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Acta Biochim Biophys
Sin 2008, 40: 7884
doi:10.1111/j.1745-7270.2008.00371.x
Novel cotton homeobox gene and
its expression profiling in root development and in response to stresses and
phytohormones
Yongxiang Ni, Xiulan Wang,
Dengdi Li, Yajie Wu, Wenliang Xu, and Xuebao Li*
Hubei Key Laboratory
of Genetic Regulation and Integrative Biology, College of Life Sciences,
Huazhong Normal University, Wuhan 430079, China
Received: July 24,
2007
Accepted: August
18, 2007
This work was supported
the grants from National Program for Basic Research of China (No.
2004CB117304), and the National Natural Sciences Foundation of China (No.
30470930)
*Corresponding
author: Tel/Fax, 86-27-67862443; E-mail, [email protected]
Homeodomain-leucine
zipper (HD-Zip) proteins are transcriptional factors involved in plant
development. In this study, one cDNA clone (Gossypium hirsutum
homeobox1, designated GhHB1) encoding HD-Zip protein was isolated from
a cotton root cDNA library. The GhHB1 cDNA is 1132 bp in length,
including an 828 bp open reading frame that encodes a peptide with 275 amino
acids, and 5-/3-untranslated regions. The predicted GhHB1 protein
containing a homeodomain and a leucine-rich zipper motif shares relatively high
identity with other plant HD-Zip proteins. Analysis using quantitative
real-time RT-PCR indicated that the GhHB1 gene is predominantly
expressed in roots and hypocotyls. Furthermore, GhHB1 transcripts were
largely accumulated in early root development, and significantly reduced to
very low levels as roots further developed, suggesting that the gene might
function in the early development of roots. Under treatment with 1% NaCl, the
expression level of the GhHB1 gene was dramatically increased in roots.
Likewise, GhHB1 activity in roots was up-regulated by abscisic acid.
These results imply that GhHB1 might play an important role in response to salt
stress and to abscisic acid signaling.
Keywords cotton; homeodomain protein; gene expression; development;
stress
Homeodomain-leucine zipper
(HD-Zip) proteins are transcriptional factors involved in plant development,
and can be classified into four groups, HD-Zip I, HD-Zip II, HD-Zip III, and
HD-Zip IV [1]. HD-Zip I and II are very similar in their domain structures, and
might be related to the signal transduction networks of light,
dehydration-induced abscisic acid (ABA), and auxin [2–5],
whereas HD-Zip III and IV display slightly different sequences in their domains
[6,7].
The homeobox (HB) genes
encoding HD-Zip proteins are identified only in plants [8–10]. The expression of the HB genes is
regulated by different external factors [11–13].
For example, the expression of AtHB-2/HAT4 is regulated by far-red
light [14]. The antisense-transgenic plants of AtHB-2/HAT4 were shorter
and developed more slowly than wild-type plants, whereas AtHB-2/HAT4
sense-transgenic plants showed a shade-avoidance phenotype with elongated
hypocotyls and petioles, as well as earlier flowering, compared with the
wild-type [14–17]. Sunflower Hahb-10
gene is predominantly expressed in mature leaves and up-regulated by etiolation
and gibberellin in seedlings [18]. Overexpression of the Hahb-10 gene
in Arabidopsis showed altered responses to illumination quality and
intensity, indicating that Hahb-10 plays a role in light-dependent
responses of plants [18]. The study revealed that ATHB7 expression in Arabidopsis
is induced by drought as well as by ABA [19]. Although some HB genes have been
well characterized in a few plant species (such as Arabidopsis), little
is known about cotton HB genes.
In this study, we isolated a
novel homeobox gene (Gossypium hirsutum homeobox1, designated GhHB1)
in cotton and we report its expression profiling in cotton root development and
in response to stresses and phytohormones.
Materials and Methods
Collection of plant materials
Cotton (Gossypium hirsutum)
seeds were surface-sterilized with 70% (V/V) ethanol for 60 s
and 10% (V/V) H2O2 for 2 h, followed by washing
with sterile water. The sterilized seeds were germinated on one-half Murashige
and Skoog (MS) medium under a 16/8 h
light/dark cycle at 28 ? for 5–6
d. Roots, cotyledons, and hypocotyls were cut from sterile seedlings. Other
tissues for RNA extraction were derived from cotton plants grown in a
greenhouse.
Isolation of GhHB1 cDNA
To identify the genes that might
be involved in regulation of root development, over 1000 cDNA clones were
randomly selected from a root cDNA library of cotton for sequencing. Some cDNA
clones, including GhHB1, encoding the HD-Zip proteins were identified
for further characterization.
DNA sequencing and protein
analysis
Nucleotide and amino acid
sequences were analyzed using DNAstar (DNAstar, Madison, USA). The GhHB1
protein structure was analyzed by Motifscan (http://myhits.isb-sib.ch/cgi-bin/motif_scan).
The HD-Zip peptide sequences were aligned with the ClustalW program (http://www.ebi.ac.uk),
and phylogenetic analysis was used to investigate the evolutionary
relationships between GhHB1 and other plant HD-Zip proteins. A neighbor-joining
tree was generated in MEGA3.1 [20]. A bootstrap analysis with 1000 replicates
was carried out to assess the statistical reliability of the tree topology.
Treatments with NaCl,
polyethylene glycol (PEG), and phytohormones, and at 4 ?C
After seeds germinated and
grew on basic MS semisolid medium with 0.4% agar without phytohormone at 28 ?C
in light for 5 d, the cotton seedlings were transferred to a cold environment,
at 4 ?C, for 12 h, or into MS liquid medium containing 1% NaCl, 16% PEG, 10 mM zeatin, 10 mM
gibberellin3, 10 mM indole acetic acid, or 10 mM ABA for 12 h. For further experiments,
the 5-d-old cotton seedlings were treated with various concentrations of ABA
for 12 h, or with 10 mM ABA and 1% NaCl for 3, 6, 12,
24, or 48 h in MS liquid medium. Roots were collected from the seedlings for
total RNA isolation.
Real-time RT-PCR
Total RNA was isolated from
fibers, ovules, anthers, petals, leaves, stems, cotyledons, hypocotyls, and
roots of cotton using modified CTAB acerbic phenol and hot phenol methods.
Concentration of the isolated total RNA was determined using a NanoDrop
spectrophotometer (NanoDrop, Wilmington, USA) and agarose gel electrophoresis.
The real-time RT-PCR reaction was carried out according to our previous
method, using a cotton polyubiquitin gene (GhUBI) as a standard control
[21]. First, total RNA samples (2 mg per
reaction) from fibers, ovules, anthers, petals, leaves, stems, cotyledons,
hypocotyls, and roots were reversely transcribed into cDNAs by AMV reverse
transcriptase (Roche, Nutley, USA) according to the manufacturer’s
instructions. The cDNAs were used as templates in real-time PCR reactions with
gene-specific primers. The specific primers of GhHB1 were GhHB1-Up
(5-GCATGACTCAACTCCTTCAAG-3) and GhHB1-Down (5-GCCAACCAACTCTCCATATTG-3).
The real-time PCR reaction was carried out using Real-time PCR Master Mix
(Toyobo, Osaka, Japan) according to the manufacturer’s instructions. The
amplification of the target genes was monitored every cycle by SYBR-Green
fluorescence. The Ct, defined as the PCR cycle at which a statistically
significant increase of reporter fluorescence is first detected, was used as a
measure for the starting copy numbers of the target gene. Relative quantification
of the target GhHB1 expression level was carried out using the
comparative Ct method. The relative value for the expression level of the GhHB1
gene was calculated using the equation:
Eq.
PCR products were confirmed on
an agarose gel. The primer efficiency was detected using GhHB1 cDNAs as
the standard template, and the RT-PCR data were normalized with the relative
efficiency of the primer pair.
Results
Isolation and characterization
of GhHB1 gene
To isolate the genes that
might be involved in regulation of root development, we randomly sequenced over
1000 cDNA clones from a root cDNA library of cotton. Clones likely to be
involved in regulation of root development were chosen for further study. Of these,
one cDNA clone (designated GhHB1; GenBank accession No. EF151309) was
identified to encode an HD-Zip protein. It was 1132 bp in length, including an
828 bp open reading frame encoding an HD-Zip protein with 275 amino acids
(molecular weight 31.28 kDa; pI 6.665). Protein structure analysis revealed
that the deduced GhHB1 protein contains the typical homeodomain and leucine
zipper motif located at Ser73–Lys133 and
Leu135–Leu177, respectively (Fig. 1).
Similarity comparison between GhHB1
and three known HD-Zip proteins is shown in Fig. 2. GhHB1 shares 51%
identity with HAHB1, 56% identity with HAT7, and 49% identity with ATHB13. All
of the proteins contain the conserved homeodomain and leucine zipper motif.
Although the homeodomain is quite conserved, there are 13 positions in which
amino acids substitutions occurred among the homeodomains of the four HD-Zip
proteins. In the homeodomain of GhHB1, the amino acid substitutions are
involved in seven positions (74, 75, 84, 89, 93, 106, and 110), and five out of
the seven substitution locations belong to conservative interchanges (Leu75/Ala/Met,
Leu84/Met,
Ala89/Thr,
Val106/Met/Ile,
and Lys110/Arg).
However, there are two positions at which dissimilar amino acid substitutions
(His74/Gln
and Ser93/Asn)
occurred on the homeodomains between GhHB1 and the other HD-Zip proteins. Such
interchanges could affect the HD-Zip protein structure and its function.
Phylogenetic analysis of GhHB1
To analyze the phylogenetic
relationships of GhHB1 with the known HD-Zip I proteins in plants, 11 HD-Zip
proteins from different plant species were used for constructing a
phylogenetic tree with MEGA3.1. As shown in Fig. 3, these HD-Zip
proteins could be divided into two clades. The first clade contains six members
(ATHB7, ATHB12, ATHB13, HAT7, HAHB1, and GhHB1) and the second clade possesses
five members (Oshox4, Oshox5, ATHB5, ATHB6, and ATHB1). All the 11 HD-Zip I
proteins might have common provenance, and each member located in the same
subgroup might have diverged relatively late during evolution. The cotton GhHB1
belongs to the first clade, and has the closest evolutionary relationship with
HAHB1 (Fig. 3).
GhHB1 gene preferentially expressed
in young roots and hypocotyls
To investigate the expression
pattern of GhHB1 in cotton, we carried out real-time quantitative
RT-PCR. The experimental results indicated that GhHB1 was strongly
expressed in roots and hypocotyls, but its transcripts were detected at very
low levels in other tissues (such as leaves, stems, cotyledons, petals,
anthers, fibers, and ovules), suggesting that GhHB1 might function mainly in
root and hypocotyl development [Fig. 4(A)]. Furthermore, the highest
accumulation of GhHB1 transcripts was detected in 3-day old roots, then
the gene expression declined significantly to a relatively low level in 6-day
old roots and gradually to much lower levels in 9- and 11-day old roots,
suggesting that GhHB1 expression is regulated during the development of
cotton roots [Fig. 4(B)].
GhHB1 expression is up-regulated by
NaCl stress in roots
To investigate GhHB1
expression under various stresses, we treated the cotton seedlings with 1%
NaCl, cold (4 ?C), and 16% PEG. The experimental results revealed that GhHB1
expression was significantly increased in roots under NaCl stress, but was only
slightly changed under cold and PEG treatments [Fig. 5(A)]. Furthermore,
the level of GhHB1 expression in the roots of the NaCl-treated seedlings
was 2.5 to 3 folds higher than in the control seedlings, and its highest
activity was detected in the roots under 1% NaCl treatment for 6–12 h [Fig. 5(B)], suggesting that
the GhHB1 gene might be involved in response to salt signaling in early
development of roots.
GhHB1 expression is induced by ABA
in roots
To investigate whether
phytohormones influence GhHB1 expression, several exogenous
phytohormones were added in MS medium for cultivating cotton seedlings, and
then GhHB1 expression activity in roots of the seedlings was detected by
real-time RT-PCR. The experimental results revealed that under ABA treatment,
the accumulation of GhHB1 mRNAs was significantly increased in roots.
Unlike ABA, however, indole acetic acid and zeatin treatments did not affect GhHB1
activity in roots (data not shown). Compared with the control seedlings, the
expression of GhHB1 was significantly enhanced by over 2-fold in roots
when the seedlings were treated by ABA for 3 h, and kept its high expression
levels in the roots till 48 h [Fig. 6(A)]. Furthermore, all the
treatments with 2–25 mM
ABA were effective on promoting GhHB1 expression activity in roots of
cotton seedlings [Fig. 6(B)]. The experimental results suggest that GhHB1
might be involved in the ABA signaling pathway.
Discussion
HD-Zip proteins are
characterized by the presence of a DNA-binding homeodomain and an adjacent
leucine zipper motif mediating protein-dimer formation [10]. HD-Zip proteins,
which are unique to plants, might play important roles in the development of
high plants. In this study, an HB gene GhHB1 encoding an HD-Zip protein
was identified in cotton. Structural analysis of the deduced protein showed
that GhHB1 contains an HD and a Zip motif (Fig. 1). Comparison of
protein sequences showed that GhHB1 shares relatively high identity with the
other plant HD-Zip I proteins (Fig. 2). The phylogenetic analysis also
showed that GhHB1 has a close evolutionary relationship with the HD-Zip I
proteins (Fig. 3). Thus, cotton GhHB1 belongs to the HD-Zip I subfamily.
The expressions of many plant
HB genes are regulated by internal and external factors, and are involved in
ABA signaling pathway. A previous study revealed that the expression of ATHB5,
as a positive regulator of ABA responsiveness, is developmentally controlled
and dependent on ABA signal transduction in Arabidopsis seedlings
[22]. Similarly, the expression of ATHB7 and ATHB12 genes are
induced by exogenous ABA or stimuli (such as water deficit or osmotic stress)
that increase the endogenous levels of ABA, indicating that these genes might
act in signaling pathways mediating responses to drought in Arabidopsis
[23–25]. Sunflower Hahb-4 is
up-regulated by water stress, suggesting that it might function in signaling
cascades of ABA-dependent responses to water stress [13]. Arabidopsis
ABI1 and ABI2, the type 2C protein serine/threonine phosphatases, act as key
regulators in response to ABA, whereas the transcriptional regulator ATHB6, as
a target of ABI1, linked the protein phosphatase 2C for the regulation of ABA
signaling [4]. The study showed that CpHB6 and CpHB7 are up-regulated by
dehydration, whereas CpHB3, CpHB4, and CpHB5 are down-regulated by dehydration
in both leaves and roots of Craterostigma plantagineum [26]. In oilseed
rape (Brassica napus), BnHB6 expression in shoots is significantly
up-regulated by ABA, mannitol, NaCl, cold, H2O2, and salicylic acid
treatments, implying that it plays a positive role as a regulator of biotic and
abiotic stresses on seedling growth [27]. In this study, likewise, the
presented data indicated that GhHB1 expression was up-regulated by ABA
and NaCl treatments (Figs. 5 and 6), implying that GhHB1
might play a role in response to salt stress and to ABA signaling. Furthermore,
the GhHB1 transcripts were largely accumulated in 3-day old roots, but
its expression activity gradually declined to very low levels as roots further
developed (Fig. 4), suggesting that it might function in early root
development of cotton.
In summary, the isolated GhHB1 gene
is a new member of the plant HB gene family, and its expression is regulated
in root development of cotton and in response to stresses and phytohormone
signaling. Thus, the results of this study contribute to the understanding of
the regulation of GhHB1 expression in cotton. However, the role of this
gene in cotton development still remains to be explored in the future.
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