinsic disorder predictions The standardized threshold and lters used by Archak and Nagaraju
Alignment of the bHLH144-like protein was performed using the (2007) for miRNA target identication in rice were used for the hit
ClustalW program in the BioEdit sequence editor software, version detection.
7.0.9.0 (Hall, 1999). The phylogenetic relationship of the amino acid se-
quence was built with other structurally related bHLH144-like and bHLH 2.7. Expression analysis in different tissues
proteins using the neighbor joining (NJ) method with p-distance correc-
tion (Saitou and Nei, 1987). Bootstrap values were derived from 1000 To examine the expression of MabHLH144a-like and MabHLH144b-
replicate runs, and phylogenetic tree was constructed using the pro- like genes in different tissues and organs of mulberry plant, total RNA
gram MEGA 4.1 (Tamura et al., 2007). Further, functional domains/mo- was isolated from 100 mg tissue of meristem, very young leaves (from
tifs of the cloned gene were predicted using the expert protein analysis top 2nd), half-matured leaves (from top 5th), fully matured leaves
system (ExPASy, http://swissmodel.expasy.org/), and proteomics serv- (from top 8th), inorescence, fruit, stem, and root using the modied
er of the Swiss Institute of Bioinformatics (SIB) (Gasteiger et al., 2003). protocol by Sajeevan et al. (2014). The cDNA was prepared as men-
The domain and three-dimensional (3D) structure prediction of the tioned in Section 2.1, and quantitative RT-PCR (qRT-PCR) was

Fig. 3. Alignment of Morus alba bHLH144-like with related bHLH144 proteins from Hevea brasiliensis bHLH1, Vitis vinifera bHLH144, Glycine max bHLH144-like, Medicago truncatula bHLH144,
Arabidopsis thaliana bHLH144, Brachypodium distachyon bHLH144-like, Zea mays bHLH, Solanum lycopersicum bHLH144-like, Aegilops tauschii bHLH144, Setaria italica bHLH144-like, Oryza
sativa bHLH, Cicer arietinum bHLH144-like, Cucumis sativus bHLH144-like, and Prunus mume bHLH144-like. The amino acid residues shaded with black and gray are having 100 and 80%
sequence similarity with other species. The bHLH domain comprising amino acid residues is marked with red box. (For interpretation of the references to color in this gure legend,
the reader is referred to the web version of this article.)
114 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117

Fig. 5. Molecular modeling of bHLH144-like gene. The HLH domain (a); predicted 3D structure of full-length protein (b).

performed with the gene-specic forward MabHLH144a-F6 and compared to its corresponding control was calculated using the
MabHLH144b-F7 (Table 1) and reverse MabHLH144-R4 (Table 1) 2CT method (Livak and Schmittgen, 2001).
primers using the SYBR Premix Ex Taq (TAKARA BIO INC.; www.
takara-bio.com) in a real-time PCR machine (MJ Research, USA, and
MJ Bioworks, Inc.) at an annealing temperature of 58 C. The melting 3. Results
curve analysis was carried out from 50 to 90 C with a hold of 1 min
for every 1 C. The threshold cycle (Ct) values of the triplicate PCR's 3.1. Cloning of bHLH gene from mulberry
were averaged, and relative expression levels of the MabHLH144a-
and MabHLH144b-like genes were calculated using the 2CT method The goal of this study was to clone and characterize bHLH144-like
(Livak and Schmittgen, 2001). gene associated with abiotic stress tolerance from perennial tree mul-
berry. Using sequence information of predicted and hypothetical bHLH
2.8. Stress imposition and expression analysis or bHLH144-like genes, primers were designed. Initial PCR yielded a par-
tial length gene fragment of 615 bp and sequence homology search
Healthy plants of mulberry, variety Dudia white, were raised in bat- showed 80% similarity with predicted gene Prunus mume bHLH144-
tery pots containing 20 kg of 2:1 garden soil and sand mixture, by like transcript variant 1 and 2 (XM 008247713.1, XM 008247714.1).
adopting all plant protection measures. Drought was imposed to 2- The 3 RACE-amplied two products of 950 and 860 bp contained an ad-
month-old plants by controlled irrigation, and soil eld capacities (FC) ditional sequence of 437 and 344 bp, respectively. The 5 RACE generat-
of 80, 60, and 40% were maintained by gravimetric approach (Karaba ed 800 bp product which had an additional sequence of 460 bp. Using
et al., 2007), thereby simulating eld drought conditions. Leaf samples the sequence information generated, full-length cDNA of sizes 1512
were collected from both stressed and non-stressed plants 8 days post and 1419 bp were cloned and analyzed (Fig. 1a). The full-length
stress imposition. MabHLH144-like gene cloned from leaf gDNA was 2304 bp (Fig. 1b)
For creating salinity and oxidative stresses, 15 cm long young twigs and sequence analysis revealed the presence of a 786 bp intron at the
of healthy mulberry plants were collected and immersed in water with- 5 untranslated region (UTR) region (Fig. 1c). BLASTn analysis showed
out any air bubble at the cut end. After 3 h, cut ends were dipped in 75% sequence identity with predicted Prunus mume bHLH144-like tran-
250 mM NaCl and 10 M methyl viologen for inducing the salinity and script variant 1 and 2 (XM 008247713.1, XM 008247714.1) followed by
oxidative stresses, respectively. All stresses were imposed without 74% identity with predicated pyrus X bretschneideri bHLH144-like tran-
changing light intensity, humidity, or photoperiod at 25 C. A set of script variant 1 and 2 (XM 009362587.1, XM 009362588.1). We desig-
twigs were maintained in water and treated as control. Leaf samples nated these clones as Morus alba (Ma) bHLH144a- and MabHLH144b-
were collected at 0, 6, 12, 24, and 48 h interval, immediately frozen in like genes. The full-length MabHLH144-like gene transcript variants a
liquid nitrogen, and stored at 70 C until use. Total RNA was isolated and b contain an open reading frame (ORF) of 726 bp, which encodes
from 100 mg leaf tissue, cDNA synthesized as mentioned in a putative polypeptide of 241 amino acid residues, with a predicated
Section 2.1 and used as the template for qRT-PCR. The qRT-PCR was per- MW of 26.94 kDa and a theoretical pI of 6.08. The 5 UTR of
formed with the gene-specic forward MabHLH144a-F6 and MabHLH144-like gene transcripts is 401 bp. However, in the 3 UTR,
MabHLH144b-F7 (Table 1) and reverse MabHLH144-R4 (Table 1) we detected 385 and 292 bp, regions in variant a and b, respectively.
primers. The relative expression levels under a given stress condition We also noticed 93 bp deletion in the transcript variant b (Fig. 2). The

Table 2
Predicated miRNA that have targets sequences in MabHLH144a- and MabHLH144b-like genes. The miRNA in bold italics is unique to the MabHLH144a-like and does not target
MabHLH144b-like gene.

Predicted miRNA Sequence (5 3) Position Nucleotide Length Energy (kCal/Mol)

Start End

mtr-miR2661 UCCGUCAGUUGCUCAAACCUG 5 UTR 132 152 21 20.47

osa-miR6253 AAUCCAUCUUUCCUACUUUCCUU 5 UTR 362 384 23 26.24
mtr-miR2673a/b CAAAAAUAUGAUGAGGAAGAGG CDS 800 822 22 20.86
osa-miR528-3p UGAGGAAGAGGUAAGCACAGC CDS 812 832 21 33.36
osa-miR529b GUGUUAGUUUUUUCUCUCUUCC 3 UTR 1199 1220 22 20.30
osa-miR396f-5p UCUUUAGGAAAGUGUGGAGA 3 UTR 1387 1406 20 22.92
 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117 115

cDNA and gDNA sequences have been deposited in GenBank under the range of native conformation of other experimentally determined pro-
accession numbers KP732521, KP732522, and KP732520. tein structures of the same size. The MabHLH144-like gene showed
128 out of 241 amino acids (53.11%) disordered spanning in six regions
3.2. Sequence analysis and phylogeny of the deduced MabHLH144-like (Supplementary Fig. S2a and b). The longest disordered region consists
protein of 48 amino acids toward the C-terminal region that is intercalated with
the -helical structure of the bHLH domain.
The MabHLH144-like amino acid sequence shares 91, 69, 66, and 65%
identity with hypothetical protein of Morus notabilis (XP 010097603.1), 3.4. Prediction of microRNA targets
predicted bHLH144-like protein of Prunus mume (XP 008245935.1),
Hevea brasiliensis (ACZ04445.1), and Malus domestica (XP0083 Attempts for predicting the possible miRNA binding sites in the
83502.1), respectively. The alignment of amino acid sequence of MabHLH144a- and MabHLH144b-like genes resulted in 6 and 5 binding
MabHLH144-like with some of the selected bHLH and bHLH144-like pro- sites, respectively. The 5 UTR and ORF of the MabHLH144a- and
teins from other plant species is shown in Fig. 3. The MabHLH144-like MabHLH144b-like genes have two miRNA binding sites each. The 3
gene encodes a protein of 241 amino acids, which contains a bHLH UTR of the MabHLH144a-like gene contains two miRNA binding sites
domain region (Supplementary Fig. S1), E-box/N-box specicity while the MabHLH144b-like gene has only one (Table 2). The 3 UTR
site and a dimerization domain (amino acid 176234, Fig. 2). Based of the MabHLH144b-like gene has a 93 bp deletion that occupies Osa-
on motif and domain analysis, the MabHLH144-like protein is pre- miR529b miRNA binding site. The sequence alignment of different tar-
dicted to contain Big-1 (bacterial Ig-like domain 1), and multiple get miRNA predicted is provided in Supplementary Table S1.
sites for N-glycosylation, casein kinase II phosphorylation, N-
myristoylation, and also protein kinase C-phosphorylation. Addi-
tionally, the MabHLH144-like protein contains a serine rich region
(SSSPESCSNYGSKTKKNRSSSSIHRSSDGASSS) and has two unique 11
and 15 amino acid long low complexity regions in between 130
140 and 170184 amino acids, respectively. The phylogenetic analy-
sis of different bHLH144-like protein sequences yielded two different
clades. All the monocot bHLH144 proteins grouped in clade I and
dicots grouped into clade II. The MabHLH144-like protein is closely
related to Hevea brasiliensis bHLH1 followed by Vitis vinifera
bHLH144 protein (Fig. 4).

3.3. Prediction of structure and intrinsic disorder

According to BLASTp analysis, the bHLH domain is the only segment

of the MabHLH144-like gene that presented a hit for proteins with
known 3D structure. The structure of the MAD protein (PDB code
1nlw.1.C) was taken as the template for the comparative modeling of
the bHLH domain. The predicted structure of DNA-binding domain
consists of two -helixes connected by a loop (Fig. 5a). To create a 3D
structure, BLAST search was performed in protein databases for proteins
with similar sequence and known 3D structure; however, no template
was found with signicant homology to model the complete sequence.
Thus, modeling of MabHLH144-like gene was done with the help of I-
TASSER server which is a hierarchical protein structure modeling
approach based on the secondary structure enhanced ProleProle
threading alignment. The MabHLH144-like gene predicted model struc-
ture had an estimated TM score of 0.40 0.14 and root mean square
deviation (RMSD) of 12.1 4.4 (Fig. 5b). Further, the predicted struc-
ture was validated with ProSA (z score = 5.32) which was in the

Fig. 7. qRT-PCR analysis of MabHLH144a and MabHLH144b-like genes under different abi-
Fig. 6. Expression analysis of MabHLH144a and MabHLH144b-like genes using qRT-PCR in otic stresses. Drought stress (40, 60, and 80% soil eld capacity) (a); salinity (b); and oxi-
different tissue types of mulberry. dative stresses (6, 12, 24, and 48 h) (c).
116 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117

3.5. Expression analysis ne-tuned regulation probably at mRNA levels, as there are miRNA tar-
gets identied in the gene. The gene was also found to be responsive to
To understand the temporal and spatial distribution of expression of different abiotic stresses. As mentioned earlier, differential expression
the two variants of MabHLH144-like genes in mulberry, qRT-PCR was of bHLH genes under different abiotic stresses have been reported
carried out. Under normal growth conditions, the expression level of (Chinnusamy et al., 2003; Seo et al., 2011; Dong et al., 2014). Abe et al.
MabHLH144b-like gene was highest in stem, followed by 8th, 5th, and (2003) reported the up-regulation of a bHLH gene, AtMYC2, at the early
2nd leaf. The levels of mRNA were comparatively low in inorescence, stages of drought stress and showed a steady increase under salinity
root, and fruit tissues (Fig. 6). The mRNA level of MabHLH144a-like stress. The MabHLH144a- and MabHLH144b-like were early drought
was very low compared to MabHLH144b-like and the expression was (80% FC) responsive with 62 and 30 folds increase respectively, and the
maximum in 8th leaf and stem, compared to other parts tested (Fig. 6). transcript abundance decreased with increase in drought stress level
The MabHLH144a- and MabHLH144b-like genes were signicantly (Fig. 7a). We predicted nuclear localization (Supplementary Fig. S3) of
upregulated at early stages of drought stress. Under 80% FC, the MabHLH144-like gene, which suggests its possible role in transcrip-
MabHLH144b-like gene showed 62-fold increase in mRNA levels over tion process similar to the related TF reported by others (Chinnusamy
control as compared to 31-fold in mRNA of MabHLH144a-like gene. et al., 2003; Xu et al., 2014). The TFs seem to be essential for activation
Further, the mRNA levels of both MabHLH144-like genes declined at of many downstream functional genes under stress and analysis of its
40 and 20% FC (Fig. 7a). Under salinity stress, MabHLH144a- and targets could be useful in identifying the specic genes linked to stress
MabHLH144b-like genes were upregulated, although varying expres- tolerance pathways. The expression pattern observed in MabHLH144-
sion pattern at different levels of stress were noticed. About 1.5-fold like genes was comparable to those of OsbHLH148, where transcript
increase in transcript level over control was noticed at 6 h in level was rapidly and transiently induced by drought and salinity stress-
MabHLH144a-like and the level declined over time. However, the es (Seo et al., 2011). Under salinity and oxidative stresses, MabHLH144a-
MabHLH144b-like expression was increased from 0.3-fold at 6 h to like expression was induced at early stages of stress (6 h) and reduced
2.1 at 12 h, while reduction in mRNA level was noticed at 24 and 48 h thereafter. However, up-regulation of MabHLH144b-like was observed
(Fig. 7b). Under methyl viologen-induced oxidative stress, the expres- at 6 and 12 h of oxidative and salinity stresses, respectively (Fig. 7b
sion of the genes was signicantly higher during the early stage. The and c). It is interesting to note that the MabHLH144-like gene variants
MabHLH144a-like gene mRNA showed 5.5-fold increase and the levels are differentially regulated under abiotic stresses implying diverse roles
of MabHLH144b-like mRNA were 3.6 fold more in 6 h. Further, mRNA of the gene under specic stresses.
levels of both MabHLH144a- and MabHLH144b-like reduced at 12 h Alternative splicing events were reported in bHLH gene family. For
and MabHLH144a-like gene expression remained unchanged at 24 and example, BIGPETAL (BPE), a gene that is involved in the control of
48 h, while the transcript levels of MabHLH144b-like reduced at 24 petal size, seems to exhibit alternative splicing control (Szecsi et al.,
and 48 h post methyl viologen treatment (Fig. 7c). 2006). The bpE-1 transcript is expressed ubiquitously, whereas the
bpE-2 transcript is preferentially expressed in petals that produced larg-
4. Discussion er petals as a result of increased cell size. The MabHLH144-like transcript
is noticed in all types of tissues studied and is also highly upregulated
The basic helixloophelix (bHLH) TFs are considered to be the larg- under different abiotic stresses. The sequence-specic interaction of
est group in plants and are induced under different abiotic stresses. To miRNAs to their target mRNAs can bring differential expression of
clone novel bHLH gene from abiotic stress-tolerant perennial crop, mul- genes in response to environment stimuli and these miRNAs are consid-
berry, we used PCR-based cloning approach using heterologous ered as important players in majority of plant stress responses (Sunkar
primers. The MabHLH144-like protein shares 69 and 66% homology et al., 2007). The MabHLH144b-like gene transcript is not a target for the
with predicted bHLH144-like proteins of Prunus mume and Hevea miR529b due to the 93 bp deletion in the 3 UTR. The miRNA Osa-
brasiliensis, respectively, with bHLH domain at the C-terminal region, a miR529b coming under the miR529 family was reported for its involve-
characteristic feature of the bHLH proteins. Besides this domain, there ment in regulating various developmental events and also under
are other amino acid conserved regions such as the serine rich region drought, salinity, and heavy metal such as cadmium (Cd), mercury
and also unique low complexity regions which is similar to Gossypium (Hg), and aluminum (Al) stresses (Sunkar et al., 2008; Zhou et al.,
hirsutum bHLH1 protein having C-terminal bHLH domain and several 2010; Khraiwesh et al., 2012). In rice, Osa-miR529 was signicantly
low-complexity regions (Meng et al., 2009). We cloned two transcript downregulated in response to drought stress and the pattern was con-
variants of MabHLH144-like comprising 1512 and 1419 bp and the rele- tradictory to the results obtained in cowpea (Barrera-Figueroa et al.,
vance of these transcript variations is yet to be understood. 2011). The molecular basis of differential expression of the
The MabHLH144-like protein has some features similar to the other MabHLH144a- and MabHLH144b-like genes in different plant parts and
TFs. Majority of the TFs do not adopt a xed folded structure but exist also under different abiotic stress conditions need to be further
as a exible form to interact with multiple partners and perform their examined.
function (Pazos et al., 2013). In Arabidopsis, genome-wide analysis of This is the rst study reporting isolation and characterization of
protein disorderness revealed that 8294% of the TFs contain intrinsic bHLH144a- and bHLH144b-like gene from mulberry. The plant bHLH
disordered regions (Liu et al., 2006). Intrinsic disorder is enriched in constitute one of the largest TF families; however, only a few members
proteins associated with cell signaling and transcription. The have been functionally characterized for stress response. This study re-
MabHLH144-like protein was found to be 53.11% disordered, spanning ports another novel TF belonging to bHLH family linked to stress re-
in 6 regions with a maximum length of 48 amino acid residues (Supple- sponse. Further functional validation is required to precisely examine
mentary Fig. S2). Similarly, Kragelund et al. (2012) reported that the role of the gene in developmental processes and abiotic stress
AtbHLH011 and bHLH (PIF3) are highly disordered indicating the plastic- response.
ity of TFs to interact with different partners at varying conditions to per- Supplementary data to this article can be found online at http://dx.
form diverse function. doi.org/10.1016/j.plgene.2016.01.004.
Since the bHLH is known to be associated with plant developmental
processes, we examined the pattern of expression of MabHLH144-like in Compliance with ethical standards
different parts of mulberry plant. The mRNA level of MabHLH144b-like
was high in all parts of plant tissue compared to MabHLH144a-like The authors declare that they have no conict of interest and the re-
gene, indicating differential regulation of expression of these variants. search has been conducted with the consent of the authorities and that
The low levels of expression of MabHLH144a-like could be due to a the research does not involve human participants and/or animals.
 R.S. Sajeevan, K.N. Nataraja / Plant Gene 5 (2016) 109117 117

Acknowledgments Li, F., Guo, S., Zhao, Y., Chen, D., Chong, K., Xu, Y., 2010. Overexpression of a homopeptide
repeat-containing bHLH protein gene (OrbHLH001) from Dongxiang Wild Rice con-
fers freezing and salt tolerance in transgenic Arabidopsis. Plant Cell Rep. 29, 977986.
We gratefully acknowledge the Department of Biotechnology (DBT), Li, X., Duan, X., Jiang, H., Sun, Y., Yang, Y., Yuan, Z., Guo, J., Liang, W., et al., 2006. Genome-
Government of India (GOI), New Delhi, for providing the nancial sup- wide analysis of basic/helix loophelix transcription factor family in rice and
Arabidopsis. Plant Physiol. 141, 11671184.
port (D.O. No. BT/PR13457/PBD/19/214), to carry out this work. We Liu, J., Perumal, N.B., Oldeld, C.J., Su, E.W., Uversky, V.N., Dunker, A.K., 2006. Intrinsic
wish to thank Mr. Vairavan Lakshmanan, JRF, inStem, National Centre disorder in transcription factors. Biochemistry 45, 68736888.
for Biological Sciences (NCBS), Bangalore, Karnataka, for the support ex- Liu, W., Tai, H., Li, S., Gao, W., Zhao, M., Xie, C., Li, W.X., 2013. bHLH122 is important for
drought and osmotic stress resistance in Arabidopsis and in the repression of ABA
tended during miRNA target prediction analysis. catabolism. New Phytol. 2, 17.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-
References time quantitative PCR and the 2CT method. Methods 25, 402408.
Meng, C.M., Zhang, T.Z., Guo, W.Z., 2009. Molecular cloning and characterization of a
Abe, H., Urao, T., Ito, T., Seki, M., Shinozaki, K., Yamaguchi-Shinozaki, K., 2003. Arabidopsis novel Gossypium hirsutum L. bHLH gene in response to ABA and drought stresses.
AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic Plant Mol. Biol. Report. 27, 381387.
acid signaling. Plant Cell 15, 6378. Monte, E., Tepperman, J.M., Al-Sady, B., Kaczorowski, K.A., Alonso, J.M., Ecker, J.R., Li, X.,
Ambrish, R., Alper, K., Yang, Z., 2010. I-TASSER: a unied platform for automated protein Zhang, Y.L., et al., 2004. The phytochrome-interacting transcription factor, PIF3, acts
structure and function prediction. Nat. Protoc. 5, 725738. early, selectively, and positively in light-induced chloroplast development. Proc.
Archak, S., Nagaraju, J., 2007. Computational prediction of rice (Oryza sativa) miRNA Natl. Acad. Sci. U. S. A. 101, 1609116098.
targets. Genomics Proteomics Bioinformatics 5, 34. Ni, M., Tepperman, J.M., Quail, P.H., 1998. PIF3, a phytochrome-interacting factor neces-
Atchley, W.R., Terhalle, W., Dress, A., 1999. Positional dependence, cliques, and predictive sary for normal photoinduced signal transduction, is a novel basic helixloophelix
motifs in the bHLH protein domain. J. Mol. Evol. 48, 501516. protein. Cell 95, 657667.
Babitha, K.C., Vemanna, R.S., Nataraja, K.N., Udayakumar, M., 2015. Overexpression of Ohno, S., Hosokawa, M., Hoshino, A., Kitamura, Y., Morita, Y., Park, K.I., Nakashima, A.,
EcbHLH57 transcription factor from Eleusine coracana L. in tobacco confers tolerance Deguchi, A., et al., 2011. A bHLH transcription factor, DvIVS, is involved in regulation
to salt, oxidative and drought stress. PLoS ONE 10, e0137098. of anthocyanin synthesis in dahlia (Dahlia variabilis). J. Exp. Bot. 62, 51055116.
Barrera-Figueroa, B.E., Gao, L., Diop, N.N., Wu, Z., Ehlers, J.D., Roberts, P.A., Close, T.J., Zhu, Pazos, F., Pietrosemoli, N., Garcia-Martn, J.A., Solano, R., 2013. Protein intrinsic disorder in
J.K., Liu, R., 2011. Identication and comparative analysis of drought-associated plants. Front. Plant Sci. 4, 363.
microRNAs in two cowpea genotypes. BMC Plant Biol. 11, 127. Romero, P., Obradovic, Z., Kissinger, C.R., Villafranca, J.E., Dunker, A.K., 1997. Identifying
Carretero-Paulet, L., Galstyan, A., Roig-Villanova, I., Martinez-Garcia, J.F., Bilbao-Castro, J.R., disordered proteins from amino acid sequences. Proc. IEEE Int. Conf. Neural Netw.
Robertson, D.L., 2010. Genome-wide classication and evolutionary analysis of the 1, 9095.
bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing
Plant Physiol. 153, 13981412. phylogenetic trees. Mol. Biol. Evol. 4, 406425.
Castilhos, G., Lazzarotto, F., Spagnolo-Fonini, L., Bodanese-Zanettini, M.H., Margis- Sajeevan, R.S., Shivanna, M.B., Nataraja, K.N., 2014. An efcient protocol for total RNA iso-
Pinheiro, M., 2014. Possible roles of basic helixloop-helix transcription factors in ad- lation from healthy and stressed tissues of mulberry (Morus sp.) and other species.
aptation to drought. Plant Sci. 223, 17. Am. J. Plant Sci. 20572065.
Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B.H., Hong, X., Agarwal, M., Zhu, J.K., 2003. ICE1: Seo, J.S., Joo, J., Kim, M.J., Kim, Y.K., Nahm, B.H., Song, S.I., Cheong, J.J., Lee, J.S., et al., 2011.
a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. OsbHLH148, a basic helixloophelix protein, interacts with OsJAZ proteins in a
Genes Dev. 17, 10431054. jasmonate signaling pathway leading to drought tolerance in rice. Plant J. 65,
Dong, Y., Wang, C., Han, X., Tang, S., Liu, S., Xia, X., Yin, W., 2014. A novel bHLH transcrip- 907921.
tion factor PebHLH35 from Populus euphratica confers drought tolerance through reg- Song, X., Huang, Z., Duan, W., Ren, J., Liu, T., Li, Y., Hou, X., 2014. Genome-wide analysis of
ulating stomatal development, photosynthesis and growth in Arabidopsis. Biochem. the bHLH transcription factor family in Chinese cabbage (Brassica rapa ssp.
Biophys. Res. Commun. 450, 453458. pekinensis). Mol. Genet. Genomics 289, 7791.
Enright, A.J., John, B., Gaul, U., Tuschl, T., Sander, C., Marks, D.S., 2003. MicroRNA targets in Sunkar, R., Chinnusamy, V., Zhu, J., Zhu, J.K., 2007. Small RNAs as big players in plant
Drosophila. Genome Biol. 5, R1. abiotic stress responses and nutrient deprivation. Trends Plant Sci. 12, 301309.
Fairchild, C.D., Schumaker, M.A., Quail, P.H., 2000. HFR1 encodes an atypical bHLH protein Sunkar, R., Zhou, X., Zheng, Y., Zhang, W., Zhu, J.K., 2008. Identication of novel and can-
that acts in phytochrome A signal transduction. Genes Dev. 14, 23772391. didate miRNAs in rice by high throughput sequencing. BMC Plant Biol. 8, 25.
Feng, X.M., Zhao, Q., Zhao, L.L., Qiao, Y., Xie, X.B., Li, H.F., Yao, Y.X., You, C.X., Hao, Y.J., 2012. Szecsi, J., Joly, C., Bordji, K., Varaud, E., Cock, J.M., Dumas, C., Bendahmane, M., 2006.
The cold-induced basic helixloophelix transcription factor gene MdCIbHLH1 BIGPETALp, a bHLH transcription factor is involved in the control of Arabidopsis
encodes an ICE-like protein in apple. BMC Plant Biol. 12, 22. petal size. EMBO J. 25, 39123920.
Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D., Bairoch, A., 2003. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics
ExPASythe proteomics server for in-depth protein knowledge and analysis. Nucleic Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 15961599.
Acids Res. 31, 37843788. Toledo-Ortiz, G., Huq, E., Quail, P.H., 2003. The Arabidopsis basic/helixloophelix tran-
Godiard, L., Lepage, A., Moreau, S., Laporte, D., Verdenaud, M., Timmers, T., Gamas, P., scription factor family. Plant Cell 15, 17491770.
2011. MtbHLH1, a bHLH transcription factor involved in Medicago truncatula nodule Wiederstein, M., Sippl, M.J., 2007. ProSA-web: interactive web service for the recognition
vascular patterning and nodule to plant metabolic exchanges. New Phytol. 191, of errors in three-dimensional structures of proteins. Nucleic Acids Res. 35, 407410.
391404. Wu, H., Chen, C., Du, J., Liu, H., Cui, Y., Zhang, Y., He, Y., Wang, Y., et al., 2012. Co-
Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis overexpression FIT with AtbHLH38 or AtbHLH39 in Arabidopsis-enhanced cadmium
program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 9598. tolerance via increased cadmium sequestration in roots and improved iron homeo-
Ito, S., Song, Y.H., Josephson-Day, A.R., Miller, R.J., Breton, G., Olmstead, R.G., Imaizumi, T., stasis of shoots. Plant Physiol. 158, 790800.
2012. FLOWERING BHLH transcriptional activators control expression of the photo- Xu, W., Zhang, N., Jiao, Y., Li, R., Xiao, D., Wang, Z., 2014. The grapevine basic helixloop
periodic owering regulator CONSTANS in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. helix (bHLH) transcription factor positively modulates CBF-pathway and confers
109, 35823587. tolerance to cold-stress in Arabidopsis. Mol. Biol. Rep. 41, 53295342.
Jin, J.P., Zhang, H., Kong, L., Gao, G., Luo, J.C., 2014. PlantTFDB 3.0: a portal for the function- Yang, F., Wang, Q., Schmitz, G., Mller, D., Theres, K., 2012. The bHLH protein ROX acts in
al and evolutionary study of plant transcription factors. Nucleic Acids Res. 42, concert with RAX1 and LAS to modulate axillary meristem formation in Arabidopsis.
D1182D1187. Plant J. 71, 6170.
Kanaoka, M.M., Pillitteri, L.J., Fujii, H., Yoshida, Y., Bogenschutz, N.L., Takabayashi, J., Zhu, Yin, Y., Vafeados, D., Tao, Y., Yoshida, S., Asami, T., Chory, J., 2005. A new class of transcrip-
J.K., Torii, K.U., 2008. SCREAM/ICE1 and SCREAM2 specify three cell-state transitional tion factors mediates brassinosteroid regulated gene expression in Arabidopsis. Cell
steps leading to Arabidopsis stomatal differentiation. Plant Cell 20, 17751785. 120, 249259.
Karaba, A., Dixit, S., Greco, R., Aharoni, A., Trijatmiko, K.R., Marsch-Martinez, N., Krishnan, Yuan, Y.X., Wu, H.L., Wang, N., Li, J., Zhao, W.N., Du, J., Wang, D.W., Ling, H.Q., 2008. FIT
A., Nataraja, K.N., et al., 2007. Improvement of water use efciency in rice by expres- interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression
sion of HARDY, an Arabidopsis drought and salt tolerance gene. Proc. Natl. Acad. Sci. U. for iron homeostasis in Arabidopsis. Cell Res. 18, 385397.
S. A. 104, 1527015275. Zheng, L., Ying, Y., Wang, L., Wang, F., Whelan, J., Shou, H., 2010. Identication of a novel
Karas, B., Amyot, L., Johansen, C., Sato, S., Tabata, S., Kawaguchi, M., Szczyglowski, K., 2009. iron regulated basic helixloophelix protein involved in Fe homeostasis in Oryza
Conservation of lotus and Arabidopsis basic helixloophelix proteins reveals new sativa. BMC Plant Biol. 10, 166.
players in root hair development. Plant Physiol. 151, 11751185. Zhou, J., Li, F., Wang, J.L., Ma, Y., Chong, K., Xu, Y.Y., 2009. Basic helixloophelix transcrip-
Khraiwesh, B., Zhu, J.K., Zhu, J., 2012. Role of miRNAs and siRNAs in biotic and abiotic tion factor from wild rice (OrbHLH2) improves tolerance to salt and osmotic stress in
stress responses of plants. Biochim. Biophys. Acta 1819, 137148. Arabidopsis. J. Plant Physiol. 166, 12961306.
Kiribuchi, K., Sugimori, M., Takeda, M., Otani, T., Okada, K., Onodera, H., Ugaki, M., Tanaka, Zhou, L., Liu, Y., Liu, Z., Kong, D., Duan, M., Luo, L., 2010. Genome-wide identication and
Y., et al., 2004. RERJ1, a jasmonic acid-responsive gene from rice, encodes a basic analysis of drought responsive microRNAs in Oryza sativa. J. Exp. Bot. 61, 41574168.
helixloophelix protein. Biochem. Biophys. Res. Commun. 325, 857863.
Kragelund, B.B., Jensen, M.K., Skriver, K., 2012. Order by disorder in plant signaling. Trends
Plant Sci. 17, 625632.