Sequence analysis and expression of the calmodulin gene, MCaM-3,
in mulberry (Morus L.)
Rongjun Fang ‧ ‧ Dongqing Hu ‧ ‧ Yinghua Zhang ‧ ‧ Long Li ‧ ‧ Weiguo Zhao ‧ ‧ Li Liu ‧ ‧ Jialin Cheng ‧ ‧
Jinliang Qi ‧ ‧ Yonghua Yang
Received: 20 September 2010 / Accepted: 18 November 2010 / Published online: 30 April 2011
© The Genetics Society of Korea and Springer 2011
Genes & Genomics (2011) 33: 97-103
A full-length cDNA sequence encoding CaM from mulberry,
which we designated MCaM-3 (GenBank accession No:
GQ303247), was cloned based on mulberry expressed se-
quence tags(ESTs). Sequence analysis showed that MCaM-3
is 951 base pairs in length, encoding 149 amino acids with
a predicted molecular weight of 16.85 kD and an isoelectric
point of 3.95. Online SMART analysis showed that the
MCaM-3 protein has four EFh functional domains, which can
bind calcium. The expression level of MCaM-3at different de-
velopmental stages in mulberry leaves and flowers and in dif-
ferent tissues was investigated. The results showed that
MCaM-3 transcripts are most abundantly expressed in mature
tissues, such as mature female flowers and climax leaves, and
the expression level of the mRNA could be increased sig-
nificantly under low temperature, drought, and salt stress con-
ditions compared to the normal growth environment. This re-
search will help us understand the resistance mechanism of
functional genes in mulberry.
Keywords Morus L. MCaM-3 gene; Molecular cloning; Gene
R. Fang ‧ D. Hu ‧ Y. Zhang ‧ L. Li(?) ‧ W. Zhao(?) ‧
L. Liu ‧ J. Cheng
Sericultural Research Institute, Chinese Academy of Agricultural
Sciences, Zhenjiang Jiangsu 212018, China;
e-mail: firstname.lastname@example.org ; email@example.com
R. Fang‧J. Qi ‧ Y. Yang
School of Life Sciences, Nanjing University, Nanjing Jiangsu
D. Hu ‧ Y. Zhang ‧ L. Li ‧ W. Zhao
College of biological and Environmental Engineering, Jiangsu Uni-
versity of Science and technology, Zhenjiang Jiangsu 212018, China
In higher plants, the calcium ion (Ca2+) plays crucial roles
as a second messenger mediating the actions of many hor-
mones and responses to environmental factors, including biotic
and abiotic stresses (Shao et al., 2008). Calmodulin (CaM),
as a calcium sensor, is a multifunctional Ca2+-binding protein
in eukaryotic cells, which plays important roles in regulating
growth and development, environmental adaptation, and stress
tolerance in plants (Hamada et al., 2009). Plants possess many
unique, putative Ca2+ sensors, including a large family (50 in
Arabidopsis thaliana) of calmodulin-like proteins implicated
in Ca2+-based stress responses. To transmit the Ca2+signal,
CaMs interact with target proteins (i.e., protein kinases, meta-
bolic enzymes, cytoskeleton-associated proteins) and regulate
their activity (Vanderbeld and Snedden, 2007).
CaM is a highly conserved Ca2+-binding protein in eukary-
otic cells in both composition and length (149 amino acids)
(Gonzalez-Andrade et al., 2009). Considered a ubiquitous in-
tracellular Ca2+ receptor involved in the transduction of a vari-
ety of extracellular signals in eukaryotes (Li et al., 2006), CaM
contains four conserved canonical motifs (calcium-binding do-
mains) called EF-hands with a helix-loop-helix structure. Each
hand can bind a single Ca2+ ion that transduces Ca2+ signals
into downstream effects influencing a range of cellular proc-
esses, including Ca2+ homeostasis (Gao et al., 2009).
Abiotic stresses including salinity, drought, and cold are im-
portant causes of crop loss worldwide (Mahajan and Tuteja,
2005). Higher plants adapt to environmental stresses by acti-
vating cascades of molecular networks involved in stress per-
ception, signal transduction, and expression of specific
stress-related genes. Stress perception and signaling pathways
are critical components of the adaptive response that are vital
for survival under extreme environmental constraints (Cao et
al., 2006). Under these conditions, a number of signal path-
98 Genes & Genomics (2011) 33: 97-103
ways including the unique Ca2+/CaM-mediated signal net-
works may be activated, resulting in transcriptional reprogram-
ming in response to these stresses (Gut et al., 2009).
Mulberry (Morus L.), a perennial tree or shrub, is an eco-
nomically important plant used not only for sericulture as the
sole food plant for the domesticated silkworm (Bombyx mori)
but also for a variety of other purposes such as the production
of edible fruits or useful timber (Rai et al., 2009). The growth
and productivity of mulberry is adversely affected by abiotic
and biotic stresses (Pan and Lou, 2008), but only a few studies
have investigated the role of CaM in mulberry, and the exact
function of CaMgenes and encoded proteins in the stress re-
sponse is still not fully understood. Therefore, efforts to inves-
tigate the molecular adaptation mechanisms to stresses and to
strengthen stress tolerance in this plant are of fundamental im-
portance to mulberry production.
In this study, we cloned the full-length sequence of a novel
CaM gene, MCaM-3, based on the expressed sequence tags
(ESTs) from a previously constructed mulberry cDNA library,
and compared its molecular characters to that of other identi-
fied CaMs. The expression levels of MCaM-3 at different de-
velopmental stages in mulberry leaves and flowers and in dif-
ferent tissues were investigated. Furthermore, changes in the
transcription level of MCaM-3 under cold, drought, and salt
stress were detected by semi-quantitative RT-PCR.The results
may help to provide a molecular basis for understanding the
complicated signal transduction mechanism underlying the
stress response and present new strategies for improving mul-
berry production through higher stress tolerance.
Materials and methods
Plant materials and reagents
To analyze gene expression under different conditions, the
mulberry variety Yu71-1 (Morus multicaulis) was grown under
standard conditions in the National Mulberry Gene Bank of
the Sericultural Research Institute, Chinese Academy of
Agricultural Sciences, Zhenjiang, Jiangsu Province, China.
The mulberry shoots were grafted, and then the grafted plants
were transferred into an incubator, and maintained at 25℃
and a 12-h photoperiod to induce burgeoning until the winter
buds grew to about 30 cm in length (50 d).
For the study and analysis of the expression level of
MCaM-3 at different developmental stages and in different tis-
sues, samples were collected from a 10-year-old mulberry tree
of the same variety and grown under the same conditions as
the plants mentioned above.
RNAiso Plus, Reverse Transcriptase M-MLV (RNaseH-),
RNase inhibitor, dNTP, LA-Taq polymerase, T4 DNA ligase,
the agarose Gel DNA Purification Kit, and pMD18-T vectors
were purchased from Takara (TAKARA Bio Co., Ltd). All
PCR primers were synthesized by the Shanghai Sangong
Biological Engineering Technology & Services Co. Ltd., and
all chemicals used were analytical grade reagents.
RNA isolation and synthesis of first-strand cDNA
Total RNA was isolated from browses (net weight about 100
mg) of grafted mulberry seedlings using RNAiso Plus reagent
following the manufacturer’s protocol, resuspended in
DEPC-treated water, and stored at -80℃. The quality of the
total RNA was determined by ultraviolet spectrophotometer
combined with electrophoresis.
The first-strand cDNA was synthesized from the total RNA
by Reverse Transcriptase M-MLV (RNaseH-) at 42℃ for 60
min with oligo-dT-adaptor primer following the manu-
facturer’s protocol. The first-strand cDNA was used as the
template for PCR in gene cloning.
Cloning of the MCaM-3 cDNA fragment and experimental
To obtain the MCaM-3 cDNA fragment, a bioinformatics-based
approach was used and BLAST (http://www.ncbi.nlm.nih.
gov/BLAST) analysis of the cDNA fragment was carried out.
The EST sequences from the mulberry cDNA library were used
to search the EST sequence database. Some ESTs for the puta-
tive CaM protein-encoding gene were selected and the putative
cDNA sequences of the CaM genes were assembled. Gene-spe-
cific primers were designed according to the assembled cDNA
sequences of the CaM genes. Browses from mulberry were
used for the detection of MCaM-3 by RT-PCR with the specific
primers MCaM-3-F: 5'- AGCGACAAACAGATTTACAGA -3'
and MCaM-3-R: 5'- GGTTCATAATATCAAAGGGAGA -3'.
The RT-PCR reactions were performed in a total volume
of 50 μL including 1 μL first-strand cDNA, 41 μL ddH2O,
1 μL each of the gene-specific primers, 0.5 μL dNTP, 5 μL
buffer, and 0.5 μL LA-Taq DNA polymerase (5 U/mL). The
RT-PCR amplifications were performed using the following
parameters: DNA was denatured at 94℃ for 5 min followed
by 26 amplification cycles (94℃ for 30s, 59℃ for 1 min,
72℃ for 1 min), with a final extension step of 7 min at 72℃.
The RT-PCR products were analyzed in 1% agarose gels and
purified using the Takara Agarose Gel DNA Purification Kit
(TAKARA Bio Co., Ltd.) following the manufacturer’s
protocol. The purified fragment, which was confirmed to have
the predicted length, was then cloned into the pMD18-T vector
and sequenced to confirm the presence of an open reading
Genes & Genomics (2011) 33: 97-103 99
frame (ORF) related to the tentative consensus sequence.
Isolation and analysis of the full-length cDNA sequence of
To study the corresponding gene’s biological functions, we
cloned the full-length cDNA using the partial cDNA fragment
as a probe to screen the mulberry cDNA library. The promoter
sequencing primers T3 (5'-AATTAACCCTCACTAAAGGG-3')
and T7 (5'-TAATACGACTCACTATAGGG-3') were used to
obtain the full-length cDNA of MCaM-3 based on the se-
quence of the vector (pBluescript II SK*) used for the cDNA
library constructed from mulberry leaves, which contained the
full-length cDNA. The sequence encoding MCaM-3 was de-
termined by homology searches in the NCBI databases
(http://www.ncbi.nlm.gov/) using the BLAST program. The
basic properties were accomplished using tools at the
http://www.expasy.org website and DNAStar software,such as
searching the ORF and translation of the nucleotide sequence
as well as the isoelectric point prediction, and determining the
molecular weight of MCaM-3.
Semi-quantitative analysis of MCaM-3 transcript levels at dif-
ferent developmental stages ofleaves and flowers and in differ-
To investigate MCaM-3mRNA content in leaves and flowers
at different developmental stages and in different tissues,
semi-quantitative RT-PCR was performed. Total RNA was
isolated from spires, tender leaves (3 days old), climax leaves
(10 days old), phloem, xylem, roots, flourishing female flowers
(unpollinated stigma), withered female flowers (pollinated
stigma), and mature female flowers (black mulberry fruits).
All of these RNAs were converted into cDNAs with reverse
transcriptase to a constant volume of 20 μl. Semi-quantitative
PCR was carried out with 1 μL cDNA as the template, and
the mulberry Maactingene
DQ785808), a house-keeping gene, was used as an internal
control to allow for normalization by visual inspection of
mRNA levels. The following primers were used for the
Maactin gene: sense, 5'-CAGTGCTTCTCACTGAGGCTC-3';
anti-sense, 5'-GGAAGAGGACTTCTGGGCATC-3'. The pri-
mers for MCaM-3 and the parameters of semi-quantitative
RT-PCR amplification were the same as for the RT-PCR
analysis. Quantitative analysis of the gel was performed using
LabImage software v. 2.7.1. (Kapelan GmbH Co, Germany).
The target band density (gray level) was used to represent the
relative expression level of the target gene, MCaM-3. All ex-
periments were repeated six or more times.
(GenBank accession No.
Expression patterns of MCaM-3 under different abiotic stresses
To reveal the putative biological function of the MCaM-3 pro-
tein, semi-quantitative RT-PCR was performed to detect the
expression level of MCaM-3 mRNA under various abiotic
stress-induced conditions in mulberry.
When the winter buds grew to about 30 cmin length (50
d), the grafted seedlings were transferred into a series of stress
treatments, including cold, drought, and salt. Stresses were ad-
ministered starting from the beginning of the photoperiod
without changing light intensity, humidity, or photoperiod.
Cold stress-induced seedlings were transferred into a low-tem-
perature incubator and exposed to 15℃ for 2 d, 8℃ for 1
d, 3℃ for 1 d, 0℃ for 3 d, -1℃ for 2 d, or -3℃ for 1 d,
with a 12/12-h artificial light/dark cycle. Salt stress was in-
duced with 300 mM NaCl. The roots of the plants were di-
rectly submerged into a container of 300 mM NaCl for 12
d, and the salt stress-induced seedlings were grown in a con-
trolled environment at 25℃ with a 12/12-h artificial light/dark
cycle. To induce drought stress, mulberry seedlings were care-
fully transferred to a dry growth chamber, under an artificial
environment at 25℃ with a 12/12-h artificial light/dark cycle
and allowed to desiccate for 11 d. Control plants were main-
tained at 25℃ and a 12-h photoperiod in the incubator, without
changing light intensity or humidity before RNA extraction.
The browses subjected to different stress conditions were col-
lected for RNA extraction until the appearance of symptoms.
All tissues harvested for nucleic acid extraction were weighed,
immediately frozen in liquid nitrogen, and stored at -70℃ until
Total RNA isolated from browses of mulberry under differ-
ent stress conditions were converted into cDNAs with reverse
transcriptase to a constant volume of 20 μL. Semi-quantitative
RT-PCR amplification and the methods of quantitative analy-
sis of the gel were the same as in above.
Sequence analysis of the MCaM-3 gene
To clone the full-length cDNA of the mulberry CaM gene, a
cloning strategy combining bioinformatics analysis and the mul-
berry cDNA library screening technique was used. Sequence
analysis showed that the isolated cDNA, designated MCaM-3
(mulberry CaM gene 3, GenBank accession No: GQ303247),
is 951 base pairs (bp) in length and contains a 143 bp
5'untranslated region (5'-UTR) and a 358 bp 3'-UTR. Its ORF
is 450 bp encoding 149 amino acids with a predicted molecular
weight of 16.85 kD and an isoelectric point of 3.95 (Fig. 1).
100 Genes & Genomics (2011) 33: 97-103
Figure 1. The full-length cDNA sequence of the mulberry calmodulin
gene MCaM-3.The amino acid sequence is displayed in a one-letter
code under the coding sequence, with the underlined amino acids
representing the EFh binding domain. The translation start codon
is framed. The asterisk denotes the stop codon.
Figure 2. Molecular modeling of the EFh domain of the MCaM-3
protein using SWISS-MODEL. The calcium-binding loops and flank-
ing helices are marked in different colors.
To further understand the protein structure of MCaM-3, we
predicted the structure domain of MCaM-3 using the online
SMART program (http://smart.embl-heidelberg.de/). The de-
duced protein of MCaM-3 contained four Ca2+-binding motifs,
known as the helix-loop-helix EF-hands, with four functional
conserved amino acid residues inside the ORF of MCaM-3,
one in position 12–40, and the others in positions 48–76, 85–
113, and 121–149. Transient Ca2+ elevation may be sensed
by several Ca2+ sensors or Ca2+-binding proteins, which usually
contain the EF-hand motif(s) (Gu et al., 2008). As the proto-
typical eukaryotic Ca2+ sensor and evolutionarily conserved
protein, the typical CaM consists of two globular regions, each
possessing a pair of Ca2+-binding domains (called an EF hand,
or EFh). Upon binding of Ca2+, CaM can shift to an open
conformation exposing two hydrophobic surfaces that enable
interactions with proteins or a Ca2+-sensitive target down-
stream (Grabarek, 2006). The solution structure of the EFh
domain revealed that MCaM-3 is a typical CaM gene, con-
sistent with a Ca2+-induced conformational change that is char-
acteristic of true Ca2+ sensors or Ca2+-binding proteins in mul-
berry (Aravind et al., 2008).
To better understand the molecular modeling of the
Ca2+-binding domains in the MCaM-3 protein, we established
the three-dimensional structure of the MCaM-3 protein using
SWISS-MODEL (http://swissmodel.expasy.org/) and the crys-
tal structure of calcium-bound dimeric GCAMP2 (PDB ID:
3evv) as the model. As shown in Figure 2, each EFh domain
of MCaM-3 is characterized by a helix-loop-helix structure
and consists of a loop flanked by two helices with the inter-
helical loop able to bind a calcium ion through structural varia-
tion (Capozzi et al., 2006). According to studies of the EFh
protein family, MCaM-3 belongs to the four EFh subfamily
(Gifford et al., 2007). The structure of the predicted EFh of
MCaM-3 suggests that it likely has Ca2+-binding properties,
like other Ca2+ sensors in mulberry.
Expression levels of MCaM-3 at different developmental
stages of mulberry leaves and flowers and in different tissues
CaM is a ubiquitous Ca2+-binding protein that regulates many
Ca2+-dependent cellular processes in both plant and animal
cells and is expressed in many cell types with different sub-
cellular locations (Orojan et al., 2006). CaM gene expression
also varies with the developmental stage of plant growth
(Folzer et al., 2005).
To elucidate the mechanisms underlying Ca2+-regulated
gene expression in mulberry, the expression of MCaM-3 was
further analyzed in roots, flowers, and leaves at different de-
velopmental stages using semi-quantitative RT-PCR (Fig. 3).
We investigated the relative levels of MCaM-3 transcripts
from nine samples, comparing five different tissues: leaf,
phloem, xylem, root, and flower. MCaM-3 transcripts were
detected in all samples and seemed to be most abundant on
the spire with the lowest expression level in the phloem.
During the three different leaf developmental stages, the ex-
pression level of MCaM-3mRNA was highest in the spire, low-
er in the climax leaf, and even lower in the tender leaf. During
Genes & Genomics (2011) 33: 97-103 101
Figure 3. Expression profiles of the MCaM-3 gene in mulberry. (A)
Samples of mulberry leaves and flowers at different development
stages and various tissues of mulberry: (1) spire,(2) tender leaf (3
days old), (3) climax leaf (10 days old), (4) phloem, (5) xylem, (6)
root, (7) flourishing female flower (unpollinated stigma), (8) withered
female flower (pollinated stigma), (9) mature female flower (black
mulberry fruit). (B) MCaM-3 gene expression in mulberry leaves,
flowers, and tissues. Lanes 1-9 represent the samples in Figure 3A
(1-9), respectively. The mulberry Maactingene (DQ785808) was used
as an internal standard to normalize the template amount from the
different samples. (C) Signal values of the gel were scanned using
LabImage software v. 2.7.1. The target band density was used to
represent the relative expression level of the target gene MCaM-3.
Figure 4. Expression patterns of MCaM-3 under different stress treat-
ments in mulberry. (A) The symptoms of mulberry seedlings induced
by abiotic stresses compared to controls (non-stress treatment). (B)
Expression patterns of MCaM-3 under different stress treatments
compared to controls. TotalRNA was isolated from 50-day-old graft-
ed seedlings from control plants (1) and seedlings exposed to cold
(2), drought (3), and NaCl (4). The mulberry Maactin gene
(DQ785808) was used to normalize the template amount from the
different samples. (C) Signal values of the gel were scanned using
LabImage software v. 2.7.1. The target band density was used to
represent the relative expression level of the MCaM-3 target gene.
the three different developmental stages of the female flower,
the expression level of MCaM-3 mRNA was highest in the
mature flower, appreciably higher than in the flourishing flow-
er, and lowest in the withered flower. The expression of
MCaM-3 exhibits a tissue-specific and developmental
stage-specific pattern even in the same tissues in mulberry.
These results suggest that MCaM-3 expression may be pre-
dominantly associated with tissue aging or with particular
Stress-induced expression patterns of MCaM-3
Ca2+ions play a major role in mediating plant growth and de-
velopment and in response to biotic and abiotic stresses (Galon
et al., 2008). As a small protein molecule with a molecular
weight of about 17 kDa, CaM is universally found in plant
cells and cell walls. Cellular CaM serves as the most important
Ca2+ receptor protein for initiating downstream Ca2+ signal
events (Ma et al., 2009). The Ca2+/CaM complex plays vital
roles in plants in sensing various environmental biotic and
abiotic signals and triggering appropriate cellular responses by
modulating the activities or functions ofa wide range of
CaM-binding proteins, including metabolic enzymes and tran-
scription factors, as well as ion channels and pumps; moreover,
there is a growing body of evidence that CaM plays a crucial
role in plant defense signaling (Koo et al., 2009). Expression
pattern analysis can help to reveal the possible biological func-
tions of the target gene. CaMgenes show differential tis-
sue-specific expression and differential temporal and spatial
expression in response to external stimuli (Gao et al., 2009).
To further investigate whether the expression of MCaM-3
was induced by abiotic stresses, we monitored the mRNA tran-
script level of the MCaM-3gene under different abiotic stress
treatments, including low temperature, drought, and salt, by
semi-quantitative RT-PCR until the appearance of symptoms
(Fig. 4A). As shown in Figure 4B, the expression level of
the MCaM-3 transcripts increased significantly under cold,
drought, and NaCl stress compared to the normal growth
environment. As shown in Figure 4C, the expression level of
MCaM-3was highest under drought treatment, followed by the
cold and NaCl stress treatments, and lowest in the control
(non-stress conditions). These results revealed that MCaM-3
was constitutively expressed under non-stress conditions, but
its expression level increased to different degrees under differ-
ent abiotic stresses.
These results suggest that the protein encoded by MCaM-3
may be involved in multiple abiotic stress tolerances in
mulberry. Clearly, the nature of MCaM-3 proteins under vari-
ous environmental stresses will be an important area for future
102 Genes & Genomics (2011) 33: 97-103
A prominent feature of CaM in higher plants is the expression
of multiple isoforms within a single species. For example,
CaM is encoded by five genes in soybean, eight genes in pota-
to, and nine genes in Arabidopsis thaliana (Maghuly et al.,
2008). MCaM-3 is the third CaM isoform we cloned from
mulberry and is different from MCaM-1 and MCaM-2. The
mRNAs encoding the three mulberry CaM proteins differ in
their sequences, suggesting a particular role for each isoform,
and the predominant isoform of CaMin mulberry is still not
In plants, the CaM isoforms not only differ from one anoth-
er by some acid substitutions, but also by the high CaM protein
accumulationin specific tissues (Al-Quraan et al., 2010). In
general, the main expression of these CaM clones was detected
in young tissues and meristems, with a very high expression
in the root apex, which is considered a perception organ
(Camas et al., 2002). It isimportant to note that, as opposed
to other plant species, the MCaM-3 protein was most abundant
in mature mulberry tissues, such as mature female flowers and
climax leaves. This suggests that the main role of MCaM-3
is related to maintenance rather than development. However,
more studies are needed to ascertain whether this CaM protein
accumulation in mature tissues is associated with all CaM iso-
forms or is specific to one in mulberry.
In general, CaMmRNAs are developmentally regulated and
signal-responsive (Wang et al., 2008). Ca2+ is essential for sig-
nal transductions that ultimately result in a variety of physio-
logical responses in plants. Changes in cytosolic Ca2+ concen-
tration are triggered by many external stimuli, and various
physiological responses are associated with these changes (Lee
et al., 2007). Ca2+-induced conformational changes in
CaMaffect the interactions among target proteins and modulate
target protein activity (McCormack et al., 2005). The
Ca2+/CaM messenger system is involved in stress-induced sig-
naling transduction, and CaMproteins are involved in the re-
sponse to environmental stimuli in plants (Hong-Bo et al.,
2008). The basic functional EFh domain of CaMconsists of
29 amino acids arranged in a helix-loop-helix conformation
and it is often present in pairs (Duplat et al., 2006). The
MCaM-3 has four EFh domains and may be an important
Ca2+-binding protein that binds to other proteins, affecting their
activities under environmental stimuli in mulberry (Wang et
In addition, during growth and development, plants respond
to a wide range of environmental stresses through multiple
defense mechanisms, such as transcriptional activation of de-
fense genes, cell wall enforcement, induction of programmed
cell death, and production ofantibiotic compounds. Plant stress
responses involve the expression of a battery of genes encod-
ing proteins with an adaptive function. These genes are thought
to function not only in protecting cells from stress by the pro-
duction of important metabolic proteins, but also in the regu-
lation of genes for signal transduction in stress responses
(Roche et al., 2009). The mulberry is the sole source of food
for silkworms (Bombyx mori) and its growth and productivity
are adversely affected by stresses such as drought, salt, and
cold (Mamrutha et al., 2010). By understanding the mecha-
nisms of plant tolerance to environmental stress, it is possible
to provide new tools and strategies for improving plants in
this respect. In our research, MCaM-3 was differentially ex-
pressed in response to abiotic stresses, indicating that the
MCaM-3 protein might have important biological functions
in stress acclimation, and the variation in the level of MCaM-3
may reflect the coping response in mulberry. The results of
this study may contribute to our understanding of the molec-
ular mechanisms of abiotic stress tolerance in the mulberry.
In summary, it is likely that MCaM-3 is an important and
common component of the response to abiotic stresses in
mulberry. Additional studies are underway to further elucidate
the biological function of MCaM-3, especially in the context
of the possible crosstalk between biotic and abiotic stress
Acknowledgments We thank three anonymous reviewers and
the editor for critically reviewing the manuscript. This work
was support by China (20060400926), Jiangsu (0602004C)
Postdoctoral Science Foundation Project, and PublicIndustry
(Agriculture) Specific Research Program (nyhyzx07-020), and
the sericulture industry technology in China program（ny-
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