Characterization and Chromosome Location of ADP-ribosylation Factors (ARFs) in Wheat
Guoyue Chen ,
Jirui Wang ,
Yaxi Liu ,
Qiantao Jiang ,
Wei Li ,
Xiujing Lan ,
Shoufen Dai ,
In this study, the ARF genes were cloned, sequenced and located
on the chromosomes. The gene expression of various stress conditions were analyzed
through RT-PCR. Two important features of ARF in wheat were found: (1) High
sequences homology among species in mammalian and plant and (2) Four exons and
three introns were conserved in Poaceae. In this study the coding genes of ADP-ribosylation
Factors (ARF) were characterized and they were located on chromosomes 3AL and
2DL in common wheat and its diploid progenitors. Forty-seven candidate SNPs
in ARF were detected which were located in exons (17 SNPs) and introns (30 SNPs),
respectively. As expected, most of the SNPs (66.34%) in ARF were transitions
and the rest (33.66%) were transversions. The expression difference of ARF under
various environmental stresses (low-temperature, Abscisic Acid (ABA), Polyethylene
Glycol (PEG), NaCl, stripe rust), in two stages (seedling and maturity) and
in different tissues (root, stem, flag leaf and immature embryo) of 15 days
post-flowering were investigated. The results revealed that the expression levels
of ARF were affected by environmental stresses. PEG stress induced the highest
level of ARF expression, followed by the stripe rust and ABA stresses.
to cite this article:
Zhien Pu, Guoyue Chen , Jirui Wang , Yaxi Liu , Qiantao Jiang , Wei Li , Xiujing Lan , Shoufen Dai , Yuming Wei and Youliang Zheng, 2014. Characterization and Chromosome Location of ADP-ribosylation Factors (ARFs) in Wheat. Pakistan Journal of Biological Sciences, 17: 792-801.
Received: January 30, 2013;
Accepted: August 31, 2013;
Published: January 29, 2014
ADP-ribosylation Factors (ARF) were GTP-binding proteins (Brown
et al., 1993; Memon, 2004). ARF1 has been
known that it plays an important role in vesicular transport between the ER
and the Golgi and maintains the stability of the Golgi and ER organization (Takeuchi
et al., 2002; Lee et al., 2002).
ARF are a critical component in the regulation of vesicle-mediated secretary
pathway in eukaryotic cells. The functions of ARF proteins played an important
role in keeping the membrane traffic and organelle integrity, at the same time,
the ARF proteins were tied to its reversible association with membranes and
specific interactions with membrane phospholipids (Donaldson,
2003; Nickel and Wieland, 1997; Bonifacino
and Glick, 2004). The activity of ARF is regulated by the binding and hydrolysis
of GTP, as they cycled between active GTP-bound and inactive GDP-bound conformational
states. The active GTP-bound state is regulated by the guanine nucleotide exchange
factors (GEFs) which promote the exchange of GDP to GTP. The form change between
inactive GDP-bound and active GTP-bound were selectively regulated by ARF cycle
(Jackson and Casanova, 2000). Based on phylogenetic
lines, ARF are abundant, ubiquitous and highly conserved in eukaryotes. Although
the ARFS have been mainly studied in mammalian and yeast, several orthologues
have been identified in plants.
In recent studies, most of ARFs were cloned and identified from various plant
species, such as rice (Higo et al., 1994), wheat
(Kobayashi-Uehara et al., 2001), carrot (Asakura
et al., 2007), Arabidopsis (Regad et al.,
1993) and so on. The features and functions of ARF genes in plant, yeast
and mammalian are similar. They are found to be mainly active in roots and flowers
(Kobayashi-Uehara et al., 2001). In Arabidopsis,
overexpression of antisense ARFA1 reduces cell division and cell expansion which
lead to antisense plants being severely stunted (Gebbie
et al., 2005). ARF is important for wheat growth and development
and the upregulated expression of this gene might contribute to the heterosis
observed in wheat root and leaf growth in wheat (Yao et
al., 2009). Although these studies on ARFs in different plants are not
complete, the findings have so far provided a reliable view on the general functions
of ARFs. In this study, ARF gDNA sequences, chromosomes location, splicing pattern
in the wheat were determined. The results also revealed that the ARF expression
was affected under various environmental stresses.
MATERIALS AND METHODS
Plant materials: It was thought that the genomes of cultivated wheat
(Triticum aestivum, AABBDD) were derived from T. urartu (A), Aegilops
speltoides (B) and Ae. tauschii (D), respectively. In this study,
the cultivated wheat and its diploid putative progenitors were chosen to characterize
ADP-ribosylation factor. Ten T. monococcum (2n = 2x = 14, AmAm),
10 T. urartu (2n = 2x = 14, AuAu), 5 Ae. tauschii
(2n = 2x = 14, DD), 5 Ae. speltoides (2n =2x =14, SS), 5 spelt wheat
and 6 cultivated wheat varieties were used (Table 1). These
diploid wheat and Aegilops accessions were kindly supplied by the USDA-ARS.
Plants grown in normal condition were used for gDNA cloning, cDNA cloning and
DNA isolation and PCR amplification: CTAB method was used for the extraction
of genomic DNA as previously described. Based on the alignment of 7ARF gene
sequences, primers were designed to amplify the complete Open Reading Frames
(ORF) of ARF gene. PCR amplification was performed with a GeneAmp PTC-200 cycler
(MJResearch,USA) in 50 μL volume which included of 50 ng of genomic DNA,
0.2 mmol L-1 of dNTPs, 150 ng of oligonucleotide primers, 1.5 U ExTaqTM
polymerase (TaKaRa), 2 mmol/MgCl2 and 5 μL 10xPCR buffer. PCR
amplifications were conducted using the following thermal cycle: 95°C for
300 sec, followed by 35 cycles of 60 sec at 95°C, 45 sec at 57-60°C
(depending on the primer sets and 45 sec at 72°C. The desired DNA fragments
were recovered from gels and ligated to the pBluescript SK (+) T-vector plasmid
(Stratagene). The positive clones were screened and sequenced.
The expression level of ARFs was analyzed by RT-PCR. RNA was prepared from
the roots, leaves, stems and immature embryo of the cultivated wheat plants.
Seedlings were treated separately with NaCl (200 mM), Abscisic Acid (ABA) (50
mM) and Polyethylene Glycol (PEG) (150 mM) for 48 h, low temperature (4°C)
for 24 h and yellow rust for 72 h at the same time. At regular intervals, 100
mg of the seedlings were harvested and the total RNA was isolated from the samples
as described above. The RNA extraction was checked by electrophoresis. Aliquots
of RNA solutions (approximately 0.5 mg RNA equivalent) were added to the RT-PCR
mixture prepared from the one step RT-PCR Kit (TakaRa).
ARFs sequence analysis: More than 90 complete and partial ARF Expressed
Sequence Tag (EST) sequences retrieved from wheat, barley, maize, rice, human
and Arabidopsis were obtained from the GenBank. To concentrate on the coding
region alone, 5 and 3
non-translated regions and several ultra-short sequences were omitted. A total
of 54 ARF sequences were left for alignment analysis using MEGA 3.0. The alignment
results showed that ARFs were highly conserved.
The analysis of full-length sequence and the construction of subsequent nucleotide
sequences were carried out under DNAman 4.0. The multiple sequence alignment
software Clustal W (http://www.ebi.ac.uk/clustalw)
was used for the SNP evaluation. ARFs were translated into amino acid sequences
using cDNA sequences. The phylogenetic trees were constructed by MEGA, version
2 (Wang et al., 2004). ARF sequences were subject
to nucleotide and amino acids alignment using CLUSTAL X program (ver.1.83).
Structure predictions: The three secondary structure elements are a
helical region known as a-helix (H), a coil/loop and an extended region known
as h-stand which joins a-helix together to form h-sheet (E). Fold recognition,
disulfide bridges analysis and structural alignments were performed with ANTHEPROT
software (Wang et al., 2004). To compare the
secondary structure of ADP-ribosylation factors, 3D-PSSM protein fold recognition
(threading) server (http://www.emblheidelberg.de/predictprotein)
was used to assume the predicted secondary structure of these ADP-ribosylation
|| Material source and name
|CN16: Chuannong 16; LM2: Liangmai 2; LM3: Liangmai 3. They
are common wheat
Sequence analysis and ARF gene classification: In order to identify
ARFs in wheat, a DNA segment 1195 bp was isolated from all samples. A number
of gDNA clones was randomly selected and sequenced. A total of 73 sequences
were obtained. The comparative analysis of the partial nucleotide sequences
was carried out by BLAST for further characterization of gDNA encoding ARFs
or ARF-like small GTP binding proteins. The sequencing results showed that these
DNA amplified segments were indeed ARFs. A sample of gDNA exhibited a high homology
(88-100%) among these clones. Compared with ARF sequences isolated from wheat
and other species, such as yeast, human, carrot, rice, maize, A. thaliana
and so on. The ARFs were isolated from barley and rice had higher sequence homology,
up to 98%. The ARFs are also highly conserved in Arabidopsis (83%) and Brassica
(84%). The results showed that the ARFs were conserved across species and also
indicated that they may have important functional roles.
The sequences isolated from diploid species and hexaploid wheat also were analyzed.
The results showed that the ARFs had two and three different sequences in diploid
species and hexaploid wheat, respectively, so the ARFs might have two copies
in diploid and three copies in hexaploid wheat which is consistent with previous
studies (Kobayashi-Uehara et al., 2001).
Sixty six of the 73 sequences were not the same and the rest of the sequence
is identical. The 66 different sequences were selected for further analysis
using the Paup software. Phylogenetic analysis based on these sequences indicated
that the 66 sequences could be divided into two groups. Most of sequences derived
from A genome were clusted in Group I and Group I could be further branched
into two subgroups again (Fig. 1). T. monococcum, Triticum
urartu and part of the sequences isolated from hexaploid chromosome which
had same haplotype with A chromosome were clustered in Group I.2. It is worth
mentioning that the Iran and Europe spelt wheat did not differ from each other.
Group I.1 included the sequences from PI225295 (Iran) and PI347862 (Europe).
Group II included all sequences isolated from D and S chromosome, as well as
sequences which had similarities with haplotype from the hexaploid chromosome.
Furthermore, the deduced amino acid sequences were divided into two subgroups.
Intron variations among different species: These DNA clones had three
introns and four exons compared with the AY736124 sequence that was isolated
from common wheat. Due to sequence mutations, the total length of the introns
varied between 754 and 779 bp. The three introns were located on 162-480, 539-731,
852-1118 bp and the length were 319, 192 and 266, respectively. A number of
ADP-ribosylation factor genes from cultivated wheat and its diploid putative
progenitors had the high sequence homology. Thus, 66 different ADP-ribosylation
factor genes were selected for further analysis. The results showed that intron
splicing had three different patterns. Intron 1 splicing occurred between GU-GA,
intron 2 splicing occurred between AG-AC and intron 3 occurred between AG-AU.
These three splicing patterns were fixed and not consistent with typical splicing
patterns which were splicing between GU-AG and special splicing site AG in intron
2 and 3. Analyzing the splice site revealed that the sequence 2-3 bp upstream
the splicing site was conserved. The sequence isolated from eukaryotes such
as Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila
melanogaster, Giardia lamblia and other lower organisms, mice, cows
and other mammals and humans had demonstrated that the structure and function
of the ARFs in plants and animals and evolution are highly conserved.
The ARF sequences of Arabidopsis thaliana, rice, Zea maize and
Brachypodium distachyon and analyzed them using online BLAST. The results
showed that the ARF in Arabidopsis thaliana also had three introns and
four exons. A comparison of published gene ARFs sequences across species revealed
an important variation in the number of introns (and consequently of exons)
present in the gene (Fig. 2): A. thaliana, Zea
maiz, Oryza sativa and Triticum aestivum L. contained 3 introns
while Brachypodium distachyon contained 2 introns. Sequence alignment
of the different exons was performed using ClustalW (data not shown) and the
result suggested a reduction in the number of exons. Exon1 in Triticum aestivum
L. could be aligned to exons (1+2) from Brachypodium distachyon (Fig.
The intron splicing patterns observed in different species were listed in Table
2. The results showed that the patterns were different among different species.
However, some common features in these patterns were found which were that intron
2 and 3 had the same splice sites AG and intron 2 had the splice sites C, intron
3 had splice sites U. That ADP-ribosylation factor might have the similar splice
pattern in common wheat and its diploid ancestors.
gDNA SNP analysis in wheat: Although the ARFs in wheat were much conserved
in their lengths and sequence homology, there were about 47 candidate SNPs in
those sequences. Among the 47 SNPs, 17 were located in exons and 30 located
|| Homologous tree based on the ARF sequences
||ARF genomic DNA structure inferred from published sequences
from different species and alignments between the exons using Clustal W
|| ARF gDNA intron splicing patterns across species
|| Information of Group-specific primer sets of each group of
|ARF gene: ADP-ribosylation factors (ARFs)
|| Haplotypes of ARF in the plant materials
The SNPs in ARFs were further investigated and the results showed that the
transition was more common than the transversions among SNPs (Cooper
and Krawczak, 1990). About 41% of the substitutions were found in the accurate
CpG site, as adenine nucleotide was known for its high mutability (Cooper
and Youssoufian, 1988). The distribution and proportion of transitions and
transversions in the ARF SNPs were investigated (Table 4).
As expected, most of the SNPs (66.34%) in ARFs were transitions and the rest
(33.66%) were transversions. In the substitution type, the transversion type
T→C was the most common while the transversions type A→G was second
The phylogenetic results and the substitution analysis showed that the SNPs
were clustered into six haplotype and three kinds of haplotypes appeared only
once. Haplotypes were associated with chromosomes. D chromosome had the haplotype
type I, Au, Am and S chromosome had the same haplotype
type 2. The only difference between type 2 and 3 was at site 371bp (T/A). There
was only 1bp difference in the remaining type.
The variations deduced by insertion, deletion and SNPs in the introns were
less than the exons. Introns had more variations and intron 1 was more conserved
than the intron 2 and intron 3.119 SNPs in about 750 bp intron sequences and
a part of the SNPs showed genome specificity.
ARF chromosome assignment by group-specific primer sets: Specific-primers
based PCR assay was carried out to identify the chromosome locations of each
group of ARFs. A total of 10 specific primer sets were designed on A and D chromosomes.
The forward primers annealed at the beginning part of the SNP domain and the
reverse primer annealed at the C-terminal domain. Due to the high sequence homology
of ARFs across species, an additional primer-template mismatched base was added
in a few primer sequences at their position 3
end (Table 3). The gDNA sequence of A and D chromosomes in
the ditelosomic lines of CS were used to carry out the specific PCR amplifications.
Therefore, each primer set should yield uniformly sized products in ditelosomiclines.
Furthermore, the chromosomal location would be consequently recognized by the
absence of the PCR products from one particular ditelosomicline which had lost
specific chromosome arms.
Two out of ten ARF groups were successfully assigned to specific chromosome
arms on the A and D chromosomes (Table 3). The ARFs isolated
from A chromosome were assigned to the long arm of chromosome 2A and the ARFs
isolated from D chromosome were assigned to the long arm of chromosome 3D. The
specific primer sets for S chromosome did not found. All primer had the same
ARF amino acid sequence analysis and protein structure prediction: The
three conserved characteristic motifs (Kahn et al.,
1995) unique to the GTP-binding protein super family involved in GTP-binding
and/or hydrolysis are: GLDAAGKT (Fig. 1), DVGGQ and ANKQD.
The structure of the ARFs includes the effector region binding to the GAPs (Amor
et al., 1994; Greasley et al., 1995;
Vitale et al., 1997); the switch 1 and switch
2 regions binding to GEF Sec7 domains (Mossessova et
al., 1998) a potential myristoylation site at Gly-2 (Kahn
et al., 1992; Antonny et al., 1997)
the N-terminal 17 amino acids which impart ARF properties to a Drosophila (Kahn
et al., 1992) residues 35-94 which activate phospholipase D and recruit
adaptor protein AP-1 (Liang et al., 1997).
|| Partial alignment of deduced amino acids sequence
The putative amino acid sequences of these ARFs showed significant sequence
homology to the ARFs from Arabidopsis thaliana, rice and other species.
The ARFs also were conserved in other functional site, such as the asparagine
glycosylation site at position 61.
The correlation between SNPs and the amino acid alteration in the ARFs was
limited. It was clear that the 73 SNPs only resulted in four amino acid variations.
As 94.5% of the candidate SNPs resulted synonymous changes (Fig.
3). Most of these variations were a potential way to obtain information
about the functional constraint via evolutionary comparisons, as it is generally
believed that conserved residues are more likely to be functionally significant
than non-conserved residues. The SNPs did not alter the amino acid sequences
of the GTP-binding and the hydrolysis domain. The results illustrated the GTP-binding
and hydrolysis functions of the ARFs were conserved. The secondary structure
of the ARFs in wheat deduced from the Swiss-Model was not altered and remained
similar with the secondary structure of the ARFs in human.
ARF expression under various stresses in wheat: In this study, the effects
of various environmental stresses, including low-temperature, ABA, PEG, NaCl,
stripe rust, on the accumulation of ARF transcription in wheat were investigated.
At the same time, the ARF expression level of 15 days post-flowering in root,
stem, flag leaf and immature embryo were analyzed. The ARF expression level
during the seedling stage and the maturity phase also were measured.
The ARF expression level was higher in root than other issue in the seeding
stage and was higher in root and embryo in the maturity phase which suggested
that the ARFs were differentially expressed in different tissue Fig.
4 (Kobayashi-Uehara et al., 2001). The results
indicated that ARFs could be more active in the parts of wheat that had higher
metabolism rate and the ARFs gene may be had higher expression in the meristem,
such as root tip and young embryo. The ARF expression in the root was also up-regulated
by 4.0 fold under the ABA stress and 2.3 fold under the PEG stress (Fig.
5 and 6). During the seeding stage, the ARF expression
level was upregulated under various environmental stresses, such as low temperature,
yellow stripe, ABA, PEG, NaCl treatment. PEG stress induced the highest ARF
expression level compared to other environmental stresses, maybe the ARF gene
regulate and control the growth of wheat in drought conditions. The ARFs expression
level was higher in leafs and roots during the seeding stage than the maturity
stage. The results showed that ARFs could be induced by several environmental
stresses (Kobayashi-Uehara et al., 2001).
||ARFs expression level of different stage in different issue.
Seeding-root, seeding-stem, seeding-leaf represent the tissue originated
from root, stem and leaf while the wheat was in seeding stage
||ARFs expression level of root under different stresses. The
ARFs expression level of root under salt, ABA and PEG stresses
||ARFs expression level of leaf under different stresses. The
ARFs expression level of leaf under salt, ABA, PEG, low temperature and
yellow rust stresses
ARFs genomic characterization in wheat: In this study, two important
features of the ARFs in wheat were found. The first feature was that ARF sequences
in wheat had high homology with other species, such as mammalian, carrot, maize
and so on. The second feature was that the ARFs in wheat contained four exons
and three introns, consistent with the ARFs in A. thaliana, H. vulgare,
Brachypodium, O. sativa and other species (Fig.
2). The genomic structure of the ARFs therefore, seemed to be very simple
and conserved in the plant kingdom. The presence of intron 2 and 3 might be
a common feature of Poaceae. Intron-1 was found in all species, but its bp length
varied, even within the same Poaceae. The Poaceae family could be further divided
based on the number of introns present: Three introns only in the sub-families
Poaceae (Wheat, Arabidopsis thaliana, Oryza) and two introns in the sub-family
Poaceae (Brachypodium). Interestingly, the length of intron-1 in Brachypodium
distachyon was equal to the total length of intron 2 and 3 in Arabidopsis
thaliana and wheat (Fig. 1, Table 2).
Intron structure in wheat and Arabidopsis all contained three introns.
The ancient species T. monococcum, T. urartu had three introns
which implied that the three introns feature could represent an ancestral state.
The introns were removed precisely which is dictated by a splicing code integrated
into the pre-mRNA. The 5 splice site (GU) 3 splice site (AG), polypyrimidine
tract and the branch point adenosine residue near the 3 splice site are
core element of the splicing code during the splicing (Wang
and Burge, 2008). Here, an alternative splicing site for ARFs which provided
an interesting model for understanding different mechanisms involved in splice
site selection. Though the cDNA sequences of the ARFs in Poaceae were conserved,
the intron splicing sites were variable. The splicing sequences of the ARFs
also were conserved. Conserved sequences in these introns are essential for
the regulation of alternative splicing in response to stress (Singh
et al., 2009). AG-rich enhancer sequence downstream of the three
introns are defined and needed for efficient recognition of the exon splice
site (Gallego et al., 1996). In this study,
the splicing was chromosome-specific.
SNPs analysis in ARFs: In this study, sequence alignment for 73 genes
derived from hexaploid and their closely related diploid wheat species were
analyzed (Triticum and Aegilops). The alignment analysis revealed
a relatively high sequence homology between different ARFs. Extron sequences
were more variable than intron terminal domain. The ARFs were divided into two
groups (Fig. 1) based on the exon sequences (Fig.
1). The ARFs also were classified into two groups based on the intron sequences
(data not shown). A marker based PCR was employed to assign the chromosome location
of each ARF group. The results indicated that two groups of ARFs were successfully
assigned to specific chromosome arms. Moreover, two subgroups with relatively
high-sequence identity were identified. The amplification results from diploid
wheat and Aegilops accessions were in agreement with those in the Chines
Spring ditelosomiclines. These results further supported the validity of ARF
classification results. Therefore, the results in this study provided the possibility
to predict the chromosome location of a new ARF using its cDNA sequence. The
classification based on SNPs could simplify the characterization of ARF sequences.
This was probably due to the limited number of genotypes of diploid species
from which cultivated wheat was derived. Wheat is a hexaploid with genome constitution
AABBDD which generally contains triplicated homologous genes derived from three
diploid ancestral species (Feldman, 2001). There could
be three possibilities for three chromosome-specific markers of homoeological
genes in the polyploidy (Wendel, 2000). Although the
chromosomal location of S-genome because S genome-specific PCR primer was unavailable,
maybe the ARFs of B genome is homologous to ARFs of D genome located on based
on the following three facts: Firstly, the ARFs of S genome showed high homology
to D genome. Secondly, there were three homologous ARFs in the wheat genome
(Kobayashi-Uehara et al., 2001) which was consistent
to this results. Similar situations in WFLs in wheat have also been shown (Shitsukawa
et al., 2006). Lastly, the phylogenetic analysis could not differentiate
S and D genome and the conclusion also tested and verified the molecular marker
A new molecular marker system termed Intron Length Polymorphism (ILP) has been
developed in rice by comparing the draft genomic sequences of indica cultivar
and japonica cultivar (Wang et al., 2006). ILP
is a codominant marker and can be conveniently detected by PCR with a pair of
primers annealing at the flanking exons. ILPs are compatible among different
species (Wang et al., 2006) because the exon-intron
structures of ARFs are highly conserved (Rogozin et
al., 2003; Roy and Gilbert, 2005; Lin
et al., 2006; Yang et al., 2007).
In addition, ILPs mainly exist in low frequency among (sub) species or higher
taxonomic ranks. It is possible that these subspecies-specific ILP (SSILP) markers
could well preserve the genetic information from wild progenitors and therefore
could reflect the original genetic differentiation between the ancestors of
wheat and its diploid putative progenitors. Moreover, introns usually have no
biological functions. The above desirable features suggest that ILP markers
should be quite suitable for studying wheat evolution. In the work described
here, SSILP markers could be utilized to investigate the genetic differentiation
of wheat in relation to the genetic differentiation among other species. Through
the ARFs sequence analysis, that ARF genome-specific markers had two important
features. First, they reflected the genetic differentiation between the two
subspecies (Table 1). Second, the two subspecies-specific
alleles were generally conserved. New markers might be developed for ARFs to
investigate the evolution of wheat. In this study, the results indicated that
the A- and D-genome had been clearly differentiated into two distinct groups.
This suggested that genome-specific extron sequences could be found as neutral
marker. Now Subspecies-specific Intron Length Polymorphism (SSILP) is developed
as a neutral marker for selection in general because introns usually do not
have biological functions in rice (Zhao et al.,
ARF expression: The expressions of the ARFs were probed by antisense
suppression. Antisense suppressed plants showed lowered stature and some plants
with severe phenotypes died or were infertile (Gebbie et
al., 2005). As for wheat, the protein level of ARFs was higher in root
and flowers than in shoots, suggesting that wheat roots and flowers accumulated
a relatively abundant amount of ARFs (Kobayashi-Uehara
et al., 2001). The disruption of ARF1 could cause cold sensitivity
and retarded growth which indicated that ARF1 played important roles in promoting
cell survival (Stearns et al., 1990). Northern
analysis of total RNA showed that ARFs were highly expressed in young seedlings
and in seeds at the early developmental stage, but to a lesser extent in cultured
cells (Higo et al., 1994). In this regard, the
effects of various environmental stresses on ARF expression were focused and
found that ARF expression level could be altered by different conditions, including
cold stress. These results were not consistent with an earlier finding that
the ARFs wheat was not cold inducible (Kobayashi-Uehara
et al., 2001).
In this study, PEG stress induced the highest ARF expression level, followed
by the stripe rust and ABA stress. RT-PCR revealed that ARFs were expressed
at 2.0, 1.8 and 1.4 fold higher under PEG, stripe rust and ABA stressed, respectively.
The ARFs could play an important role in DNA repair by self-modifying the transcripts.
Cell division and cell expansion both depend on vesicle trafficking to deposit
new wall material, to secrete proteins (e.g., expansins) that promote cell wall
biogenesis and to deliver new plasma membrane proteins for cellulose synthesis
and other needs (Samuels et al., 1995; Thiel
and Battey, 1998; Cosgrove, 2000).
The relative abundance of ARF mRNA in roots and flag-leafs may indicate a high
level of vesicular transporting activity in these tissues of wheat plants. The
ARFs have been proposed to function through direct activation of enzyme activities,
local alterations in lipid composition, or recruitment of proteins to the membrane
(Donaldson et al., 1992). Therefore, the relative
abundance of ARF mRNA in roots and embryos may indicate a high level of vesicular
transporting activity in these tissues (Yao et al.,
2009). These theories can explain why the expression level of ARFs was upregulated
under ABA and PEG stresses, since cell production rate and final cell size were
both reduced. Changed time-to-flowering, apical dominance and fertility may
reflect alterations to hormonal and other signaling pathways with which ARFs
may be associated with (Gebbie et al., 2005).
In conclusion, the ARFs gene sequences, SNP site, splicing pattern and gene
expressions level under stresses were investigated in this study. These results
showed that the ARFs gene sequences were conserved in the Poaceae family and
the introns might have the similar splice pattern in common wheat and its diploid
ancestors. The chromosome locations of the gene were located on 3AL and 2DL.
The GTP-binding area of the ARFs was conserved and the secondary structure of
ARFs in wheat was not altered with the nucleotide changing. Under environmental
stresses, the gene expression level was higher than control. The ARFs were more
active in the parts of wheat that had higher metabolism rate, such as root tip
and young embryo.
This study was supported by the National Basic Research Program of China (973
Program 2010CB134400) and China Transgenic Research Program (2011ZX08002).
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